drug discovery and evaluation || analgesic, anti-inflammatory, and anti-pyretic activity

Chapter HAnalgesic, Anti-inflammatory, and Anti-pyretic Activity1

H.1 Central Analgesic Activity . . . . . . 984H.1.0.1 General Considerations . . . . . . . . . . 984H.1.1 In Vitro Methods

for Central Analgesic Activity . . . . 985H.1.1.1 Survey . . . . . . . . . . . . . . . . . . . . . . . . . . 985H.1.1.2 3H-Naloxone Binding Assay . . . . . 989H.1.1.3 3H-Dihydromorphine Binding

to μ Opiate Receptorsin Rat Brain . . . . . . . . . . . . . . . . . . . . . 990

H.1.1.4 3H-Bremazocine Bindingto κ Opiate Receptorsin Guinea Pig Cerebellum . . . . . . . . 991

H.1.1.5 Inhibition of Enkephalinase . . . . . . 993H.1.1.6 Nociceptin . . . . . . . . . . . . . . . . . . . . . . 994H.1.1.6.1 General Considerations

on Nociceptin . . . . . . . . . . . . . . . . . . . 994H.1.1.6.2 Receptor Binding of Nociceptin . . 995H.1.1.6.3 Bioassays for Nociceptin . . . . . . . . . 996H.1.1.7 Vasoactive Intestinal Polypeptide

(VIP) and Pituitary AdenylateCyclase-Activating Peptide(PACAP) . . . . . . . . . . . . . . . . . . . . . . . . 998

H.1.1.8 Cannabinoid Activity . . . . . . . . . . . . 1000H.1.1.8.1 General Considerations

on Cannabinoids . . . . . . . . . . . . . . . . . 1000H.1.1.8.2 Receptor Binding

of Cannabinoids . . . . . . . . . . . . . . . . . 1003H.1.1.9 Vanilloid (Capsaicin) Activity . . . . 1005H.1.1.9.1 General Considerations

on Vanilloids . . . . . . . . . . . . . . . . . . . . 1005H.1.1.9.2 Vanilloid Receptor Binding . . . . . . . 1007H.1.1.9.3 Evaluation of Vanilloid

Receptor Antagonists . . . . . . . . . . . . 1008H.1.2 In Vivo Methods for Testing

Central Analgesic Activity . . . . . . . 1010H.1.2.1 General Considerations . . . . . . . . . . 1010H.1.2.2 Haffner’s Tail Clip Method . . . . . . . 1010H.1.2.3 Radiant Heat Method . . . . . . . . . . . . 1011

1Contributions to earlier editions by R. Schleyerbach, K.U. Wei-thmann and R.R. Bartlett.

H.1.2.4 Hot Plate Method . . . . . . . . . . . . . . . . 1013H.1.2.5 Tail Immersion Test . . . . . . . . . . . . . . 1014H.1.2.6 Electrical Stimulation of the Tail . . 1016H.1.2.7 Grid Shock Test . . . . . . . . . . . . . . . . . 1017H.1.2.8 Tooth Pulp Stimulation . . . . . . . . . . . 1018H.1.2.9 Monkey Shock Titration Test . . . . . 1019H.1.2.10 Formalin Test in Rats . . . . . . . . . . . . 1020H.1.2.11 Neuropathic Pain . . . . . . . . . . . . . . . . 1022H.1.2.11.1 General Considerations . . . . . . . . . . 1022H.1.2.11.2 Chronic Nerve Constriction Injury 1022H.1.2.11.3 Peripheral Nerve Injury Model . . . 1024H.1.2.11.4 Spared Nerve Injury Model. . . . . . . 1025H.1.2.11.5 Spinal Cord Injury . . . . . . . . . . . . . . . 1026H.1.2.11.6 Chemotherapy-Induced Pain . . . . . 1028H.1.2.11.7 Trigeminal Neuropathic

Pain Model . . . . . . . . . . . . . . . . . . . . . . 1029H.1.2.11.8 Migraine Model in Cats . . . . . . . . . . 1030H.1.3 Side Effects

of Central Analgesic Drugs . . . . . . . 1030H.2 Peripheral Analgesic Activity . . . 1030H.2.0.1 General Considerations . . . . . . . . . . 1030H.2.0.2 Writhing Tests . . . . . . . . . . . . . . . . . . . 1031H.2.0.3 Pain in Inflamed Tissue

(RANDALL-SELITTO-Test) . . . . . 1032H.2.0.4 Mechanical Visceral Pain Model

in the Rat . . . . . . . . . . . . . . . . . . . . . . . . 1035H.2.0.5 Antagonism Against Local

Effects of Bradykinin . . . . . . . . . . . . 1036H.2.0.6 Effect of Analgesics

on Spinal Neurons . . . . . . . . . . . . . . . 1038H.2.0.7 Antagonism to Nerve

Growth Factor . . . . . . . . . . . . . . . . . . . 1041H.2.0.7.1 General Considerations

on Nerve Growth Factor . . . . . . . . . . 1041H.2.0.7.2 In Vitro Assays

of Nerve Growth Factor . . . . . . . . . . 1041H.2.0.7.3 In Vivo Assays of Nerve

Growth Factor Antagonism . . . . . . . 1043H.3 Anti-Inflammatory Activity . . . . . 1047H.3.0.1 General Considerations . . . . . . . . . . 1047

984 Chapter H · Analgesic, Anti-Inflammatory, and Anti-Pyretic Activity

H.3.1 In Vitro Methodsfor Anti-Inflammatory Activity . . . 1047

H.3.1.1 General Considerations . . . . . . . . . . 1047H.3.1.2 3H-Bradykinin Receptor Binding . 1048H.3.1.3 Substance P

and the Tachykinin Family . . . . . . . 1051H.3.1.3.1 General Considerations . . . . . . . . . . 1051H.3.1.3.2 3H-Substance P Receptor Binding 1053H.3.1.3.3 Neurokinin Receptor Binding . . . . 1053H.3.1.3.4 Characterization of Neurokinin

Agonists and Antagonistsby Biological Assays . . . . . . . . . . . . . 1055

H.3.1.4 Assay of PolymorphonuclearLeukocyte Chemotaxis In Vitro . . . 1058

H.3.1.5 Polymorphonuclear LeukocytesAggregation Induced by FMLP . . . 1059

H.3.1.6 Constitutive and InducibleCellular Arachidonic AcidMetabolism Metabolism In Vitro . 1060

H.3.1.6.1 Formation of Leukotriene B4

in Human White Blood CellsIn Vitro . . . . . . . . . . . . . . . . . . . . . . . . . 1061

H.3.1.6.2 Formation of LipoxygenaseProducts from 14C-ArachidonicAcid in Human Polymorpho-nuclear Neutrophils (PMN)In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . 1061

H.3.1.6.3 Formation of Eicosanoidsfrom 14C-Arachidonic Acidin Human Platelets In Vitro . . . . . . . 1062

H.3.1.6.4 Stimulation of InducibleProstaglandin Pathwayin Human PMNL . . . . . . . . . . . . . . . . 1062

H.3.1.6.5 COX-1 and COX-2 Inhibition . . . . 1063H.3.1.7 Influence of Cytokines . . . . . . . . . . . 1069H.3.1.7.1 Induced Release of Cytokines

(Interleukin-1α, IL-1β, IL-6, IL-8and TNFα) from Human WhiteBlood Cells In Vitro . . . . . . . . . . . . . 1069

H.3.1.7.2 Flow Cytometric Analysisof Intracellular Cytokines . . . . . . . . 1071

H.3.1.7.3 Screening for Interleukin-1Antagonists . . . . . . . . . . . . . . . . . . . . . 1072

H.3.1.7.4 Inhibition of Interleukin-1βConverting Enzyme (ICE) . . . . . . . . 1074

H.3.1.7.5 Nuclear Factor-κB . . . . . . . . . . . . . . . 1076H.3.1.8 TNF-α Antagonism . . . . . . . . . . . . . . 1081H.3.1.8.1 General Considerations . . . . . . . . . . 1081H.3.1.8.2 Inhibition of TNF-α Release . . . . . 1083H.3.1.8.3 Effect of TNF-α Binding . . . . . . . . . 1084H.3.1.9 Binding to Interferon Receptors . . 1087H.3.1.10 Chemokine Antagonism. . . . . . . . . . 1089

H.3.1.11 Influence of PeroxisomeProliferator-ActivatedReceptors (PPARs)on Inflammation . . . . . . . . . . . . . . . . . 1091

H.3.1.12 Binding to Histamine H4 Receptor 1093H.3.2 In Vivo Methods

for Anti-inflammatory Activity . . . 1094H.3.2.1 General considerations . . . . . . . . . . . 1094H.3.2.2 Methods for Testing Acute

and Subacute Inflammation . . . . . . . 1095H.3.2.2.1 Ultraviolet Erythema

in Guinea Pigs . . . . . . . . . . . . . . . . . . . 1095H.3.2.2.2 Vascular Permeability . . . . . . . . . . . . 1096H.3.2.2.3 Inhibition of Leukocyte Adhesion

to Rat Mesenteric VenulesIn Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . 1098

H.3.2.2.4 Oxazolone-Induced Ear Edemain Mice . . . . . . . . . . . . . . . . . . . . . . . . . . 1099

H.3.2.2.5 Croton-oil Ear Edemain Rats and Mice . . . . . . . . . . . . . . . . . 1100

H.3.2.2.6 Paw Edema . . . . . . . . . . . . . . . . . . . . . . 1103H.3.2.2.7 Pleurisy Test . . . . . . . . . . . . . . . . . . . . . 1106H.3.2.2.8 Granuloma Pouch Technique . . . . . 1107H.3.2.2.9 Urate-Induced Synovitis . . . . . . . . . 1109H.3.2.3 Methods for Testing

the Proliferative Phase(Granuloma Formation) . . . . . . . . . . 1110

H.3.2.3.1 Cotton Wool Granuloma . . . . . . . . . 1110H.3.2.3.2 Sponge Implantation Technique . . 1111H.3.2.3.3 Glass Rod Granuloma . . . . . . . . . . . . 1113H.3.3 Side Effects

of Anti-inflammatory Compounds 1113H.4 Antipyretic Activity . . . . . . . . . . . . . 1113H.4.0.1 General Considerations . . . . . . . . . . 1113H.4.0.2 Antipyretic Testing in Rats . . . . . . . 1114H.4.0.3 Antipyretic Testing in Rabbits . . . . 1115

H.1Central Analgesic Activity

H.1.0.1General Considerations

Pain is a symptom of many diseases requiring treat-ment with analgesics. Severe pain due to cancer metas-tases needs the use of strong analgesics, that meansopioid drugs. The addiction liability of opioids led tointensive research for compounds without this side ef-fect. Many approaches have been used to differentiatethe various actions of strong analgesics by developinganimal models not only for analgesic activity but also

H.1 · Central Analgesic Activity 985

for addiction liability. Several types of opioid recep-tors have been identified in the brain allowing in vitrobinding tests. However, the in vitro tests can only par-tially substitute for animal experiments involving pain.Pain is a common phenomenon in all animals, at leastin vertebral animals, similar to that felt by man. Anal-gesic effects in animals are comparable with the thera-peutic effects in man. Needless to say, that in every in-stance painful stimuli to animals must be restricted asmuch as possible. Painful stimuli can consist of directstimulation of the efferent sensory nerves or stimula-tion of pain receptors by various means such as heator pressure. The role of endogenous peptides such asenkephalins and endorphins gives more insight intobrain processes and the action of central analgesics.

Pain can also be elicited by inflammation. Progresshas been made in elucidating the role of various en-dogenous substances such as prostaglandins and pep-tides in the inflammatory process. Most of the so callednon-steroidal anti-inflammatory agents have also anal-gesic activity. Lim and Guzman (1968) differentiatedbetween antipyretic analgesics causing analgesia byblocking impulse generation at pain receptors in theperiphery while the narcotic analgesics block synaptictransmission of impulses signaling pain in the centralnervous system. An old but excellent survey on meth-ods being used to test compounds for analgesic activityhas been provided by Collier (1964). Today, the classi-fication into central and peripheral analgesics is defini-tively too simplified (Bannwarth et al. 1993) but pro-vides a guide for differentiation by pharmacologicalmethods.

REFERENCES AND FURTHER READINGBannwarth B, Demotes-Mainard F, Schæverbeke T, Dahais J

(1993) Where are peripheral analgesics acting? AnnRheum Dis 52:1–4

Besson JM, Chaouch A (1987) Peripheral and spinal mecha-nisms of nociception. Physiol Rev 67:67–186

Collier HOJ (1964) Analgesics. In: Laurence DR, Bacharach AL(eds) Evaluation of Drug Activities: Pharmacometrics.pp 183–203. Academic Press London, New York

Lim RKS, Guzman F (1968) Manifestations of pain in analgesicevaluation in animals and man. In: Soulairac A, Cahn J,Charpentier J (eds) Pain. Academic Press, London, NewYork, pp 119–152

H.1.1In Vitro Methods for Central Analgesic Activity

H.1.1.1Survey

In 1973, high-affinity stereospecific binding of radio-labeled opiate compounds by CNS membrane prepa-

rations was reported (Pert and Snyder 1973; Simonet al. 1973; Terenius 1973). The in vivo pharma-cological potency of opiate agonists and antagonistsparallels the in vitro displacement of 3H-naloxone,a potent narcotic antagonist. Based on these find-ings, the 3H-naloxone binding assay was introducedfor evaluation of potential analgesics with opiate-like properties. According to different pharmacolog-ical profiles of opiates, several receptor types havebeen identified designated as μ, κ , δ, and σ recep-tor (μ for morphine = MOP receptor, κ for ketocy-clazocine = KOP receptor, δ for deferens because itwas first identified in mouse vas deferens = DOP re-ceptor). The σ receptor (σ for SKF10047) was onlyinitially classified as an opioid receptor (see below).Several reviews on opioid receptors have been pub-lished: Knappe et al. (1995), Mansour et al. (1995),Satoh and Minami (1995), Dhawan et al. (1996), Singhet al. (1997), Standifer and Pasternak (1997), Lawet al. (2000), Snyder and Pasternak (2003), Janeckaet al. (2004), Eguchi (2004), and Waldhoer et al.(2004).

The opioid receptors were reclassified according torecommendations of the International Union of Physi-ological Sciences, the International Union of Pharma-cology (IUPHAR; Dhawan et al. 1996, 1998; Alexan-der and Peters 2000). This nomenclature applies anabbreviation of the generic term for the family (OPfor opioid) and a subscript number. OP1 stands forδ, OP2 for κ , and OP3 for μ receptor.

For the μ receptor, subtypes named μ1 and μ2have been described (Fowler and Fraser 1994; Traynor1994; Pasternak 2001). Analgesia is thought to in-volve activation of μ receptors (largely at supraspinalsites) and κ receptors (principally within the spinalcord); δ receptors may also be involved at the spinaland supraspinal level. Other consequences of μ acti-vation include respiratory depression, miosis, reducedgastrointestinal motility, and euphoria. The μ1 recep-tors are postulated to mediate the supraspinal anal-gesic action and the μ2 receptors to mediate respi-ratory depression and suppression of gastrointestinalmotility. Moreover, different effects on heart rate weredescribed (Paakkari et al. 1992). Two endogenous pep-tides were described, named endomorphins, as ag-onists with high specific affinity for the μ-receptor(Hackler et al. 1997; Zadina et al. 1997, 1999; Horvath2000).

Several studies provide evidence for the existenceof δ-opioid receptor subtypes (Sofuoglu et al. 1991;Porreca et al. 1992; Horan et al. 1993; Miyamoto et al.1993; Tiseo and Yaksh 1993; Burkey et al. 1998).


Binding studies with δ opioid receptors have been per-formed by Mosberg et al. (1983). Simonin et al. (1994)reported the genomic organization, cDNA cloning, thefunctional expression in COS cells, and the distribu-tion in human brain of the human δ-opioid recep-tor.

Endogenous ligands for δ receptors are enkepha-lins.

A rat κ opioid receptor has been cloned (Menget al. 1993). Evidence for different subtypes of the κ-opioid receptor is available (Zukin et al. 1988; Clarket al. 1989; Rothman et al. 1989, 1992, 1993; Wolle-mann et al. 1993). Simonin et al. (1995) describedcDNA and genomic cloning, chromosomal assign-ment, functional expression, pharmacology, and ex-pression pattern in the central nervous system of theδ-opioid receptor in humans.

Salvinorin A – derived from Salvia divinorum,a hallucinogenic plant used by Mazatec Indiansof Mexico for traditional spiritual ceremonies – isa highly selective κ-opioid receptor agonist withantinociceptive effects (Yan and Roth 2004; Johnet al. 2006; Rothman et al. 2006; Stewart et al. 2006;Vortherms and Roth 2006).

Endogenous ligands for κ receptors are dynor-phins.

With the development of highly selective ligands ithas become possible to label selectively each of theμ-,δ-, and κ-opioid binding sites.

The μ-binding sites are labeled with [3H]-[Tyr-D-Ala2,MePhe4,Gly-ol5]enkephalin (Kosterlitz andPaterson 1981), 125I-FK 33–824 (Moyse et al.1986), [3H]-Tyr-Pro-MePhe-D-Pro-NH2 (PL O17;Hawkins et al. 1987), or [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] (CTOP); Hawkins et al.1989), the δ-binding sites with [3H]-[D-Pen2,D-Pen5]enkephalin (Akiyama et al. 1985; Cotton et al.1985; Mosberg et al. 1987); [3H]-D-Ser2 (O-tert-butyl),Leu5]enkephalyl-Thr6 (Delay-Goyet et al.1988) or [3H]-[D-Pen2-pClPhe4,D-Pen5]enkephalin(Vaughan et al. 1989); [3H]TIPP (Nevin et al. 1993),and the κ-sites with [3H]-U-69593 (Lahti et al.1985; Maguire et al. 1992), [3H]-PD117302 (Clarket al. 1988), or [3H]-CI-977 (Boyle et al. 1990) or[3H]norBNI (Marki et al. 1995).

Cloning and molecular biology of opioid receptorshas been reviewed (Reisine and Bell 1993).

Advances in research on non-peptide opioid recep-tor ligands were published by Kaczor and Matusiuk(2002).

Exploring the opioid system by gene knockout wasdescribed by Kieffer and Gaveriaux-Ruff (2002).

Oligomerization of opioid receptors and the gener-ation of novel signaling units was discussed by Levacet al. (2003).

OTHER RECEPTORSThere is some evidence that other opioid receptorsmay exist, such as a β-endorphin-sensitive ε recep-tor (Wüster et al. 1981). The ζ receptor (Zagon et al.1991) and a high affinity binding site referred to as theλ site (Grevel et al. 1985) may also be part of the opi-oid receptor system.

The existence of a σ receptor was first postu-lated by Martin et al. (1976) to account for the psy-chotomimetic effects of N-allylnormetazocine (SKF10,047) in the chronic spinal dog. σ Binding sites wereproposed to be identical to phencyclidine binding sitesbased on the finding that phencyclidine generalized to(+)-SKF 10,047 in drug discrimination tests. Furtherwork led to the application of the term σ to an uniqueclass of non-opiate, non-phencyclidine sites that mayserve as receptors for an as yet unidentified neuro-modulator or neurotransmitter (Monnet et al. 1994). Atleast two subtypes of binding sites, σ 1 and σ 2, are pro-posed (Bowen et al. 1989; Itzhak and Stein 1991; Kar-bon et al. 1991; Knight et al. 1991; Connick et al. 1992;Quirion et al. 1992; Leitner et al. 1994). Radioligandsfor σ receptors (Weber et al. 1986; de Costa et al.1989) and for subtype σ 1 (Matsuno et al. 1996) andsubtype σ 2 (Mach et al. 1999) were described. Phar-macological studies indicate a role of σ receptors notonly in analgesia (Mach et al. 1999), but also in motorfunction (Walker et al. 1993), schizophrenia (Debon-nel and de Montigny 1996; Guitard et al. 1998; Taka-hashi et al. 1999) and learning and memory (Mauriceet al. 1999).

The heterogeneity of opioid receptors has beenstudied in isolated tissue preparations in which neu-rotransmission is sensitive to inhibition by opioids.The relative potencies of opioid agonists are assessedby their ability to inhibit the electrically evoked con-tractions of isolated tissue preparations from five dif-ferent species: the contractions of the mouse vas def-erens are inhibited by μ-, δ-, and κ-agonists (Maguireet al. 1992), those of the guinea-pig myentericplexus-longitudinal muscle preparation by μ- and κ-agonists (Berzetei-Gurske 1992), those of the rabbitvas deferens by κ-agonists, and those of the ham-ster vas deferens by δ-agonists (Sheehan et al. 1986).The contractions of the rat vas deferens are inhibitedmainly, but not exclusively, by δ-agonists. The actionsof β-endorphin in the rat vas deferens are mediated bya further type of opioid receptors, termed ε-receptor


(Wüster et al. 1981; Corbett et al. 1992; Smith andLeslie 1992).

REFERENCES AND FURTHER READINGAkiyama K, Gee KW, Mosberg HI, Hruby VJ, Yamamura

HI (1985) Characterization of [3H][2-D-penicillamine,5-D-penicillamine]-enkephalin binding to δ-opiate receptorsin the rat brain and neuroblastoma-glioma hybrid cell line(NG 108–15). Proc Natl Acad Sci USA 82:2543–2547

Alexander SPH, Peters JA (2000) 2000 Receptor and IonChannel Nomenclature Supplement. Trends Pharmacol Scipp 70–71

Berzetei-Gurske IP, Troll L (1992) The μ-opioid activity of κ-opioid receptor agonist compounds in the guinea pig ileum.Eur J Pharmacol 212:283–286

Bowen WD, Hellewell SB, McGarry KA (1989) Evidence fora multi-site model of the rat brain σ receptor. Eur J Phar-macol 163:309–318

Boyle SJ, Meecham KG, Hunter JC, Hughes J (1990) [3H]-CI-977: a highly selective ligand for the k-opioid receptor inboth guinea-pig and rat forebrain. Mol Neuropharmacol1:23–29

Burkey TH, Ehlert FJ, Hososhata Y, Quok RM, Cowell S, Hoso-hata K, Stropova VD, Li X, Slate C, Nagase H, Porreca F,Hruby VJ, Roeske WR, Yamamura HI (1998) The efficacyof opioid receptor-selective drugs. Life Sci 62:1531–1536

Clark CR, Birchmore B, Sharif NA, Hunter JC, Hill RG,Hughes J (1988) PD117302: a selective agonist for the κ-opioid receptor. Br J Pharmacol 93:618–626

Clark JA, Liu L, Price M, Hersh B, Edelson M, Pasternak GW(1989) Kappa opiate receptor multiplicity: Evidence fortwo U50,488 sensitive k1 subtypes and a novel κ3 subtype.J Pharmacol Exp Ther 251:461–468

Corbett AD, Paterson SJ, Kosterlitz HW (1992) Selectivity ofligands for opioid receptors. In: Herz A, Akil H, Simon EJ(eds) Opioids I, Handbook of Experimental PharmacologyVol 104/I, Chapter 26, pp 645–679. Springer Berlin, Hei-delberg, New York

Connick JH, Hanlon G, Roberts J, France L, Fox PK, Nichol-son CD (1992) Multiple σ binding sites in guinea-pig andrat brain membranes: G-protein interactions. Br J Pharma-col 107:726–731

Cotton R, Kosterlitz HW, Paterson SJ, Rance MJ, Traynor JR(1985) The use of [3H]-[D-Pen2,D-Pen5]enkephalin asa highly selective ligand for the δ-binding site. Br J Phar-macol 84:927–932

Bebonnel G, de Montigny C (1996) Modulation of NMDA anddopaminergic neurotransmissions by sigma ligands: possi-ble implications for the treatment of psychiatric disorders.Life Sci 58:721–733

de Costa BR, Bowen WD, Hellewell, Walker JM, Thurkauf A,Jacobson AE, Rice KC (1989) Synthesis and evaluation ofoptically pure [3H]-(+)-pentazocine, a highly potent and se-lective ligand for σ receptors. FEBS Lett 251:53–58

Delay-Goyet P, Seguin C, Gacel G, Roques BP (1988) [3H]-[-D-Ser2 (O-tert-butyl),Leu5]enkephalyl-Thr6 and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6(O-tert-butyl). Two newenkephalin analogs with both a good selectivity and highaffinity towards δ-opioid binding sites. J Biol Chem263:4124–4130

Dhawan BN, Cesselin F, Rhaghubir R, Reisine T, Bradley PB,Portoghese PS, Hamon M (1996) International Unionof Pharmacology. XII. Classification of opioid receptors.Pharmacol Rev 48:567–592

Dhawan BN, Raghubir R, Hamon M, NC-IUPHAR Subcom-mittee on Opioid Receptors (1998) Opioid receptors. The

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Eguchi M (2004) Recent advances in selective opiod agonistsand antagonists. Med Res Rev 24:182–212

Fowler CJ, Fraser GL (1994) μ-, δ-, κ-Opiod receptors and theirsubtypes: a critical review with emphasis on radioligandbinding experiments. Neurochem Int 24:401–426

Goldstein A, Naidu A (1989) Multiple opioid receptors: ligandselectivity profiles and binding site signatures. Mol Phar-macol 36:265–272

Grevel J, Yu V, Sadee W (1985) Characterization of a la-bile naloxone binding site in rat brain. J Neurochem44:1647–1656

Guitard X, Codony X, Ballarin N, Dordal A, Farre AJ (1998)E-5842: A new potent and preferential sigma ligand. Pre-clinical pharmacological profile. CNS Drug Rev 4:201–224

Hackler L, Zadina JE, Ge L-G, Kastin AJ (1997) Isolation of rel-atively large amounts of endomorphin-1 and endomorphin-2 from human brain cortex. Peptides 18:1635–1639

Hawkins KN, Morelli M, Gulya K, Chang KJ, Yamamura HI(1987) Autoradiographic localization of [3H][MePhe3,D-Pro4] morphiceptin ([3H]PL O17) to μ-opioid receptors inrat brain. Eur J Pharmacol 133:351–352

Hawkins KN, Knapp RJ, Lui GK, Gulya K, Kazmier-ski W, Wan YP, Pelton JT, Hruby VJ, Yamamura HI(1989) [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP), a potent and highly selective peptidefor μ-opioid receptors in rat brain. J Pharmacol Exp Ther248:73–80

Horan PJ, Wild KD, Misicka A, Lipkowski A, Haaseth RC,Hruby VJ, Weber SJ, Davis TP, Yamamura HI, Por-reca F (1993) Agonist and antagonist profiles of [D-Ala2,Glu4]deltorphin and its [Cys4]- and [Ser4]-substitutedderivatives: further evidence for opioid delta receptor mul-tiplicity. J Pharmacol Exp Ther 265:896–902

Horvath G (2000) Endomorphin-1 and endomorphin-2: pharma-cology of the selective endogenous μ-opioid receptor ago-nists. Pharmacol Ther 88:437–463

Itzhak Y, Stein I (1991) Regulation of σ receptors and respon-siveness to guanine nucleotides following repeated expo-sure of rats to haloperidol: further evidence of multiple σbinding sites. Brain Res 566:166–172

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H.1.1.23H-Naloxone Binding Assay

PURPOSE AND RATIONALEA good correlation between the in vivo pharmacolog-ical potency of opiate agonists and antagonists withtheir ability to displace radiolabeled naloxone has beenreported. The later discovery that Na+ (100 mM) en-hances the binding of antagonists and reduces the bind-ing of agonists has led to the development of an as-say which is used to classify compounds as opiate ag-onists, mixed agonist-antagonists and antagonists bydetermining the IC50 values for 3H-Naloxone in thepresence or absence of Na+.

PROCEDUREReagents[N-allyl-2,3-3H] Naloxone (38–58 Ci/mmol) is ob-tained from New England Nuclear.

For IC50 determinations 3H-naloxone is made up toa concentration of 100 nM and 50 µl is added to eachtube yielding a final concentration 5 nM in the assay.

Levorphanol tartrate is obtained from HoffmannLaRoche. A stock solution of 1 mM levorphanol ismade up in distilled water. This stock is diluted1:200 in distilled water and 20 µl is added to 3 tubes todetermine stereospecific binding yielding a final con-centration of 0.1 µM in the assay.

Dextrorphan tartrate is obtained from HoffmannLaRoche. A stock solution of 1 mM dextrophan ismade up in distilled water. This stock is diluted 1:200in distilled water and 20 µl is added to the tubes con-taining the various concentrations of test drug and thetubes for total binding.

Test compounds: For most assays, a 1 mM stocksolution is made up in a suitable solvent and seriallydiluted, such that the final concentration in the assay


ranges from 10−5 to 10−8 M. At least 7 concentrationsare used for each assay. Higher or lower concentrationsmay be used, depending on the potency of the drug.

Tissue PreparationMale Wistar rats are decapitated and their brainsrapidly removed. Whole brains minus cerebella areweighed and homogenized in 50 volumes of ice-cold0.05 M Tris buffer with a Tekmar tissue homogenizer.The homogenate is centrifuged at 40,000 g for 15 min,the supernatant is decanted and the pellet resuspendedin fresh buffer and recentrifuged at 40,000 g. The finalpellet is resuspended in the original volume of fresh0.05 M Tris buffer. This yields a tissue concentrationin the assay of 10 mg/ml.

Assay

310 µl H2O

20 µl 5 µM dextrorphan (total binding) or

5 µM levorphanol (non-specific binding)

50 µl 2 M NaCl or H2O

50 µl 0.5 M Tris buffer, pH 7.7

20 µl drug or vehicle

50 µl 3H-naloxone

500 µl tissue suspension.

The tubes are incubated for 30 min at 37°C. The as-say is stopped by vacuum filtration through WhatmanGF/B filters which are then washed 3 times with ice-cold 0.05 M Tris buffer, pH 7,7. The filters are thencounted in 10 ml of Liquiscint liquid scintillation cock-tail. Stereospecific binding is defined as the differencebetween binding in the presence of 0.1 µM dextrorphanand 0.1 µM levorphanol. Specific binding is roughly1% of the total added ligand and 50% of the totalbound in the absence of Na+ and 2% of the total addedligand and 65% of the total bound ligand in the pres-ence of Na+ (100 mM). The increase in binding is dueto an increase in specific binding.

EVALUATIONData are converted into % stereospecific 3H-naloxonebinding displaced by the test drug. IC50 values are de-termined from computer-derived log-probit analysis.The sodium shift is calculated from IC50 values withand without NaCl. High sodium shifts are found withagonists, low values with antagonists and medium val-ues with mixed agonists-antagonists.

Data can be analyzed using a computer program asdescribed by McPherson (1985).

REFERENCES AND FURTHER READINGHubbard JW, Locke KW, Forster HV, Brice AG, Pan LG,

Lowry TF, Forster AML, Forster MA, Cornfeldt M,Vanselous CL, Hamer RRL, Glamkowski EJ, Fielding S(1992) Cardiorespiratory effects of the novel opioid anal-gesic HP 736 in the anesthetized dog and conscious goat.J Pharmacol Exp Ther 260:1268–1277

McPherson GA (1985) Analysis of radioligand binding experi-ments. A collection of computer programs for the IBM PC.J Pharmacol Meth 14:213–228

Mini-Symposium (1981) The in vivo differentiation of opiate re-ceptors. Life Sci 28:1543–1584

Pert CB, Snyder SH (1973) Properties of opiate-receptor bindingin rat brain. Proc. Natl. Acad. Sci, USA 70:2243–2247

Pert CB, Snyder SH (1974) Opiate receptor binding of agonistsand antagonists affected differentially by sodium. MolecPharmacol 10:868–879

Pert CB, Snyder SH (1975) Differential interactions of agonistsand antagonists with the opiate receptor. In: Snyder andWatthysse (eds) Opiate Receptor Mechanisms. MIT PressCambridge. pp 73–79

Pert CB, Pasternak G, Snyder SH (1973) Opiate agonists andantagonists discriminated by receptor binding in brain. Sci-ence 182:1359–1361

Wolozin BL, Nishimura S, Pasternak GW (1982) The binding ofκ- and σ -opiates in rat brain. J Neurosci 2:708–713

H.1.1.33H-Dihydromorphine Binding to μ Opiate Receptorsin Rat Brain

PURPOSE AND RATIONALEμ Receptors are considered to mediate the supraspinalactivity of opioids. 3H-Dihydromorphine (3H-DHM)exhibits some selectivity for the μ receptor, a highaffinity opiate binding site. The test is used to de-tect compounds that inhibit binding of 3H-DHM ina synaptic membrane preparation obtained from ratbrain.

PROCEDUREReagents[1,7,8-3H]Dihydromorphine (3H-DHM) (specific ac-tivity 69 Ci/mmol) is obtained from Amersham.

For IC50 determinations a 20 nM stock solution ismade up. Fifty µl are added to each test tube to yielda final concentration of 0.5 nM in the 2 ml assay.

Levallorphan tartrate is used for the determinationof nonspecific binding. A 0.1 mM stock solution is pre-pared in deionized water. Twenty µl added to each of3 tubes yields a final concentration of 0.1 µM in the2 ml assay.

A 1 mM stock solution is made up of the test com-pounds in a suitable solvent and serially diluted, suchthat the final concentrations in the assay range from10−6 to 10−9 M. At least 7 concentrations are used foreach assay.


Tissue PreparationMale Wistar rats are sacrificed by decapitation. Wholebrains minus cerebella are removed, weighed and ho-mogenized in 30 volumes of ice-cold 0.05 M Trisbuffer, pH 7.7. The homogenate is centrifuged at48,000 g for 15 min, the supernatant is decanted andthe pellet resuspended in the same volume of buffer.This homogenate is then incubated for 30 min at 37°Cto remove the endogenous opiate peptides and cen-trifuged again as before. The final pellet is resuspendedin 50 volumes of 0.05 M Tris buffer, pH 7.7.

Assay

1850 µl tissue suspension

80 µl distilled water

20 µl vehicle, or levallorphan, or appropriate con-centration of drug

50 µl [3H]DHM.

Tubes are incubated for 30 min at 25°C. The assay isstopped by vacuum filtration through Whatman GF/Bfilters which are washed twice with 5 ml of 0.05 MTris buffer. The filters are then placed into scintilla-tion vials with 10 ml Liquiscint scintillation cocktailand counted.

EVALUATIONSpecific binding is defined as the difference betweentotal binding and binding in the presence of 0.1 mMlevallorphan. IC50 values are calculated from the per-cent specific binding at each drug concentration.

The KD value for [3H]DHM binding was found tobe 0.38 nM by Scatchard analysis of a receptor satura-tion experiment. The Ki value may be calculated fromthe IC50 by the Cheng–Prusoff equation:

Ki = IC50/1 + L/KD .

REFERENCES AND FURTHER READINGAdler MW (1981) Mini-Symposium on Opiate Receptors. Life

Sci 28:1543–1584Cheng YC, Prusoff WH (1973) Relationship between the inhibi-

tion constant (Ki) and the concentration of inhibitor whichcauses 50 percent inhibition (I50) of an enzymatic reaction.Biochem Pharmacol 22:3099–3108

Childers S, Creese I, Snowman AM, Snyder SH (1979) Opiatereceptor binding affected differentially by opiates and opi-oid peptides. Eur J Pharmacol 55:11–18

Goldstein A (1987) Binding selectivity profiles for ligands ofmultiple receptor types: Focus on opioid receptors. TIPS8:456–459

Hubbard JW, Locke KW, Forster HV, Brice AG, Pan LG,Lowry TF, Forster AML, Forster MA, Cornfeldt M,Vanselous CL, Hamer RRL, Glamkowski EJ, Fielding S

(1992) Cardiorespiratory effects of the novel opioid anal-gesic HP 736 in the anesthetized dog and conscious goat.J Pharmacol Exp Ther 260:1268–1277

Laugwitz KL, Offermanns S, Spicher K, Schulz G (1993) μ andδ opioid receptors differentially couple to G protein sub-types in membranes of human neuroblastoma SH-SY5Ycells. Neuron 5:233–242

Locke KW, Dunn RW, Hubbard JW, Vanselous ChL, CornfeldtM, Fielding St, Strupczewski JT (1990) HP 818: A cen-trally acting analgesic with neuroleptic properties. DrugDev Res 19:239–256

Mansour A, Lewis ME, Khachaturian H, Akil H, Watson SJ(1986) Pharmacological and anatomical evidence of selec-tive μ, δ and κ opioid receptor binding in rat brain. BrainRes 399:69–79

Pasternak GW (1987) Opioid receptors. In: Meltzer HY (ed)Psychopharmacology: The Third Generation of Progress.Raven Press, New York pp 281–288

Pasternak GW, Wilson HA, Snyder SH (1975) Differential ef-fects of protein-modifying reagents on the receptor bind-ing of opiate agonists and antagonists. Mol Pharmacol11:340–351

Robson LE, Foote RW, Maurer R, Kosterlitz HW (1984) Opi-oid binding sites of the κ-type in guinea pig cerebellum.Neurosci 12:621–627

Snyder SH (1984) Drug and neurotransmitter receptors in thebrain. Science 224:22–31

Wolozin BL, Nishimura S, Pasternak GW (1982) The binding ofκ and σ opiates in rat brain. J Neurosci 2:708–713

Zukin RS, Zukin SR (1981) Multiple opiate receptors: Emergingconcepts. Life Sci 29:2681–2690

H.1.1.43H-Bremazocine Binding to κ Opiate Receptorsin Guinea Pig Cerebellum

PURPOSE AND RATIONALEκ Receptors are thought to be involved in the anal-gesic activity of opiates mainly within the spinal cord,whereas μ receptors are predominately located atsupraspinal sites. The pharmacological effects of κ ag-onists differ from the μ agonists in various analgesictests, effects on diuresis, sensitivity to naloxone andpropensity to cause respiratory depression. κ Agonistsmay induce water diuresis (Salas et al. 1992). The re-ceptor subtype selectivity can be determined by testingthe affinity of new compounds for the κ opiate recep-tor and comparing these results with the data from theμ receptor assay.

Although the benzomorphanes, such as ethylketo-cyclazocine and bremazocine, are potent κ agonists,they are not selective for this receptor subtype. Todemonstrate specific binding of these ligands to κ re-ceptors, the assay must be done in a tissue where theκ subtype predominates, such as the guinea pig cere-bellum. Moreover, binding to μ and δ receptors is pre-vented by inclusion of the peptide DAGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol) to mask the μ receptor and of


[D-Pen2,5]-enkephalin (Tyr-D-Pen-Gly-Phe-D-Pen) tomask the δ receptor.

PROCEDUREReagentsBremazocine(–)-[9-3H] (specific activity 21–28 Ci/mmol), is obtained from, New England Nuclear. ForIC50 determinations a 24 nM stock solution is madeup. Fifty µl are added to each tube to yield a final con-centration of 0.6 nM in the 2 ml assay.

U50,488H is made up to a 500 µM stock solution indeionized water. Twenty ml are added to each of the3 tubes for determination of unspecific binding yield-ing a final concentration of 5.0 µM in the 2 ml assay.

Opiate peptides of the μ- and δ-type are includedin the assay to prevent binding of the radioligand andthe test drug to these receptors. DAGO and [D-Pen2,5]-enkephalin are obtained from Penninsula Laboratories.Concentrated stock solutions of 10−3 M are made up indeionized water and further diluted to 10−5 M. Twentyµl of this solution are added to each tube to result ina final concentration of 100 nM of each in the 2 ml as-say.

For the assays a 1 mM stock solution of test com-pounds is made up in a suitable solvent and seriallydiluted, such that the final concentration in the assayranges from 10−5 to 10−8 M. At least 7 concentrationsare used for each assay.

Tissue PreparationMale guinea pigs are sacrificed and cerebella are re-moved, weighed and homogenized in 10 volumes ofice-cold 0.05 M Tris-buffer, pH 7.4. The homogenateis centrifuged at 48,000 g for 10 min, the supernatantdecanted and the pellet resuspended in 20 volumes ofbuffer. This homogenate is then incubated for 45 min at37°C to remove endogenous opiate peptides and cen-trifuged again as before. This pellet is resuspended in200 volumes of 0.05 M Tris buffer, pH 7.4.

Assay

1850 µl tissue suspension

60 µl distilled water

20 µl peptide solution

20 µl vehicle, or U50,488H, or test drug

50 µl [3H]bremazocine

Tubes are incubated for 40 min at 25°C. The assay isstopped by vacuum filtration through Whatman GF/Bfilters which are then washed 3 times with 5 ml of

0.05 M Tris buffer. The filters are then placed in scintil-lation vials with 10 ml Liquiscint scintillation cocktailand counted.

EVALUATIONSpecific binding is defined as the difference betweentotal binding and binding in the presence of 5.0 µMU50,488H. IC50 values are calculated from the percentspecific binding at each drug concentration.

The KD value for [3H]bremazocine binding wasfound to be 0.14 nM by Scatchard analysis of a recep-tor saturation experiment. The Ki value may be calcu-lated from IC50 by the Cheng–Prusoff equation:

Ki = IC50/1 + L/KD .

REFERENCES AND FURTHER READINGAbbott FV et al (1986) A dose-ratio comparison of μ and κ ag-

onists in formalin and thermal pain. Life Sci 39:2017–2024

Cheng YC, Prusoff WH (1973) Relationship between the inhibi-tion constant (Ki) and the concentration of inhibitor whichcauses 50 percent inhibition (I50) of an enzymatic reaction.Biochem Pharmacol 22:3099–3108

Goodman RR, Snyder SH (1982) Autoradiographic localizationof kappa opiate receptors to deep layers of the cerebral cor-tex may explain unique sedative and analgesic effects. LifeSci 31:1291–1294

Higginbottom M, Nolan W, O’Toole J, Ratcliffe GS, Rees DC,Roberts E (1993) The design and synthesis of kappa opi-oid ligands based on a binding model for kappa agonists.Bioorg Med Chem Lett 3:841–846

Hubbard JW, Locke KW, Forster HV, Brice AG, Pan LG,Lowry TF, Forster AML, Forster MA, Cornfeldt M,Vanselous CL, Hamer RRL, Glamkowski EJ, Fielding S(1992) Cardiorespiratory effects of the novel opioid anal-gesic HP 736 in the anesthetized dog and conscious goat.J Pharmacol Exp Ther 260:1268–1277

Inenaga K, Nagamoto T, Nakao K, Yanaihara N, Yamashita HY(1994) Kappa-selective agonists decrease postsynaptic po-tentials and calcium components of action potentials in thesupraoptic nucleus of rat hypothalamus in vitro. Neurosci58:331–340

Kosterlitz HW, Paterson SJ, Robson LE (1981) Characterizationof the κ-subtype of the opiate receptor in the guinea pigbrain. Br J Pharmacol 73:939–949

Mansour A, Lewis ME, Khachaturian H, Akil H, Watson SJ(1986) Pharmacological and anatomical evidence of se-lective μ, δ and κ opioid receptors in brain. Brain Res399:69–79

Peter GR et al (1987) Diuretic actions in man of a selectivekappa opioid agonist: U-62,066E. J Pharmacol Exper Ther240:128–131

Robson LE, Foote RW, Maurer R, Kosterlitz HW (1984) Opi-oid binding sites of the κ-type in guinea pig cerebellum.Neurosci 12:621–627

Salas SP, Roblero JS, López LF, Tachibana S, Huidobro-Toro JP (1992) [N-Methyl-Tyr1,N-methyl-Arg7-D-Leu8]-dynorphin-A-(1–8) ethylamide, a stable dynorphin analog,produces diuresis by kappa-opiate receptor activation in therat. J Pharmacol Exp Ther 262:979–986


Snyder SH (1984) Drug and neurotransmitter receptors in thebrain. Science 224:22–31

Steinfels GF, Cook L (1986) Antinociceptive profiles of μ andκ opioid agonists in a rat tooth pulp stimulation procedure.J Pharmacol Exper Ther 236:111–117

Tyers MB (1982) Studies on the antinociceptive activities ofmixtures of μ- and κ-opiate agonists and antagonists. LifeSci 31:1233–1236

Wolozin BL, Nishimura S, Pasternak GW (1982) The binding ofκ- and σ -opiates in rat brain. J Neurosci 2:708–713

Zukin RS, Zukin SR (1981) Multiple opiate receptors: Emergingconcepts. Life Sci 29:2681–2690

H.1.1.5Inhibition of Enkephalinase

PURPOSE AND RATIONALESince the discovery of brain peptides with pharmaco-logical properties similar to morphine (Hughes 1975),the metabolic breakdown of enkephalins has beenstudied (Malfroy et al. 1978; Llorens and Schwartz1981; Mumford et al. 1981; Malfroy and Schwartz1982; Roques 1982; Schwartz 1983). Roques BPet al. (1980), Costentin et al. (1986) found thatthe enkephalinase inhibitor thiorphan shows antinoci-ceptive activity in mice. A highly sensitive fluoro-metric assay for “enkephalinase”, a neutral metal-loendopeptidase that releases tyrosine-glycine-glycinefrom enkephalins has been developed by Florentinet al. (1984). A fluorogenic peptide, dansyl-D-Ala-Gly-Phe(pNO2)-Gly (DAGNPG) was synthesized asa selective substrate for the neutral metalloendopep-tidase involved in enkephalin metabolism. This en-zyme, designated “enkephalinase” cleaves the Gly-Phe(pNO2) peptide bond of DAGNPG leading to a flu-orescence increase related to the disappearance of in-tramolecular quenching of the dansyl fluorescence bythe nitrophenyl residue.

Enkephalinase induces inactivation of atrial natri-uretic factor (ANF). The protection of endogenousANF against inactivation may result in therapeutic ap-plications (Schwartz et al. 1990).

PROCEDUREFresh rat kidney is homogenized in 10 vol of cold0.05 M Tris-HCl buffer, pH 7.4, using a Polytron ho-mogenizer. The homogenate is centrifuged for 5 minat 1000 g. The pellet is discarded and the supernatantcentrifuged at 60,000 g for 60 min. The resulting pelletis resuspended in 50 mM Tris-HCl buffer, pH 7.4, andused as the enzyme source.

Standard assays for “enkephalinase” activity us-ing DAGNPG are carried out at 37°C in hemol-ysis tubes. A 0.1-ml amount of 50 mM Tris-HCl

buffer, pH 7.4, containing 50 µM DAGNPG is prein-cubated 15 min at 37°C. The reaction is initiatedby addition of 50 µl of the enzyme preparation to-gether with 0.5 µM Captopril. The tubes are incu-bated for 30 min in a water bath with constant shak-ing. The enzymatic reaction is stopped by boilingat 100°C for 5 min. The samples are then dilutedwith 1.35 ml of Tris HCl buffer and centrifuged at500 g for 30 min. An aliquot of 1 ml of the super-natant is transferred to thermostated cells of a spec-trofluorometer. Readings are performed at 562 nmwith an excitation wavelength of 342 nm. A calibra-tion curve is prepared by adding increasing concen-trations of DNS-D-Ala-Gly and decreasing concen-trations of the substrate in Tris-HCl buffer contain-ing the denaturated enzymatic preparation. For the as-say of “enkephalinase” inhibition, the test compoundor the standard thiorphan = [(R-,S-)-3-mercapto-2-benzylpropanoyl]glycine is added in various concen-trations.

EVALUATIONThe inhibitory potencies of test compounds are com-pared with the standard.

MODIFICATIONS OF THE METHODKsander et al. (1989) incubated synaptic membranesfrom rat striatum with 3H-Tyr-Leu-enkephalin for15 min at 30°C, pH 6.5, in the presence of 10−6 Mbestatin. The reaction was stopped by the additionof 30% acetic acid and the reaction product 3H-Tyr-Gly-Gly was separated from unreacted 3H-Tyr-Leu-enkephalin on a Porapak Q column followed by a Cu2+

chelex column. The 3H-Tyr-Gly-Gly was counted byliquid scintillation.

The antinociceptive effects of intrathecally admin-istered SCH32615, an enkephalinase inhibitor werestudied in the rat by Oshita et al. (1990).

REFERENCES AND FURTHER READINGChipkin RE (1986) Inhibition of enkephalinase: The next gener-

ation of analgesics. Drugs Future 11:593–606Chipkin RE, Berger JG, Billard W, Iorio LC, Chapman R, Bar-

nett A (1988) Pharmacology of SCH 34826, an orally ac-tive enkephalinase inhibitor analgesic. J Pharm Exp Ther245:829–838

Costentin J, Vlaiculescu A, Chaillet P, Natan B, Aveaux D,Schwartz JC (1986) Dissociated effects of inhibitors ofenkephalin-metabolizing peptidases or naloxone on variousnociceptive responses. Eur J Pharmacol 123:37–44

Florentin D, Sassi A, Roques BP (1984) A highly sensitive flu-orimetric assay for “enkephalinase”, a neutral metalloen-dopeptidase that releases tyrosine-glycine-glycine fromenkephalins. Anal Biochem 141:62–69


Hughes J (1975) Isolation of an endogenous compound from thebrain with pharmacologic properties similar to morphine.Brain Res 88:295–308

Ksander GM, Diefenbacher CG, Yuan AM, Clark F, Saka-ne Y, Ghai RD (1989) Enkephalinase inhibitors. I. 2,4-Di-benzylglutaric acid derivatives. J Med Chem 32:2519–2526

Llorens C, Schwartz JC (1981) Enkephalinase activity in rat pe-ripheral organs. Eur J Pharmacol 69:113–116

Malfroy B, Schwartz JC (1982) Properties of “enkephalinase”from rat kidney: comparison of dipeptidyl-carboxypepti-dase and endopeptidase activities. Biochem Biophys ResCommun 106:276–285

Malfroy B, Swerts JP, Guyon A, Roques BP, Schwartz JC (1978)High-affinity enkephalin-degrading peptidase in brain is in-creased after morphine. Nature 276:523–526

Mumford RA, Pierzchala PA, Strauss AW, Zimmerman M(1981) Purification of a membrane bound metalloendopep-tidase from porcine kidney that degrades peptide hormones.Proc Natl Acad Sci USA 78:6623–6627

Oshita S, Yaksh TL, Chipkin R (1990) The antinocicep-tive effects of intrathecally administered SCH32615, anenkephalinase inhibitor in the rat. Brain Res 515:143–148

Roques BP, Fournié-Zaluski MC, Soroca E, Lecomte LM, Mal-froy B, Llorens C, Schwartz JC (1980) The enkephalinaseinhibitor thiorphan shows antinociceptive activity in mice.Nature 288:286–288

Roques BP, Fournié-Zaluski MC, Florentin D, Waksman G,Sassi A, Chaillet P, Collado H, Ciostentin J (1982)New enkephalinase inhibitors as probes to differentiate“enkephalinase” and angiotensin-converting-enzyme activesites. Life Sci 31:1749–1752

Schwartz JC (1983) Metabolism of enkephalins and the inacti-vating neuropeptidase concept. TINS 1983:45–48

Schwartz JC, Gros C, Lecomte JM, Bralet J (1990) Enkephali-nase (EC 3.4.24.11) inhibitors: protection of endogenousANF against inactivation and potential therapeutic applica-tions. Life Sci 47:1279–1297

H.1.1.6Nociceptin

H.1.1.6.1General Considerations on Nociceptin

PURPOSE AND RATIONALEA heptadekapeptide (nociceptin or orphanin FQ) hasbeen isolated as endogenous agonist of the opioidreceptor-like ORL1 receptor (Reinscheid et al. 1995;Meunier et al. 1995; Barlocco et al. 2000; Mollereauand Mouledous 2000; Mogil and Pasternak 2001;Witta et al. 2004) which shows high structural ho-mology with opioid peptides, especially dynorphin A(Calò et al. 2000). Nociceptin activates a specific re-ceptor, which has been cloned in man and animalsand has been shown to be structurally similar to opi-oid receptors (Mollereau et al. 1994; Calò et al. 2000;Hawkinson et al. 2000). At the cellular level, thenociceptin receptor has been shown to act throughthe same mechanisms as classical opioid receptors,namely the inhibition of adenylyl cyclase, the activa-tion of potassium channels and inhibition of calcium

channels (Connor et al. 1996a, b). In vitro and in vivostudies have demonstrated that nociceptin mediatesa variety of biological actions (Civelli et al. 1998; Dar-land et al. 1998). Nociceptin induces analgesia whenadministered intrathecally (Stanfa et al. 1996; Xu et al.1996), while it causes hyperalgesia and reversal ofopioid induced analgesia when given intracerebroven-tricularly; nociceptin stimulates food intake (Polidoriet al. 2000) and produces anxiolysis. Depending on thedose, nociceptin stimulates or inhibits locomotor activ-ity. Nociceptin inhibits long-term potentiation, mem-ory processes, induces bradycardia, hypotension anddiuresis. In addition, nociceptin inhibits neurotrans-mitter release both at central and peripheral sites. In-tracavernosal injection of nociceptin induces a potentand relatively long-lasting erectile response in the cat(Champion et al. 1997, 1998). Intrathecal injection ofnociceptin elicits scratching, licking and biting in mice(Sakurada et al. 1999, 2000). Synthetic agonists andantagonists of the nociceptin receptor have been re-ported (Guerrini et al. 1998; Salvadori et al. 1999;Calò et al. 2000; Hashimoto et al. 2000; Meunier 2000;Ozaki et al. 2000).

REFERENCES AND FURTHER READINGBarlocco D, Cignarella G, Giardina GAM, Toma L (2000) The

opioid-receptor-lie 1 (ORL-1) as a potential target for newanalgesics. Eur J Med Chem 35:275–282

Calò G, Guerrini R, Bigoni R, Rizzi A, Marzola G, Okawa H,Bianchi C, Lambert DG, Salvadori S, Regoli D (2000)Characterization of [Nph1]nociceptin(1–13)NH2, a newselective nociceptin receptor antagonist. Br J Pharmacol129:1183–1193

Champion HC, Wang R, Hellstrom WJG, Kadowitz PJ (1997)Nociceptin, a novel endogenous ligand for the ORL1 re-ceptor, has potent erectile activity in the cat. Am J Physiol273 (Endocrinol Metab 36):E214–E219

Champion HC, Bivalacqua TJ, Wang R, Hellstrom WJG, Kad-owitz PJ (1998) [Tyr1]nociceptin and nociceptin have simi-lar naloxoneinsensitive erectile activity in the cat. J Androl19:747–753

Civelli O, Nothacker HP, Reinscheid R (1998) Reverse phys-iology: discovery of the novel neuropeptide, orphaninFQ/nociceptin. Crit Rev Neurobiol 12:163–176

Connor M, Vaughan CW, Chieng B, Christie MJ (1996a) No-ciceptin receptor coupling to a potassium conductance inrat locus coeruleus neurones in vitro. Br J Pharmacol119:1614–1618

Connor M, Yeo A, Henderson G (1996b) Effect of nociceptinon Ca2+ channel current and intracellular Ca2+ in the SH-SY5Y human neuroblastoma cell line. Br J Pharmacol118:205–207

Darland T, Heinricher MM, Grandy DK (1998) OrphaninFQ/nociceptin: a role in pain and analgesia, but so much more.Trends Neurosci 21:215–221

Guerrini R, Calò G, Rizzi A, Bigoni R, Bianchi C, SalvadoriS, Regoli D (1998) A new selective antagonist of the noci-ceptin receptor. Br J Pharmacol 123:163–165


Hashimoto Y, Calò G, Guerrini R, Smith G, Lambert DG (2000)Antagonistic effects of [Nphe1]nociceptin(1–13)NH2 onnociceptin mediated inhibition of cAMP formation in Chi-nese hamster ovary cells stably expressing the recombinanthuman nociceptin receptor. Neurosci Lett 278:109–112

Hawkinson JE, Acosta-Burruel M, Espitia SE (2000) Opi-oid activity profiles indicate similarities between noci-ceptin/orphanin FQ and opioid receptors. Eur J Pharmacol389:107–114

Meunier JC (2000) The therapeutic value of nociceptin agonistsand antagonists. Expert Opin Ther Pat 10:371–388

Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C,Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monser-rat B, Mazarguil H, Vassart G, Parmentier M, Costentin J(1995) Isolation and structure of the endogenous agonist ofopioid receptor-like ORL1 receptor. Nature 377:532–535

Mogil JS, Pasternak (2001) The molecular and behavioral phar-macology of the orphanin FQ/nociception peptide and re-ceptor family. Pharmacol Rev 53:381–415

Mollereau C, Mouledous L (2000) Tissue distribution of the opi-oid receptor-like (ORL1) receptor. Peptides 21:907–917

Mollereau C, Parmentier M, Mailleux P, Butour JL, Moisand C,Chalon P, Caput D, Vassart G, Meunier JC (1994)ORL1, a novel member of the opioid receptor family:cloning, functional expression and localization. FEBS Lett341:33–38

Ozaki S, Kawamoto H, Itoh Y, Miyaji M, Iwasawa Y, Ohta H(2000) A potent and highly selective nonpeptidyl noci-ceptin/orphanin FQ receptor (ORL1) antagonist: J-113397.Eur J Pharmacol 387:R17–R18

Polidori C, Calò G, Ciccocioppo R, Geurrini R, Regoli D,Massi M (2000) Pharmacological characterization ofthe nociceptin receptor mediating hyperphagia: Identi-fication of a selective antagonist. Psychopharmacology148:430–437

Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Hen-ningsen RA, Bunzow JR, Grandy DK, Langen H, Mon-sma FJ, Civelli O (1995) Orphanin FQ: a neuropeptide thatactivates an opioidlike G protein-coupled receptor. Science270:792–794

Sakurada T, Katsuyama S, Sakurada S, Inoue M, Tan No-K,Kisara K, Sakurada C, Ueda M, Sasaki J (1999) No-ciceptin-induced scratching, biting and licking in mice:Involvement of spinal NK1 receptors. Br J Pharmacol127:1712–1718

Sakurada T, Sakurada S, Katsuyama S, Hayashi T, Sakurada C,Tan No-K, Johansson H, Sandin J, Terenius L (2000) Ev-idence that N-terminal fragments of nociceptin modulatenociceptin-induced scratching, biting and licking in mice.Neurosci Lett 279:61–64

Salvadori S, Guerrini R, Calò G, Regoli D (1999) Structure-ac-tivity studies on nociceptin/orphanin FQ: From full agonistto partial agonist, to pure antagonist. Farmaco 54:810–825

Stanfa LC, Chapman V, Kerr N, Dickenson AH (1996) In-hibitory action of nociceptin on spinal dorsal horn neuronesof the rat, in vivo. Br J Pharmacol 118:1875–1877

Varani K, Rizzi A, Calò G, Bigoni R, Toth G, Guerrini R,Gessi S, Salvadori S, Borea PA, Regoli D (2000) Pharma-cology of [Tyr1]nociceptin analogs: receptor binding andbioassay studies. Naunyn-Schmiedeberg’s Arch Pharmacol360:270–277

Witta J, Palkovits M, Rosenberger J, Cox BM (2004) Distri-bution of nociception/orphanin FQ in adult human brain.Brain Res 30:24–29

Xu X-J, Hao J-X, Wiesenfeld-Hallin Z (1996) Nociceptin oranti-nociceptin: potent spinal antinociceptive effect of or-phanin FQ/nociceptin in the rat. NeuroReport 7:2092–2094

H.1.1.6.2Receptor Binding of Nociceptin

PURPOSE AND RATIONALEThe nociceptin receptor has been termed by differentgroups of investigators as ORL1, LC132, ROR (seeMeunier 1997). Based on the structural and transduc-tional similarities between receptors for nociceptin andthose for opioids, Hamon (1998) proposed to includethe nociceptin receptor in the opioid receptor familywith the name OP4.

Varani et al. (1999) tested synthetic nociceptinanalogs for their displacement at the nociceptin- andat classical opioid receptors. The displacement of[3H]NCNH2 ([3H]nociceptin amide, ORL1 site), andof the selective opioid receptor ligands [3H]DAMGO(μ site), [3H]deltorphin II (δ site), and [3H]U69593(κ site) was studied.

Wisner et al. (2006) described human opiorphin,a natural antinociceptive modulator of opioid-depen-dent pathways inhibiting enkephalin-inactivating zincectopeptidases.

PROCEDUREMembrane PreparationGuinea pigs are decapitated and the whole brain (with-out cerebellum) rapidly removed. The tissue is dis-rupted in a Polytron homogenizer (setting 5) in 50 mMTris HCl, pH 7.4, to prepare membranes for the clas-sic opioid receptor studies. The homogenate is cen-trifuged at 4000 g for 10 min and the pellet is resus-pended with a Polytron PTA 10 probe (setting 5) in thesame ice-cold buffer. To study the binding to ORL1 re-ceptor, the tissue is homogenized in 50 mM Tris HCl,2 mM EDTA and 100 µM phenylmethylsulphonylflu-oride HCl (PMSF) at pH 7.4. The suspension is cen-trifuged at 40,000 g for 10 min and the pellet is resus-pended in the same buffer. After 30 min of incubationat 37°C the membranes are centrifuged at 40,000 g for10 min and the pellets are stored at –70°C. The pro-tein concentration is determined with bovine albuminas standard.

Binding AssaysClassic opioid receptors, μ, δ and κ , are studied ac-cording to Bhargava and Zhao (1996). Saturation bind-ing experiment are carried out using 8–10 differentconcentrations of [3H]DAMGO ranging from 0.15 nMto 15 nM, [3H]deltorphin II from 0.1 nM to 10 nM,and [3H]U69593 from 0.15 nM to 15 nM, respectively.Inhibition experiments are carried out in duplicate ina final volume of 250 µl in test tubes containing ei-


ther 1.5 nM [3H]DAMGO or 1.0 nM [3H]deltorphin IIor 1.5 nM [3H]U69593, 50 mM Tris HCl at pH 7.4,guinea pig brain membranes (150–200 µg of pro-tein/assay) and at least 8–10 different concentra-tions of the ligands under study. Binding assaysto the ORL1 receptor are carried out according toVarani et al. (1998). In saturation studies, mem-branes are incubated with 8–10 different concentra-tions of [3H]NCNH2 ([3H]nociceptin amide) rangingfrom 0.1 mM to 10 mM. Inhibition experiments arecarried out in duplicate in a final volume of 250 µlin test tubes containing 1 mM [3H]NCNH2, 50 mMTris HCl, 2 mM EDTA, 100 µM phenylmethylsulpho-nylfluoride HCl (PMSF) at pH 7.4, guinea pig mem-branes, and at least 8–10 different concentration ofthe compound under examination. The incubation timeis 1 h for [3H]DAMGO and [3H]U69593 and 2 h for[3H]deltorphin II and [3H]NCNH2. Nonspecific bind-ing is defined as the binding measured in the presenceof 100 µM bremazocine for classic opioid receptorsand 10 µM NCNH2 for ORL1 receptors.

Bound and free radioactivity are separated by filter-ing the assay mixture through Whatman GF/B glass-fibre filters, previously treated with PEI 0.1%; the in-cubation mixture is diluted with 3 ml of ice-cold incu-bation buffer, rapidly filtered by vacuum, and the fil-ter washed three times with 3 ml of incubation buffer.The filter-bound radioactivity is measured in a Beck-man LS-1800 Spectrometer.

EVALUATIONThe inhibitory binding constant (Ki) values are calcu-lated from the IC50 values according to the Cheng andPrusoff equation. The weighted non-linear last-squarescurved fitting program LIGAND (Munson and Rod-bard 1980) is used for computer analysis of saturationand inhibition experiments.

MODIFICATIONS OF THE METHODArdati et al. (1997) developed two radioligands forthe orphanin FQ receptor: a tritiated OFQ peptide([3H]orphanin FQ) and radioiodinated form in whichLeu14 is substituted by tyrosine (125I-Tyr14-orphaninFQ). Both exhibit virtually identical characteristics.

Seki et al. (1999) analyzed the pharmacologicalproperties of κ-opioid receptor-selective agonist TRK-820 using Chinese hamster ovary cells expressingcloned rat μ-, δ- and κ-opioid receptors and humannociceptin receptor.

Mouledous et al. (2000) reported a site-directed mu-tagenesis study of the ORL1 receptor transmembrane-binding domain.

REFERENCES AND FURTHER READINGArdati A, Henningsen RA, Higelin J, Reinscheid RK, Cinelli O,

Monsma FJ (1997) Interaction of [3H]orphanin FQ and125I-Tyr14-orphanin FQ with the orphanin FQ receptor: ki-netics and modulation by cations and guanine nucleotides.Mol Pharmacol 51:816–824

Bhargava HN, Zhao GM (1996) Effects of competitive and non-competitive antagonists of the N-methyl-D-aspartate recep-tor on the analgesic action of δ1 and δ2 opioid receptors inmice. Br J Pharmacol 119:1586–1590

Calò G, Rizzi A, Bodin M, Neugebauer W, Salvadori S, Guer-rini R, Bianchi C, Regoli D (1997) Pharmacological char-acterization of nociceptin receptor: an in vitro study. Can JPhysiol Pharmacol 75:713–718

Hamon M (1998) The new approach to opioid recep-tors. Naunyn-Schmiedeberg’s Arch Pharmacol 358 (Suppl2):SA 5.3

Meunier JC (1997) Nociceptin/orphanin FQ and the opioidreceptor-like ORL1 receptor. Eur J Pharmacol 340:1–15

Mouledous L, Topham CM, Moisand C, Mollereau C, MeunierJC (2000) Functional investigation of the nociceptin recep-tor by alanine substitution of glutamine 286 at the C ter-minus of the transmembrane segment VI: Evidence froma site-directed mutagenesis study of the ORL1 receptortransmembrane binding domain. Mol Pharmacol 57:495–502

Munson PJ, Rodbard D (1980) LIGAND; a versatile comput-erized approach for the characterization of ligand bindingsystems. Anal Biochem 107:220–239

Seki T, Awamura S, Kimura C, Ide S, Sakano K, Minami M,Nagase H, Satoh M (1999) Pharmacological properties ofTRK-820 on cloned μ-, δ- and κ-opioid receptors and no-ciceptin receptor. Eur J Pharmacol 376:159–167

Varani K, Calò G, Rizzi A, Merighi S, Toth G, Guerrini R,Salvadori S, Borea PA, Regoli D (1998) Nociceptin re-ceptor binding in mice forebrain membranes: thermody-namic characteristics and structure-activity relationships.Br J Pharmacol 125:1485–1490

Varani K, Rizzi A, Calò G, Bigoni R, Toth G, Guerrini R,Gessi S, Salvadori S, Borea PA, Regoli D (1999) Pharma-cology of [Tyr1]nociceptin analogs: receptor binding andbioassay studies. Naunyn-Schmiedeberg’s Arch Pharmacol360:270–277

Wisner A, Dufour E, Messaoudi M, Nejdi A, Marcel A, Unge-heuer MN, Rougeot C (2006) Human opiorphin, a naturalantinociceptive modulator of opioid-dependent pathways.Proc Natl Acad Sci USA 193:17979–17984

H.1.1.6.3Bioassays for Nociceptin

PURPOSE AND RATIONALENociceptin receptors in the periphery can be character-ized by studies in isolated organs (Guerrini et al. 1998;Bigoni et al. 1999): the guinea pig ileum according toPaton (1957) (see J.4.3.1), the mouse vas deferens ac-cording to Hughes et al. (1975), the rabbit vas defer-ens according to Oka et al. (1980) (see A.1.2.3), theguinea pig renal pelvis (Giuliani and Maggi 1996) (seeC.4.2.1).


PROCEDURETissues are taken from male Swiss mice (25–30 g),guinea pigs (300–350 g) Sprague Dawley rats(300–350 g) and New Zealand albino rabbits(1.5–1.8 kg). They are suspended in 10 ml organbaths containing Krebs solution oxygenated with95% O2 and 5% CO2. The temperature is set at 33°Cfor the mouse vas deferens and at 37°C for the othertissues. A resting tension of 0.3 g is applied to themouse deferens, 1 g to the guinea pig ileum, ratsvas deferens, and rabbit vas deferens and 0.15 g tothe guinea pig renal pelvis. For experiments at themouse vas deferens a Mg2+-free Krebs solution isused and for rat vas deferens experiments a Krebssolution containing 1.8 mM CaCl2. Guinea pig renalpelvis experiments are performed in the presence ofindomethacin (3 µM).

The mouse vas deferens, guinea pig ileum, rat vasdeferens, and rabbit vas deferens are continuouslystimulated through two platinum ring electrodes withsupramaximal voltage rectangular pulses of 1 ms du-ration and 0.1 Hz frequency. The electrically evokedcontractions are measured isotonically with a straingauge transducer and recorded on a multichannel chartrecorder. After an equilibration period of about 60 minthe contractions induced by electrical field stimulationare stable; at this time, cumulative concentration re-sponse curves to nociceptin or opioid peptides are per-formed (0.5 log unit steps).

The guinea pig renal pelvis is stimulated throughtwo platinum ring electrodes with 100 V square wavepulses of 1 ms duration at a frequency of 5 Hz for10 s. The spontaneous activity and the positive in-otropic responses to electrical field stimulation aremeasured by an isotonic transducer and recorded bya two channel recorder. The experiments are startedfollowing a 60 min equilibration period. Four electri-cal field stimulation are performed with each tissueat 30 min intervals. Agonists are added to the bath5 min, and antagonists 15 min before the next stimu-lus. The contractile responses to electrical field stim-ulation are expressed as % increment the spontaneousactivity of the tissue; the biological effects of the appli-cation of agonists or antagonists are expressed as % in-hibition of electrical filed stimulation-induced contrac-tion.

EVALUATIONData are expressed as means ±SEM of n experimentsand statistically analyzed with Student two-tailedt-test of one way ANOVA plus Dunnett test. Theagonist potencies are given as pE50, which is the

negative logarithm to base 10 of the agonist mo-lar concentration that produces 50% of the max-imal possible effect of that agonist. The Emax isthe maximal effect that an agonist can elicit ina given preparation. Antagonist potencies are ex-pressed in terms of pA2, which is the negative log-arithm to base 10 of the antagonist molar concen-tration that makes it necessary to double the ago-nist concentration to elicit the original submaximal re-sponse.

MODIFICATIONS OF THE METHODRizzi et al. (1999) studied nociceptin and nociceptinanalogs in the isolated mouse colon.

Bigoni et al. (1999) used nociceptin, a se-ries of nociceptin fragments, naloxone as wellas [Phe1� (CH2-NH)Gly2]nociceptin(1–13)NH2 and[Nphe1]nociceptin(1–13)NH2 to characterize noci-ceptin receptors in peripheral organs, such as mouseand rat vas deferens (noradrenergic nerve terminals),in the guinea pig ileum (cholinergic nerves) and re-nal pelvis (sensory nerves) and in vivo by measur-ing the blood pressure and heart rate in anesthetizedrats.

Menzies et al. (1999) described the agonisteffects of nociceptin and [Phe1� (CH2-NH)Gly2]nociceptin(1–13)NH2 in the mouse and rat colon andin the mouse vas deferens.

Kolesnikov and Pasternak (1999) found an ED50 of16.3 µg after peripheral administration of nociceptin inthe tail flick test in mice.

Bertorelli et al. (1999) found anti-opioid effectsof nociceptin and the ORL1 ligand [Phe1� (CH2-NH)Gly2]nociceptin(1–13)NH2 in the Freund’s adju-vant-induced arthritic rat model of chronic pain.

Yamamoto and Sakashita (1999) studied the effectof nocistatin, a 17 amino acid peptide which is pro-cessed from prepronociceptin and its interaction withnociceptin in the rat formalin test.

Hashiba et al. (2003) measured the effects of no-ciceptin/orphanin FQ receptor ligands on blood pres-sure, heart rate, and plasma catecholamine concentra-tions in guinea pigs.

Using the forced swimming test and the tailsuspension test in rats and mice, Gavioli et al.(2004) demonstrated antidepressant-like effects ofthe nociceptin/orphanin FQ receptor antagonist UFP-101.

Varty et al. (2005) characterized a nociceptin recep-tor (ORL-1) agonist in tests of anxiety across threespecies: rat, guinea pig, and mouse.


REFERENCES AND FURTHER READINGBertorelli R, Corradini L, Rafiq K, Tupper J, Calò G,

Ongini E (1999) Nociceptin and the ORL1 ligand[Phe1� (CH2-NH)Gly2]nociceptin(1–13)NH2 exert anti-opioid effects in the Freund’s adjuvant-induced arthriticrat model of chronic pain. Br J Pharmacol 128:1252–1258

Bigoni R, Giuliani S, Calò G, Rizzi A, Guerrini R, Salvadori S,Regoli D, Maggi CA (1999) Characterization of nociceptinreceptors in the periphery: in vitro and in vivo studies.Naunyn-Schmiedeberg’s Arch Pharmacol 359:160–167

Calò G, Guerrini R, Bigoni R, Rizzi A, Marzola G, Okawa H,Bianchi C, Lambert DG, Salvadori S, Regoli D (2000)Characterization of [Nph1]nociceptin(1–13)NH2, a newselective nociceptin receptor antagonist. Br J Pharmacol129:1183–1193

Gavioli EC, Vaughan CW, Marzola G, Guerrini R, MitchellVA, Zucchini S, De Lima TC, Rae GA, Salvadori S, Re-goli D, Calo G (2004) Antidepressant-like effects of thenociceptin/orphanin FQ receptor antagonist UFP-101: newevidence from rats and mice. Naunyn-Schmiedebergs ArchPharmacol 369:547–553

Giuliani S, Maggi CA (1996) Inhibition of tachykinin re-lease from peripheral endings of sensory nerves by noci-ceptin, a novel opioid peptide. Br J Pharmacol 118:1567–1569

Guerrini R, Calò G, Rizzi A, Bigoni R, Bianchi C, SalvadoriS, Regoli D (1998) A new selective antagonist of the noci-ceptin receptor. Br J Pharmacol 123:163–165

Hashiba E, Hirota K, Kudo T, Calo G, Guerrini R, Mat-suki A (2003) Effects of nociceptin/orphanin FQ recep-tor ligands on blood pressure, heart rate, and plasmacatecholamine concentrations in guinea pigs. Naunyn-Schmiedebergs Arch Pharmacol 367:342–347

Hughes J, Kosterlitz HW, Leslie FM (1974) Assessment of theagonistic and antagonistic activities of narcotic analgesicdrugs by means of the mouse vas deferens. Br J Pharmacol51:139P–140P

Kolesnikov YA, Pasternak GW (1999) Peripheral or-phanin FQ/nociceptin analgesia in the mouse. LifeSci 64:2021–2028

Menzies JRW, Glen T, Davies MRP, Paterson SJ, Corbett AD(1999) In vitro agonist effects of nociceptin and [Phe1�

(CH2-NH)Gly2]nociceptin(1–13)NH2 in the mouse andrat colon and the mouse vas deferens. Eur J Pharmacol385:217–223

Oka T, Negishi K, Suda M, Matsumiya T, Inazu T, Ueki M(1980) Rabbits vas deferens: a specific bioassay for opioidκ-receptor agonists. Eur J Pharmacol 73:235–236

Paton WDM (1957) The action of morphine and related sub-stances on contraction and on acetylcholine output ofcoaxially stimulated guinea-pig ileum. Br J Pharmacol12:119–127

Rizzi A, Bigoni R, Calò G, Guerrini R, Salvadori S, Re-goli D (1999) [Nphe1]nociceptin(1–13)NH2 antagonizesnociceptin effects in the mouse colon. Eur J Pharmacol385:2–3

Varty GB, Hyde LA, Hodgson RA, Lu SX, McCool MF, Kaz-doba TM, DelVecchio RA, Guthrie DH, Pond AJ, GrzelakME, Xu X, Korfmacher WA, Tulshian D, Parker EM, Hig-gins GA (2005) Characterization of the nociceptin recep-tor (ORL-1) agonist, Ro64–6198, in tests of anxiety acrossmultiple species. Psychopharmacology 182:132–143

Yamamoto T, Sakashita Y (1999) Effect of nocistatin and its in-teraction with nociceptin/orphanin FQ on the rat formalintest. Neurosci Lett 262:179–182

H.1.1.7Vasoactive Intestinal Polypeptide (VIP) and PituitaryAdenylate Cyclase-Activating Peptide (PACAP)

PURPOSE AND RATIONALESeveral peptides are considered to play a role in thealtered transmission of sensory information in neu-ropathic conditions, such as neuropathic pain aris-ing from trauma or compression injury of peripheralnerves (Zhang et al. 1998).

Vasoactive intestinal polypeptide (VIP), isolated byNakajima et (1970), is a neuropeptide of 28 aminoacids with widespread distribution in both the cen-tral and peripheral nervous system (Fahrenkrug 1979;Gafvelin 1990).

Together with the structurally related pituitaryadenylate cyclase-activating peptide (PACAP) thispeptide is considered to play an important role inthe somatosensory processing of pain (Dickinson andFleetwood-Walker 1999). PACAP-38 (a 38 amino-acid polypeptide) and the C-terminally truncated formPACAP-37 share 68% amino acid homology at theirN-terminal domain with VIP. A shorter peptide with27 amino acids, named as PACAP27 was describedby Miyata et al. (1990). These peptides are membersof a superfamily of hormones that includes glucagon,glucagon-like peptide, secretin and growth hormonereleasing factor.

Three G-protein-coupled receptors are described:the VPAC1 receptor, originally described as the VIPreceptor and subsequently designated as VIP1 recep-tor; the VPAC2 receptor, previously designated VIP2;and the PAC1 receptor, previously known as PACAPtype I receptor (Buscail et al. 1990; Guijarro et al.1991; Felley et al. 1992; Calvo et al. 1994; Van Ram-pelbergh et al. 1996; Harmar et al. 1998; Robberechtet al. 1999).

Many peripheral activities of VIP/PACAP are de-scribed, such as stimulation of pancreatic secretion(Onaga et al. 1997; Ito et al. 1998; Soo Tek Leeet al. 1998); stimulation of duodenal bicarbonate se-cretion (Takeuchi et al. 1998); relaxation of smoothmuscle cells in the intestinal tract, e. g., gall blad-der (Pang and Kline 1998), cecal circular smoothmuscle (Motomura et al. 1998), internal anal sphinc-ter (Rattan and Chakder 1997); on duodenal motil-ity (Onaga et al. 1998); enhancement of insulin se-cretion (Yada et al. 1997; Filipsson et al. 1998);bronchodilation (Linden et al. 1998; Shigyo et al.1998; Okazawa et al. 1998). Centrally administeredPACAP showed an anorectic effect (Mizuno et al.1998).


Agonists (Gourlet et al. 1997a, b) and antagonists(Gozes et al. 1991; Gourlet et al. 1997c) for VIPwere described. Further studies are aimed to develop-ment of drugs for neuropathic analgesia, ultimately ofnon-peptide nature, using VPAC1, VPAC2, and PAC1,receptors as drug targets (Dickinson and Fleetwood-Walker 1999).

PROCEDURECHO cell lines expressing the rat VIP1 receptor (Cic-carelli et al. 1994), the human VIP2 receptor (Sreed-haran et al. 1993), the rat PACAP I receptor (Ciccarelliet al. 1995), and the rat secretin receptor (Ishihara et al.1991) are used.

Transfected CHO cells are harvested with a rubberpoliceman and pelleted by low speed centrifugation.The supernatant is discarded and the cell lysed in mMNaHCO3 solution and immediate freezing in liquid ni-trogen. After thawing, the lysate is first centrifugedat 4°C for 10 min at 400 g and the supernatant is fur-ther centrifuged at 20,000 g for 10 min. The pellet, re-suspended in 1 mM NaHCO3 is used immediately asa crude membrane fraction.

Binding is performed using [125I]VIP (specificradioactivity of 0.5 Ci/nmol), [125I]Tyr25 secretin(specific radioactivity of 1.0 Ci/nmol) and [125I-Ac-His1]PACAP-27 (specific radioactivity of 0.7 Ci/nmol)as tracers. In all cases, non-specific binding is definedas the residual binding in the presence of 1 µM of theunlabeled peptide corresponding to the tracer. Bindingis performed at 37°C in a 20 mM Tris-maleate, 2 mMMgCl2, 0.1 mg/ml bacitracin, 1% bovine serum albu-min (pH 7.4) buffer. Bound radioactivity is separatedfrom free by filtration through glass-fibre GF/C fil-ters presoaked for 24 h in 0.1% polyethyleneimine andrinsed three times with a 20 mM (pH 7.4) sodium phos-phate buffer containing 1% bovine serum albumin.

EVALUATIONThe IC50 values (in mM) for each peptide on each re-ceptor are calculated from complete dose-effect curvesperformed on three different membrane preparationsusing the LIGAND program.

MODIFICATIONS OF THE METHODSchmidt et al. (1993) studied the binding of PAPAC,VIP and analogues of VIP and PAPAC in rat AR4–2J pancreatic carcinoma cells and isolated pancre-atic acini to the PAPAC-1 receptor, abundantly ex-pressed in AR 4–2J pancreatic carcinoma cells, and tothe VIP/PAPAC-2 receptor. Simultaneously, biologicaleffects (lipase secretion and cAMP production) in pan-

creatic acini were determined. PAPAC was regarded asa potent ligand for both receptor types and as a potentVIP-like secretagogue.

REFERENCES AND FURTHER READINGBuscail L, Gourlet P, Cauvin A, de Neef P, Gossen D,

Arimura A, Miyata A, Coy DH (1990) Presence of highlyselective receptors for PACAP (pituitary adenylate cyclaseactivating peptide) in membranes from the rat pancreaticacinar cell line AR 4-J. FEBS Lett 262:77–81

Calvo JR, Montilla ML, Guerrero JM, Segura JJ (1994) Expres-sion of VIP receptors in mouse peritoneal macrophages:Functional and molecular characterization. J Neuroim-munol 50:85–93

Ciccarelli E, Vilardaga JP, de Neef P, di PaoloE, Warelbroeck M,Bollen A, Robberecht P (1994) Properties of the VIP-PACAP type II receptor stably expressed in CHO cells.Regul Pept 54:397–407

Ciccarelli E, Svoboda M, de Neef P, di Paolo E, Bollen A,Dubeaux C, Vilardage JP, Waelbroeck M, Robberecht P(1995) Pharmacological properties of two recombinantsplice variants of the PACAP type I receptor transferredand stably expressed in CHO cells. Eur J Pharmacol288:259–267

Couvineau A, Rousset M, Laburthe M (1985) Molecular iden-tification and structural requirement of vasoactive intesti-nal peptide (VIP) receptors in the human colon adenocarci-noma cell line, HT-29. Biochem J 213:139–143

Dickinson T, Fleetwood-Walker SM (1999) VIP and PACAP:very important in pain? Trends Pharmacol Sci 20:324–329

Fahrenkrug J (1979) Vasoactive intestinal peptide: Measure-ment, distribution and putative neurotransmitter function.Digestion 19:149–169

Felley CP, Qian JM, Mantey S, Pradhan T, Jensen RT (1992)Chief cells possess a receptor with high affinity for PACAPand VIP that stimulates pepsinogen release. Am J Physiol263 (Gastrointest Liver Physiol 26):G901–G907

Filipsson K, Pacine G, Scheurink AJW, Ahren B (1998)PACAP stimulates insulin secretion but inhibits insulinsensitivity in mice. Am J Physiol 274; Endocrinol Metab37:E834–E842

Gafvelin (1990) Isolation and primary structure of VIP fromsheep brain. Peptides 11:703–706

Gourlet P, Vertongen P, Vandermeers A, Vandermeers-Piret MC,Rathe J, de Neef P, Waelbroeck M, Robberecht P (1997)The long-acting vasoactive intestinal polypeptide agonistRO 25–1553 ins highly selective of the VIP2 receptor sub-class. Peptides 18:403–408

Gourlet P, Vandermeers A, Vertongen P, Rathe J, de Neef P,Cnudde J, Waelbroeck M, Robberecht P (1997b) Devel-opment of high affinity selective VIP1 receptor agonists.Peptides 18:1539–1545

Gourlet P, de Neef P, Cnudde J, Waelbroeck M, Robberecht P(1997c) In vitro properties of a high affinity selective an-tagonist of the VIP1 receptor. Peptides 18:1555–1560

Gozes I, McCune SK, Jacobson L, Warren D, Moody TW, Frid-kin M, Brenneman DE (1991) An antagonist to vasoactiveintestinal peptide affects cellular functions in the centralnervous system. J Pharmacol Exp Ther 257:959–966

Guijarro LG, Rodriguez-Pena MS, Prieto JC (1991) Charac-terization of vasoactive intestinal peptide receptors in ratseminal vesicle. Am J Physiol 260 (Endocrinol Metab23):E286–E291

Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M,Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedha-


ran SP, Wank SA, Wascheck JA (1998) International Unionof Pharmacology. XVIII. Nomenclature of receptors for va-soactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 50:265–270

Ishihara T, Nakamura S, Kaziro Y, Takahashi T, Taka-hashi K, Nagata S (1991) Molecular cloning and expres-sion of a cDNA encoding the secretion receptor. EMBO J10:1635–1641

Ito O, Naruse S, Kitagawa M, Ishiguro H, Ko S, Nakajima M,Hayakawa t (1998) The effect of VIP/PACAP family ofpeptides on pancreatic blood flow and secretion in con-scious dogs. Regul Pept 78:105–112

Linden A, Cardell LO, Yoshihara S, Stjarne P, Nadel JA (1998)PACAP 1–38 as an inhaled bronchodilator in guinea pigs invivo. Peptides 19:93–98

Miyata A, Jiang L, Dahl RD, Kitada C, Kubo K, Fujino M, Mi-namino N, Arimura A (1990) Isolation of a neuropeptidecorresponding to the N-terminal 27 residues of the pituitaryadenylate cyclase-activating polypeptide with 38 residues(PACAP38). Biochem Biophys Res Commun 170:643–648

Mizuno Y, Kondo K, Terashima Y, Arima H, Murase T, Oiso Y(1998) Anorectic effect of pituitary adenylate cyclase acti-vating polypeptide (PACAP) in rats: Lack of evidence forinvolvement of hypothalamic neuropeptide gene expres-sion. J Neuroendocrinol 10:611–616

Motomura Y, Chijiiwa Y, Iwakiri Y, Ochiai T, Nawata H(1998) Interactive mechanisms among pituitary adeny-late cyclase-activating peptide, vasoactive intestinal pep-tide, and parathyroid receptors in guinea pig cecal cir-cular smooth muscle cells. Endocrinology 139:2869–2878

Nakajima T, Tanimura T, Pisano JJ (1970) Isolation and struc-ture of a new vasoactive peptide Fed Proc 29:282

Okazawa A, Cui ZH, Lotvall J, Yoshihara S, Skoogh BE,Kashimoto K, Linden A (1998) Effect of a novel PACAP-27 analogue on muscarinic airway responsiveness in guineapigs in vivo. Eur Respir J 12:1062–1066

Onaga T, Okamoto K, Harada Y, Mineo H, Kato S (1997)PACAP stimulates pancreatic exocrine secretion via the va-gal cholinergic nerves in sheep. Regul Pept 72:1147–153

Onaga T, Harada Y, Okamoto K (1998) Pituitary adenylatecyclase-activating polypeptide (PACAP) induces duode-nal phasic contractions via the vagal cholinergic nerves insheep. Regul Pept 77:69–76

Robberecht P, Vertongen P, Perret J, van Rampelbergh J, Juar-ranz MG, Waelbroeck M (1999) Receptors for VIP andPACAP. Trends Pharmacol Sci; Receptor and Ion ChannelNomenclature Supplement

Schmidt WE, Seebeck J, Höcker M, Schwarzhoff R,Schäfer H, Fornefeld H, Morys-Wortmann C, Fölsch UR,Creutzfeldt W (1993) PAPAC and VIP stimulate en-zyme secretion in rat pancreatic acini via interactionwith VIP/PACAP-2 receptors: additive augmentationof CCK/carbachol-induced enzyme release. Pancreas8:476–487

Shigyo M, Aizawa H, Inoue H, Matsumoto K, Takada S, Hara N(1998) Pituitary adenylate cyclase activating peptide reg-ulates neurally mediated airway responses. Eur Resp J12:64–70

Soo Tek Lee, Kae Yol Lee, Li P, Coy D, Chang TM, Chey WY(1998) Pituitary adenylate cyclase-activating peptide stim-ulates rat pancreatic secretion via secretin and cholecys-tokinin releases. Gastroenterology 114:1054–1060

Sreedharan SP, Patel DR, Huang j-X, Goetzl EJ (1993) Cloningand functional expression of a human neuroendocrine va-soactive intestinal peptide receptor. Biochem Biophys ResCommun 193:546–553

Takeuchi K, Yagi K, Sugamoto S, Furukawa O, Kawauchi S,(1998) Involvement of PACAP in acid-induced HCO3

− re-sponse in rat duodenum. Pharmacol Res 38:475–480

Van Rampelbergh J, Gourlet P, de Neef P, Robberecht P, Wael-brouck M (1996) Properties of the pituitary adenylate cy-clase-activating polypeptide I and II receptors, vasoac-tive intestinal peptide1, and chimeric amino-terminal pi-tuitary adenylate cyclase-activating polypeptide/vasoactiveintestinal peptide1 receptors: Evidence for multiple recep-tor states. Mol Pharmacol 50:1596–1604

Yada T, Sakurada M, Ishihara A, Nakata M, Shioda S,Yaekura K, Hamakawa N, Yanagida K, Kikuchi M, Oka Y(1997) Pituitary adenylate cyclase-activating polypeptide(PACAP) is an islet substance serving as an intra-islet am-plifier of glucose-induced insulin secretion in rats. J Physiol505:319–328

Zhang Y, Danielson N, Sundler F, Mulder H (1998) Pituitaryadenylate cyclase-activating peptide in upregulated in sen-sory neurons by inflammation. NeuroReport 9:2833–2836

H.1.1.8Cannabinoid Activity

H.1.1.8.1General Considerations on Cannabinoids

In the centuries since hashish and marijuana (Cannabissativa) were used as psychoactive drugs the most sig-nificant discoveries in regard to the mechanism of ac-tion were made with the isolation of (–)-trans-�9-tetrahydrocannabinol (�9-THC) as the principal ac-tive ingredient (Mechoulam et al. 1970), the character-ization and localization of the cannabinoid receptor inthe brain (Devane et al. 1988), the cloning of its gene(Matsuda et al. 1990), and the identification of an en-dogenous ligand (Devane et al. 1992). Most cannabi-noid effects occur receptor mediated in the CNS (Mar-tin 1986; Herkenham et al. 1990; Porter and Felder2001; Howlett et al. 2002). The recognized CNS re-sponses to cannabinoids include alterations in cog-nition and memory, euphoria and sedation (Howlett1995). Ranganathan and D’Souza (2006) reviewed theacute effects of cannabinoids on memory in humans.

Cannabinoids have been shown to produce anal-gesia without the respiratory problems associatedwith opioid analgesics (Buxbaum 1972; Martin 1985;Dewey 1986; Razdan 1986; Compton et al. 1992;Meng et al. 1998; Strangman et al. 1998) whichmay be of value for therapeutic applications (Hollister1986; Izzo et al. 2000a; Pertwee 2000). Simultaneousadministration of cannabinoid receptor agonists andμ- or κ-receptor agonists indicate a cannabinoid-opi-oid interaction in anti-nociception (Manzanares et al.2000). Baker et al. (1990, 2000) described experimen-tal allergic encephalomyelitis with relapsing-remittingepisodes, spasticity and tremor similar to multiple scle-


rosis in human beings in Biozzi AB/H mice. Thesesymptoms could be antagonized by cannabinoids.

A multiple-evaluation paradigm of in vivo mouseassays is employed to test for cannabimimetic effects.This paradigm includes assays for reduction in spon-taneous activity, and the production of hypothermia,catalepsy, and antinociception measured by tail-flickassay (Compton et al. 1992; Welch et al. 1998). Thebehavioral effects of �9-THC and related cannabi-noids in mice have been termed the “popcorn” effect.That is, groups of mice are in a sedated state with lit-tle or no movement until a stimulus causes one mouseto jump (hyper-reflexia). This animal falls on anothermouse which in turn jumps so that this repeated hyper-reflexic jumping looks like corn popping in a machine.Subsequently, all mice will be sedated until anotherstimulus reinitiates the process (Dewey 1986). Like theopioids, cannabinoids inhibit electrically evoked con-tractions of the mouse vas deferens and the guinea pigileum, but unlike the opioids, these effects are not an-tagonized by naloxone (Pertwee et al. 1992; Hillardet al. 1999).

In addition to the effects in the CNS (Chaperon andThiebot 1999), peripheral effects of cannabinoids areknown (Lynn and Herkenham 1994) including actionson the endocrine system (Patra and Wadsworth 1990,(Block et al. 1991; Wenger et al. 2000), on the diges-tive tract (Rosell and Agurell 1975; Izzo et al. 1990a,b, 2000; Coutts et al. 2000; Massa et al. 2005), on in-gestive behavior (Giuliani et al. 2000), on the pul-monary and cardiovascular system (Stengel et al.1998; White and Hiley 1998; Niederhoffer and Szabo1999; Liu et al. 2000; Niederhoffer et al. 2003), and onimmune modulation (Kaminski et al. 1992; Lynn andHerkenham 1994; Achiron et al. 2000).

An endogenous cannabinoid was isolated fromporcine brain by Devane et al. (1992) and found tobe an unsaturated fatty acid ethanolamide, arachi-donylethanolamide, also called anandamide, whichactivates CB1 receptors (Devane et al. 1992; Hillardand Jarrahian 2005) and produces similar effects as�9-tetrahydrocannabinol including anti-nociception,hypothermia, hypomotility and catalepsy in mice(Smith et al. 1994). The brain enzyme hydrolyzingand synthesizing anandamide has been characterizedby Ueda et al. (1995). The human brain fatty-acidamide hydrolase was characterized by Maccarroneet al. (1998) as a single protein, which hydrolysesanandamide to arachidonate and ethanolamine.

Similar effects are produced by other polyunsat-urated N-acetylethanolamines, such as N-palmitoyl-ethanolamine, which activates the CB-2-like receptor

subtype (Hanu et al. 1993; Facci et al. 1995). Bothendogenous cannabinoids (called endocannabinoids)derive from cleavage of a precursor phospholipid,N-acylphosphatidylethanolamine, catalyzed by Ca2+-activated D-type phosphodiesterase activity (Cadaset al. 1996). 2-Arachidonylglycerol was described asa further endogenous ligand for cannabinoid receptors(Ameri and Simmet 2000; Sigiura et al. 2000)

Numerous synthetic analogs and cannabimimeticcompounds have been evaluated as agonists and an-tagonists by in vitro and in vivo pharmacological meth-ods (Martin et al. 1991; D’Ambra et al. 1992; Melvinet al. 1993; Barth and Rinaldi-Carmona 1999; Hillardet al. 1999; Palmer et al. 2000; Piomelli et al. 2000;De Petrocellis et al. 2004; Costa et al. 2005; Griebelet al. 2005; Lange et al. 2005; Makriyannis et al. 2005;Pertwee 2006).

Costa et al. (2004) described oral anti-inflammatoryactivity of cannabidiol, a non-psychoactive constituentof cannabis, in acute carrageenan-induced inflamma-tion in the rat paw.

For studies on the effects of cannabinoids andcannabinoid antagonists on digestive system and obe-sity see L.3.1.3.

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anabinol (HU-211) effect on experimental autoimmune en-cephalomyelitis. J Neuroimmunol 102:26–31

Ameri A, Simmet T (2000) Effects of 2-arachidonylglycerol, anendogenous cannabinoid, on neuronal activity in rat hip-pocampal slices. Naunyn-Schmiedeberg’s Arch Pharmacol361:265–272

Baker D, O’Neill JK, Gschmeissner SE, Wilcox CE, Butter C,Turk JL (1990) Induction of chronic relapsing experimentalallergic encephalomyelitis in Biozzi mice. J Neuroimmunol28:261–270

Baker D, Pryce G, Croxford JL, Brown P, Pertwee RG, Huff-man HW, Layward L (2000) Cannabinoids control spas-ticity and tremor in a multiple sclerosis model. Nature202:84–87

Barth F, Rinaldi-Carmona M (1999) The development ofcannabinoid antagonists. Curr Med Chem 6:745–755

Block RI, Farinpour R, Schlechte JA (1991) Effects of chronicmarijuana use on testosterone, luteinizing hormone, folli-cle stimulating hormone, prolactin and cortisol in men andwomen. Drug Alcohol Depend 28:121–128

Buxbaum DM (1972) Analgesic activity of �9-tetrahydro-cannabinol in the rat and mouse. Psychopharmacology25:275–280

Cadas H, Gaillet S, Beltramo M, Venance L, Piomelli D (1996)Biosynthesis of an endogenous cannabinoid precursor inneurons and its control by calcium and cAMP. J Neurosci16:3934–3942

Chaperon F, Thiebot MH (1999) Behavioral effects of cannabi-noid agents in animals. Crit Rev Neurobiol 13:243–281

Compton DR, Johnson MR, Melvin LS, Martin BR (1992) Phar-macological evaluation of a series of bicyclic cannabinoid


analogs: Classification as cannabimimetic agents. J Phar-macol Exp Ther 260:201–209

Costa B, Colleoni M, Conti S, Parolaro D, Franke C,Trovato AE, Giagnoni G (2004) Oral anti-inflammatoryactivity of cannabidiol, a non-psychoactive constituentof cannabis, in acute carrageenan-induced inflammationin the rat paw. Naunyn-Schmiedebergs Arch Pharmacol369:294–299

Costa B, Trovato AE, Colleoni M, Giagnoni G, Zarini E, Croci T(2005) Effect of the cannabinoid CB1 receptor antagonist,SR141716, on nociceptive response and nerve demyelina-tion in rodents with chronic constriction injury of the sciaticnerve. Pain 116:52–61

Coutts AA, Brewster M, Ingram T, Razdan RK, Pertwee RG(2000) Comparison of novel cannabinoid partial agonistand SR 141716A in the guinea pig small intestine. Br JPharmacol 129:645–652

D’Ambra TE, Estep KG, Bell MR, Eissenstat MA, JosefKA, Ward SJ, Haycock DA, Baizman ER, Casiano FM,Beglin NC, Chippari SM, Grego JD, Kullnig RK, Daley GT(1992) Conformationally restrained analogues of pravado-line: nanomolar potent, enatioselective, (aminoalkyl)indoleagonists on the cannabinoid receptor. J Med Chem35:124–135

De Petrocellis L, Cascio MG, Di Marzo V (2004) The endo-cannabinoid system: a general view and latest additions. BrJ Pharmacol 141:765–774

Devane WA, Dysarz FAI, Johnson MR, Melvin LS, Howlett AC(1988) Determination and characterization of a cannabi-noid receptor in rat brain. Mol Pharmacol 34:605–613

Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA,Griffin G, Gibson D, Mandelbaum A, Etinger A, Me-choulam R (1992) Isolation and structure of a brain con-stituent that binds to the cannabinoid receptor. Science258:1946–1949

Dewey WL (1986) Cannabinoid pharmacology. Pharmacol Rev38:151–178

Facci L, Dal Torso R, Romanello S, Buriani A, Skaper SD,Leon A (1995) Mast cells express a peripheral cannabi-noid receptor with differential sensitivity to anandamideand palmitoyletholamine. Proc Natl Acad Sci USA92:3376–3380

Giuliani D, Ottani A, Ferrari F (2000) Effects of the cannabinoidreceptor agonist, HU 210, on ingestive behavior and bodyweight in rats. Eur J Pharmacol 391:275–279

Griebel G, Stemmelin J, Scatton B (2005) Effects of thecannabinoid CB1 receptor antagonist rimonabant in mod-els of emotional reactivity in rodents. Biol Psychiatry57:261–267

Hanu L, Gopher A, Almog S, Mechoulam R (1993) Two newunsaturated fatty acid ethanolamines in brain bind to thecannabinoid receptor. J Med Chem 36:3032–3034

Herkenham M, Lynn AB, Little MD, Johnson MR, Melvin LS,de Costa BR, Rice KC (1990) Cannabinoid receptor lo-calization in brain. Proc Natl Acad Sci USA 87:1932–1936

Hillard CJ, Manna S, Greenberg MJ, DiCamelli R, Ross RA,Stevenson LA, Murphy V, Pertwee RG, Campbell WB(1999) Synthesis and characterization of potent and selec-tive agonists of the neuronal cannabinoid receptor (CB1).J Pharmacol Exp Ther 289:1427–1433

Hillard CJ, Jarrahian A (2005) Accumulation of anan-damide: evidence for cellular diversity. Neuropharmacol-ogy 48:1072–1078

Hollister LE (1986) Health aspects of cannabis. Pharmacol Rev38:1–20

Howlett AC (1995) Pharmacology of cannabinoid receptors.Annu Rev Pharmacol Toxicol 35:607–634

Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, De-vane WA, Felder CC, Herkenham M, Mackie K, Mar-tin BR, Mechoulam R, Pertwee RG (2002) InternationalUnion of Pharmacology XXVII. Classification of cannabi-noid receptors. Pharmacol Rev 54:161–202

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Izzo AA, Mascolo N, Pinto N, Capasso R, Capasso F (1999b)The role of cannabinoid receptors in intestinal motility, de-faecation and diarrhoea in rats. Eur J Pharmacol 384:37–42

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Izzo AA, Mascolo N, Tonini M, Capasso F (2000b) Modulationof peristalsis by cannabinoid CB-1 ligands in the isolatedguinea pig ileum. Br J Pharmacol 129:984–990

Kaminski NE, Abood ME, Kessler FK, Martin BR, Schatz AR(1992) Identification of a functionally relevant cannabi-noid receptor on mouse spleen cells that is involved incannabinoid-mediated immune modulation. Mol Pharma-col 42:736–742

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H.1.1.8.2Receptor Binding of Cannabinoids

PURPOSE AND RATIONALEAfter the discovery of cannabinoid receptors in brain(Howlett et al. 1988; Devane et al. 1988; Pertwee1993), two cannabinoid receptor subtypes were iden-tified: CB1 and CB2. Cannabinoid receptors and werereviewed by Felder and Glass (1998), Pertwee (1999,2001).

CB1 has an amino acid sequence consistent witha tertiary structure typical of the seven transmembrane-spanning proteins that are coupled to G proteins (Ger-ard et al. 1990, 1991; Howlett et al. 1990; Matsudaet al. 1990). The CNS responses to cannabinoid com-pounds are apparently mediated exclusively by CB1,since CB2 transcripts could not be found in brain tis-sue. CB1 transduces signals in response to CNS activeconstituents of Cannabis sativa, as well as syntheticbicyclic and tricyclic analogs, aminoalkylindole, andeicosanoid cannabi-mimetic compounds. CB1 is cou-pled to G1 to inhibit adenylate cyclase activity and toa pertussis-sensitive G protein to regulate Ca2+ cur-rents. Zimmer et al. (1999) produced a mouse strainwith a disrupted CB1 gene. These CB1 knockoutmice had a significantly increased mortality rat anddisplayed reduced locomotor activity, increased ringcatalepsy, and hypoalgesia in hot plate and formalintests.

CB2, the second cannabinoid-binding seven-transmembrane spanning receptor, exhibits 68%identity to CB1 within the helical regions, and 44%identity throughout the total protein. The CB2 clonewas derived from a human promyelocytic leukemiacell lin HL60 cDNA library (Munro et al. 1993),also expressed in human leukocytes (Bouaboula et al.1993). The gene for the rat CB2 receptor was cloned,


expressed, and its properties compared with those ofmouse and human CB2 receptors (Griffin et al. 2000).

Receptor binding of cannabinoids in correlation toin vivo activities was described by Compton et al.(1993).

PROCEDUREMembrane PreparationMale Sprague Dawley rats weighing 150–200 g aredecapitated and the brain rapidly removed. The cor-tex is dissected free using visual landmarks follow-ing reflection of cortical material from the midline andimmersed in 30 ml of ice-cold centrifugation solution(320 mM sucrose, 2 mM Tris-EDTA, 5 mM MgCl2).The process is repeated until the cortices of five rats arecombined. The cortical material is homogenized witha Potter-Elvehjem glass-Teflon grinding system. Thehomogenate is centrifuged at 1600 g for 15 min, the su-pernatant saved and combined with the two subsequentsupernatants obtained from washing and 1600 g cen-trifugation of the P1 pellet. The combined supernatantfractions are centrifuged at 39,000 g for 15 min. TheP2 pellet is resuspended in 50 ml buffer (50 mM Tris-HCl, 2 mM Tris EDTA, 5 mM MgCl2, pH 7.0), incu-bated for 10 min at 37°C, then centrifuged at 23,000 gfor 10 min. The P2 membrane is resuspended in 50 mlof buffer A, incubated again except at 30°C for 40 min,then centrifuged at 11,000 g for 15 min. The finalwash-treated P2 pellet is resuspended in assay buffer B(50 mM Tris-HCl, 1 mM Tris EDTA, 3 mM MgCl2,pH 7.4) to a protein concentration of approximately2 mg/ml. The membrane preparation is divided into4 aliquots and quickly frozen in a bath solution of dryice and 2-methylbutane and then stored at −80°C.

Binding AssayBinding is initiated by the addition of 150 mg of P2membrane to test tubes containing [3H]CP-55,940(79 Ci/mmol), a cannabinoid analog, (for displace-ment studies) and a sufficient quantity of buffer C(50 mM Tris-HCl, 1 mM Tris EDTA, 3 mM MgCl2,5 mg/ml BSA) to bring the total incubation volumeto 1 ml. The concentration of [3H]CP-55,940 indisplacement studies is 400 pM, whereas that insaturation studies varies from 25 to 2500 pM. Nonspe-cific binding is determined by the addition of 1 mMunlabeled CP-55,940. The standard CP-55,940 andother cannabinoid analogs are prepared in suspensionbuffer C from a 1 mg/ml ethanolic stock withoutevaporation of the alcohol.

After incubation at 30°C for 1 h, binding is termi-nated by addition of 2 ml ice-cold buffer D (50 mM

Tris-HCl, 1 mg/ml BSA) and vacuum filtration throughpretreated filters in a 12-well sampling manifold. Re-action vessels are washed once with 2 ml of ice-coldbuffer D, and the filters washed twice with 4 ml of ice-cold buffer D. Filters are placed into 20-ml plastic scin-tillation vials with 1 ml of distilled water and 10 ml ofBudget-Solve (RPI Corp., Mount Prospect, IL). Aftershaking for 1 h, the radioactivity present is determinedby liquid scintillation photometry.

EVALUATIONThe Bmax and Kd values obtained from Scatchardanalysis are determined via a suitable computer pro-gram. Displacement IC50 values are determined by un-weighted least squares linear regression of log concen-tration-percent displacement data and then convertedto KI values.

MODIFICATIONS OF THE METHODTo further characterize neuronal cannabinoid recep-tors, Thomas et al. (1998) compared the ability ofcannabinoid analogs to compete for receptor sites la-beled either with [3H]SR141716A or[3H]CP-55940.

Herkenham et al. (1991) characterized and localizedcannabinoid receptors in rat brain by a quantitative au-toradiographic study.

Felder et al. (1995) compared the pharmacologyand signal transduction of the human cannabinoid CB1and CB2 receptors.

Ross et al. (1998) compared cannabinoid bindingsites in guinea pig forebrain and small intestine.

Rinaldi-Camora et al. (1998) tested the affinity ofan antagonist of the CB2 cannabinoid receptor for ratspleen and cloned human CB2 receptors.

Bilkei-Gorzo et al. (2005) described early age-related cognitive impairment in mice lacking cannabi-noid CB1 receptors.

REFERENCES AND FURTHER READINGBilkei-Gorzo A, Racz I, Valverde O, Otto M, Michel K,

Sarstre M, Zimmer A (2005) Early age-related cognitiveimpairment in mice lacking cannabinoid CB1 receptors.Proc Natl Acad Sci USA 102:15670–15675

Bouaboula M, Rinaldi M, Carayon P, Carillon C, Delpech B,Shire D, Le Fur G, Casellas P (1993) Cannabinoid-re-ceptor expression in human leukocytes. Eur J Biochem214:173–180

Compton DR, Rice KC, de Costa BR, Razdan RK, Melvin LS,Johnson MR, Martin BR (1993) Cannabinoid structure-ac-tivity relationships: Correlation of receptor binding and invivo activities. J Pharmacol Exp Ther 265:218–226

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Felder CC, Joyce KE, Briley EM, Mansouri J, Mackie K,Blond O, Lay Y, Ma AL, Mitchell RL (1995) Compari-son of the pharmacology and signal transduction of the hu-man cannabinoid CB1 and CB2 receptors. Mol Pharmacol48:443–450

Felder CC, Glass M (1998) Cannabinoid receptors andtheir endogenous agonists. Ann Rev Pharmacol Toxicol38:179–200

Gerard C, Mollereau C, Vassart G, Parmentier M (1990) Nu-cleotide sequence of a human cannabinoid receptor cDNA.Nucleic Acids Res 18:7142

Gerard CM, Mollereau C, Vassart G, Parmentier M (1991)Molecular cloning of a human cannabinoid receptor whichis also expressed in testis. Biochem J 279:129–134

Griffin G, Tao Q, Abood ME (2000) Cloning and pharmacolog-ical characterization of the rat CB2 cannabinoid receptor.J Pharmacol Exp Ther 292:886–894

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Howlett AC, Bidaut-Russell M, Devane WA, Melvin LS, John-son MR, Herkenham M (1990) The cannabinoid recep-tor: biochemical, anatomical and behavioral characteriza-tion. Trends Neurosci 13:420–423Matsuda LA, Lolait SJ,Young AC, Bonner TI (1990)Structure of a cannabinoid re-ceptor and functional expression of the cloned cDNA, Na-ture 346:561–564

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Ross RA, Brockie HC, Fernando SR, Sha B, Razdan RK, Per-twee RG (1998) Comparison of cannabinoid binding sitesin guinea pig forebrain and small intestine. Br J Pharmacol125:1345–1351

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H.1.1.9Vanilloid (Capsaicin) Activity

H.1.1.9.1General Considerations on Vanilloids

PURPOSE AND RATIONALESeveral authors reviewed the recent development ofcapsaicin and vanilloid receptors (Holzer P 1991; Bíró

et al. 1997; Sterner and Szallasi 1999; Szallasi andBlumberg 1999; Caterina and Julius 2001; Piomelli2001; Gunthorpe et al. 2002). Capsaicin was isolatedby Thresh (1846). The chemical structure was deter-mined by Nelson (1919). The analgesic use of cap-saicin was reviewed by Lembeck (1987). Capsaicin ex-cites a subset of primary sensory neurons with somatain the dorsal root ganglion or trigeminal ganglion. Asa general rule, these vanilloid-sensitive neurons arepeptidergic, small diameter (50 µm) neurons, givingrise to thin, unmyelinated C fibers. Among sensoryneuropeptides, the tachykinin Substance P shows thebest correlation with vanilloid sensitivity. Vanilloid-sensitive neurons transmit noxious information (usu-ally perceived as itching or pain) to the CNS, whereasperipheral terminals rare sites of release of a variety ofpro-inflammatory neuropeptides. Among irritant com-pounds acting on primary sensory neurons, capsaicinand related vanilloids are unique in that the initial stim-ulation by vanilloids is followed by a long lasting re-fractory state. Neurotoxicity has been observed whencapsaicin as given to newborn rats (Jancsó et al. 1977;Nagy and van der Kooy 1983).

Besides capsaicin, several natural vanilloid ago-nists were described (Jonassohn and Sterner 1997;Liu et al. 1997; Sterner and Szallasi 1999; Mendeset al. 2000). The irritant principle from Euphorbiaresinifera, named resiniferatoxin, was isolated by Her-genhahn et al. (1975). In several assays, resiniferatoxinand its derivatives are several thousand-fold more po-tent than capsaicin (Szolcsanyi et al. 1990; Ács et al.1995), which is explained by specific receptor bind-ing (Szallasi and Blumberg 1990; Ács et al. 1994).Lee et al. (2001) described simplified resiniferatoxinderivatives as potent vanilloid receptor agonists withpotent analgesic activity and reduced pungency.

The high affinity of vanilloid receptors argues forthe existence of endogenous vanilloids. Hwang et al.(2000), Piomelli (2001) reported a direct activation ofcapsaicin receptors by products of lipogenases. Pain-inducing substances, such as bradykinin, may acti-vate phospholipase-linked receptors in sensory neu-rons, mobilizing arachidonic acid from phospholipidsand generating 12-HPETE. This lipid second messen-ger interacts in turn with a cytosolic domain of theVR1 receptor channel, increasing its opening proba-bility and causing the sensory neuron to become depo-larized.

The endogenous ligand of CB1 cannabinoid recep-tors, anandamide, is also a full agonist at vanilloidVR1 receptors (Zygmunt et al. 1999; Maccarrone et al.2000; Smart et al. 2000; DePetrocellis et al. 2001).


Premkumar and Ahern (2000) showed that activationof protein kinase C activates VR1 channel activity.

The first capsaicin or vanilloid receptor, termedVR1, was cloned by Caterina et al. (1997). Hayeset al. (2000) reported the cloning and functional ex-pression of a human orthologue of rat vanilloid recep-tor 1. Pharmacological differences between the humanand rat vanilloid receptor 1 were observed (McIntyreet al. 2001). VR1 functions as a molecular integratorof painful chemical and physical stimuli including cap-saicin, noxious heat and low pH (Tominaga et al. 1998;Michael and Priestley 1999; Davis et al. 2000; Welchet al. 2000). In mice lacking the capsaicin receptor im-paired nociception and pain sensation was observed(Caterina et al. 2000).

Vanilloid receptors are differently distributed in thecentral and peripheral nervous system (Szallasi 1995;Szallasi et al. 1995; Mezey et al. 2000; Ichikawa andSugimoto 2001). Bíró et al. (1998) reported charac-terization of functional vanilloid receptors expressedby mast cells. Biological and electrophysiologicaldata indicate heterogeneity within the vanilloid recep-tors. Caterina et al. (1999) described a capsaicin-re-ceptor homologue, named vanilloid-receptor-like pro-tein (VRL-1) with a high threshold for noxious heat.A novel human vanilloid receptor-like protein, namedVRL-2 was identified and characterized by Delanyet al. (2001).

Price et al. (2004) repoted modulation of trigeminalsensory neuron activity by the dual cannabinoid-vanil-loid agonists anandamide, N-arachidonoyl-dopamineand arachidonyl-2-chloroethylamide.

Several vanilloid antagonists were described, suchas capsazepine (Bevan et al. 1992; Walpole et al. 1994)or iodo-resiniferatoxin (Wahl et al. 2001).

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Thresh LT (1846) Isolation of capsaicin. Pharm J 6:941Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert

H, Skinner K, Raumann BE, Basbaum AI, Julius D (1998)The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21:531–543

Wahl P, Foged C, Tullin S, Thomsen C (2001) Iodo-resinifera-toxin, a new potent vanilloid receptor antagonist. Mol Phar-macol 59:9–15

Walpole CS, Bevan S, Boverman G, Boelsterli JJ, Brecken-ridge R, Davies JW, Hughes GA, James I, Oberer L, WinterJ (1994) The discovery of capsazepine, the first compet-itive antagonist of the sensory neuron excitants capsaicinand resiniferatoxin. J Med Chem 37:1942–1954

Welch JM, Simon SA, Reinhart PH (2000) The activation mech-anism of rat vanilloid receptor 1 by capsaicin involves thepore domain and differs from the activation by either acidor heat. Proc Natl Acad Sci 97:13889–13894

Zygmunt PM, Petersson J, Andersson DA, Chuang H-H,Sørgård M, DiMarzo V, Julius D, Högestätt ED (1999)Vanilloid receptors on sensory nerves mediate the vasodila-tor action of anadamide. Nature 400:452–457

H.1.1.9.2Vanilloid Receptor Binding

PURPOSE AND RATIONALEÁcs et al. (1994) described [3H]resiniferatoxin bind-ing by the human vanilloid (capsaicin) receptor. Re-ceptor types and species differences of the vanilloidreceptor were described by Szallasi et al. (1994, 1996).The rat vanilloid receptor (rVR1) was cloned and sta-bly expressed in HEK293 cells by Jerman et al. (2000).A detailed pharmacological characterization was con-ducted using the Ca2+-sensitive dye, Fluo3AM in a flu-orimetric imaging plate reader (FLIPR). Ross et al.(2001) studied structure-activity relationship for theendogenous cannabinoid, anadamide, and certain of itsanalogues at vanilloid receptors in transfected CHOcells.

PROCEDURECell CultureRat vanilloid receptor (rVR1) transfected CHO cellsare maintained in MEM Alpha minus media contain-ing 2 mM L-glutamine supplemented with 10% hy-clone fetal bovine serum, 350 µg/ml G418 (Sigma-Aldrich), 100 units/ml penicillin and 100 µg/ml strep-tomycin. Cells are maintained in 5% CO2 at 37°C andpassed twice a week using non-enzymatic cell dissoci-ation solution. For the radioligand binding assay, cellsare removed from flasks by scraping and then frozenas a pellet at –20°C for up to one month.


Radioligand Binding ExperimentsAssays are performed in DMEM containing HEPES(25 mM) and BSA (0.25 mg/ml). Total assay volumeis 500 µl containing 20 µg of cell membranes. Bindingis initiated by addition of [3H]resiniferatoxin ([3H]-RTX). Assays are carried out at 37°C for 1 h, be-fore termination by addition of ice-cold wash buffer(50 mM Tris-buffer, 1 mg/ml BSA, pH 7.4) and vac-uum filtration using a 12-well sampling manifold(Brandell cell harvester) and Whatman GF/B filtersthat have been soaked in wash buffer at 4°C for at least24 h. Each reaction is washed 9 times with a 1.5 mlaliquot of wash buffer. The filters are oven-dried andthen placed in 5 ml scintillation fluid. Radioactivity isquantified by liquid scintillation spectrometry. Specificbinding is determined in the presence of 1 µM unla-belled RTX. Protein assays are performed using a Bio-Rad De Kit. Unlabelled compounds are added in a vol-ume of 50 µl after serial dilution using assay bufferfrom a 10 mM stock in ethanol or DMSO. [3H]-RTXis also added in a 50 µl volume following dilution inassay buffer.

EVALUATIONThe KD value and Bmax for [3H]-RTX and the concen-tration of competing ligands to produce 50% displace-ment of the radioligand (IC50) from specific bindingsites are calculated using GraphPad Prism (GraphPadSoftware, San Diego). Dissociation constant (Ki) val-ues are calculated using the Cheng and Prussoff equa-tion.

MODIFICATIONS OF THE METHODWardle et al. (1997) used a 96-well plate assay systemto characterize pharmacologically the vanilloid recep-tor in the dorsal spinal cord of the rat.

Hayes et al. (2000) described the cloning and func-tional expression of a human orthologue of rat vanil-loid receptor-1.

REFERENCES AND FURTHER READINGÁcs G, Palkovits M, Blumberg PM (1994) [3H]resiniferatoxin

binding by the human vanilloid (capsaicin) receptor. BrainRes Mol Brain Res 23:185–190

Hayes P, Meadows HJ, Gunthorpe MJ, Harries MH, Duck-worth DM, Cairns W, Harrison DC, Clarke CE, Elling-ton K, Prinja RK, Barton AGL, Medhurst AD, Smith GD,Topp S, Murdock P, Sanger GJ, Terrett J, Jenkins O, Ben-ham CD, Randall AD, Gloger IS, Davis JB (2000) Cloningand functional expression of a human orthologue of ratvanilloid receptor-1. Pain 88:205–215

Jerman JC, Brough SJ, Prinjha R, Harries MH, Davis JB,Smart D (2000) Characterization using FLIPR of ratvanilloid receptor (rVR1) pharmacology. Br J Pharmacol130:916–922

Ross RA, Gibson TM, Brockie HC, Leslie M, Pashmi G,Craib SJ, DiMarzo V, Pertwee RC (2001) Structure-activityrelationship for the endogenous cannabinoid, anadamide,and certain of its analogues at vanilloid receptors in trans-fected cells and vas deferens. Br J Pharmacol 132:631–640

Szallasi A (1994) The vanilloid (capsaicin) receptor: receptortypes and species differences. Gen Pharmacol 25:223–243

Szallasi A, Blumberg PM (1996) Vanilloid receptors: newinsights enhance potential as a therapeutic target. Pain68:195–208

Wardle KA, Ranson J, Sanger GJ (1997) Pharmacological char-acterization of the vanilloid receptor in the rat dorsal spinalcord. Br J Pharmacol 121:1012–1016

H.1.1.9.3Evaluation of Vanilloid Receptor Antagonists

PURPOSE AND RATIONALESeveral vanilloid receptor antagonists were described,such as capsazepine (Bevan et al. 1992; Walpoleet al. 1994) or iodo-resiniferatoxin (Wahl et al. 2001).Kirschstein et al. (1999) described the inhibition ofrapid heat responses in nociceptive primary sensoryneurons of rats by vanilloid receptor antagonists.

PROCEDUREAdult Sprague Dawley rats of both sexes are deeplyanesthetized with diethyl ether and rapidly decapi-tated. The spine is chilled at 4°C in F12 Dulbecco’smodified Eagle’s medium saturated with carbogengas and additionally containing 30 mM NaHCO3,100,000 units/l penicillin and 100 mg/l streptomycin.Thoracic and lumbar dorsal root ganglions are quicklydissected and freed from connective tissue. Neuronsare dissociated in an incubation chamber enrichedwith carbogen gas at 37°C using collagenase CLS II(5–10 mg/ml, 10–12 min) and trypsin (0.2–1 mg/ml,10–12 min) dissolved in F12 medium. After trituration(4–6 times with a Pasteur pipette) neurons are platedin 35-mm culture dishes, which also serve as record-ing chambers, and stored at 37°C in a humidified 5%CO2 atmosphere before used for electrophysiologicalrecordings.

Only round or oval-shaped neurons without anyprocesses are included in the study. The average ofthe major and the minor diameter is used to mea-sure the size of oval shaped neurons. Whole cellpatch-clamp experiments are performed in carbogengas saturated F12 medium (pH 7.4) at room tempera-ture using an Axo-patch 200A amplifier (Axon Instru-ments) in voltage-clamp mode at a holding potential of–80 mV controlled by pCLAMP6 software. Data arealso registered on a chart recorder. Patch pipettes arefabricated from borosilicate glass using a horizontalmicropipette puller and filled with a solution contain-


ing (in mM) 160 KCl, 8.13 EGTA, 10 HEPES (pH 7.2,RTip = 5.3 ±0.2 M, mean ±SE). Cell diameter, crosssectional area, and membrane capacitance are mea-sured, and excitability is tested by depolarizing voltagesteps for each neuron. Cells lacking a fast inward cur-rent with a reversal potential close to the equilibriumpotential of sodium followed by a prolonged outwardcurrent are excluded from further investigation. Exper-iments in current-clamp mode are performed to mea-sure the resting membrane potential of each neuronand to investigate single action potentials elicited byshort (3 ms) depolarizing current pulses in neurons thatare hyperpolarized by constant current injection result-ing in membrane potentials between –70 and –80 mV.Inflections in the repolarizing phase are qualitativelydetected as second negative peak in the first derivative(dV/dt) of each action potential; the duration of repo-larization is quantitatively assessed by the 10–90% de-cay time.

Applications of ∼50 µl of heated extracellular solu-tion through a puffing system fixed on a micromanip-ulator is used to elicit heat-evoked currents. Controlmeasurements with a fast temperature sensor (BAT-12,Physitemp; τ = 5 ms) in place of the neurons are maderevealing an effective temperature of ∼53°C, a risetime of ∼250 ms, and a decay with a time constant of∼20 s. Effects are compared with those of applicationof the same amount of medium at room temperature.Heat stimuli with or without vanilloid receptor antago-nists and control applications at room temperature arerepeated 2–10 times, and the elicited currents are av-eraged. A neuron is considered as heat sensitive whenthe heat-evoked inward current is significantly greaterthan any fluctuations caused by superfusion of solu-tion at room temperature. Heating the buffered solu-tion may change its pH, and acid solution of pH 6.2are known to activate nociceptive dorsal root ganglionneurons (Bevan and Yeats 1991). The pH of a HEPES-buffered solution decreases while heating (e. g., pH 7.1at 50°C). In contrast, higher temperatures increase thepH of a NaHCO3/CO2 buffer, because the solubilityof CO2 is reduced and thus reverses the HEPES ef-fect. The pH of the F12 medium maximally changes ina range of 7.28–7.52 while heating to 50°C and cool-ing down to room temperature. The membrane con-ductance is measured in voltage-clamp mode by hy-perpolarizing pulses (5 mV, 10 ms, 50 s−1), and con-ductance changes are determined at the maximum am-plitude of heat evoked currents.

Reversal potentials of heat- and capsaicin-inducedcurrents are measured as described by Liu et al.(1997) using fast depolarizing ramps (–80 to +30 mV

in 22 ms every 550 ms). Patch pipettes are filledwith a potassium-free solution containing (in mM)140 CsCl, 10 HEPES, 10 EGTA, and 4 MgCl2 (ad-justed to pH 7.2). Tetrodotoxin (100 µM) and nifedip-ine (1 µM) are added to the extracellular solution toblock voltage gated Na+ and Ca2+ channels. Capsaicinis dissolved in ethanol, diluted to its final concentra-tion with F12 medium, and applied through the puff-ing system. Capsazepine (dissolved in DMSO) andruthenium red are prepared as concentrated stock so-lutions, diluted to final concentration in F12 medium,and applied either at room temperature or at ∼53°C.Reversibility of antagonist action is tested by reap-plication of heated extracellular solution without anyagents.

EVALUATIONOff-line measurements and statistical analysis is doneusing pCLAMP6 (Axon Instruments) and EXCEL 5.0(Microsoft). Data are presented as means ± SE. Treat-ment effects are statistically analyzed by Student’st-test for paired data and χ2 test for analysis of inci-dences.

MODIFICATIONS OF THE METHODNagy et al. (1983) described dose-dependent effects ofcapsaicin on primary sensory neurons in the neonatalrat.

Lopshire and Nicol (1998) performed whole-celland single-channel studies in rat sensory neurons andfound a prostaglandin E2 induced enhancement of thecapsaicin elicited current.

Jung et al. (1999) performed patch-clamp experi-ments in dorsal root ganglion neurons of neonatal ratsand concluded that capsaicin binds to the intracellulardomain of the capsaicin-activated ion channel.

Nagy and Humphrey (1999) compared the mem-brane responses of rat sensory neurons to noxious heatand capsaicin, using electrophysiological and ion fluxmeasurements.

Baumann and Martenson (2000) found that extra-cellular protons both increase the activity and reducethe conductance of capsaicin-gated channels.

Liu et al. (2001) investigated mechanisms underly-ing capsaicin-mediated inhibition of action potentialsand modulation of voltage-gated sodium channels incultured trigeminal ganglion neurons.

Gunthorpe et al. (2004) identified and characterizeda potent and selective vanilloid receptor antagonist iso-lated via high-throughput screening of a large chemi-cal library in an FLPR-based C2+ assay.


For further information on the vanilloid receptor seeA.1.1.32.5.

REFERENCES AND FURTHER READINGBaumann TK, Martenson ME (2000) Extracellular protons

both increase the activity and reduce the conductance ofcapsaicin-gated channels. J Neurosci 20: RC80 (1–5)

Bevan S, Yeats JC (1991) Protons activate a cation conduc-tance in a subpopulation of rat dorsal root ganglion neu-rons. J Physiol (Lon.) 433:145–161

Bevan S, Hothi S, Hughes G, James IF, Rang HP, Shah K,Walpole CJS, Yeats JC (1992) Capsazepine: A competitiveantagonist of the sensory neuron excitant capsaicin. Br JPharmacol 107:544–552

Gunthorpe MJ, Rami HK, Jerman JC, Smart D, Gill CH, Sof-fin EM, Luis Hannan S, Lappin SC, Egerton J, Smith GD,Worby A, Howett L, Owen D, Nasir S, Davies CH, Thomp-son M, Wyman PA, Randall AD, Davis JB (2004) Iden-tification and characterization of SB-366791, a potent andselective vanilloid receptor (VR1/TRPV1) antagonist. Neu-ropharmacology 46:133–149

Jung J, Hwang SW, Kwak J, Lee S-Y, Kang C-J, Kim W-B,Kim D, Oh U (1999) Capsaicin binds to the intracellulardomain of the capsaicin-activated ion channel. J Neurosci19:529–538

Kirschstein T, Greefrath W, Büsselberg D, Treede RD (1999)Inhibition of rapid heat responses in nociceptive primarysensory neurons of rats by vanilloid receptor antagonists.J Neurophysiol 82:2853–2860

Liu L, Lo Y-C, Chen I-J, Simon SA (1997) The response or rattrigeminal ganglion neurons to capsaicin and two nonpun-gent vanilloid receptor agonists, olvanil and glyceryl non-amide. J Neurosci 17:4101–4111

Liu L, Oortgiesen M, Li L, Simon SA (2001) Capsaicin in-hibits activation of voltage-gated sodium currents in cap-saicin-sensitive trigeminal ganglion nerves. J Neurophysiol85:745–758

Lopshire JC, Nicol GD (1998) The cAMP transduction cascademediates the prostaglandin E2 enhancement of the cap-saicin elicited current in rat sensory neurons: whole-celland single-channel studies. J Neurosci 18:6081–6092

Nagy I, Humphrey PR (1999) Similarities and differences be-tween the responses of rat sensory neurons to noxious heatand capsaicin. J Neurosci 19:10647–10655

Nagy JI, Iversen LL, Goedert M, Chapman D, Hunt SP (1983)Dose-dependent effects of capsaicin on primary sensoryneurons in the neonatal rat. J Neurosci 3:399–406

Walpole CS, Bevan S, Boverman G, Boelsterli JJ, Brecken-ridge R, Davies JW, Hughes GA, James I, Oberer L, Win-ter J (1994) The discovery of capsazepine, the first compet-itive antagonist of the sensory neuron excitants capsaicinand resiniferatoxin. J Med Chem 37:1942–1954

H.1.2In Vivo Methods for TestingCentral Analgesic Activity


Although the in vivo methods have been used more ex-tensively in the past, they are still necessary in presentresearch analgesic tests in animals before a compound

can be given to man. Mostly, rodents, such as mice orrats, are used for analgesic tests, but in some instancesexperiments in higher animals such as monkeys arenecessary.

Several methods are available for testing centralanalgesic activity, such as

• Haffner’s tail clip method in mice,• tail flick or other radiant heat methods,• tail immersion tests,• hot plate methods in mice or rats,• electrical stimulation (grid shock, stimulation of

tooth pulp or tail),• monkey shock titration,• formalin test in rats.

REFERENCES AND FURTHER READINGVon Voigtlander PF (1982) Pharmacological alteration of pain:

The discovery and evaluation of analgesics in animals.In: Lednicer D (ed) Central Analgesics. John Wiley andSons, New York, pp 51–79

H.1.2.2Haffner’s Tail Clip Method

PURPOSE AND RATIONALEThe method was described as early as 1929 by Haffnerwho observed the raised tail (Straub phenomenon) inmice treated with morphine or similar opioid drugs andfound the tail after drug treatment to be less sensitiveto noxious stimuli. He already described the high sen-sitivity of this method to morphine. Since then, themethod has been used and modified by many authors.

PROCEDUREAn artery clip is applied to the root of the tail of miceand the reaction time is noted. Male mice (CharlesRiver strain or other strains) with a weight between18 and 25 g are used. The control group consists of10 mice. The test compounds are administered subcu-taneously to fed mice or orally to fasted animals. Thetest groups and the control group consist of 7–10 mice.The drug is administered 15, 30 or 60 min prior testing.An artery clip is applied to the root of the tail (approxi-mately 1 cm from the body) to induce pain. The animalquickly responds to this noxious stimuli by biting theclip or the tail near the location of the clip. The timebetween stimulation onset and response is measuredby a stopwatch in 1/10 seconds increments.

EVALUATIONA cut-off time is determined by taking the averagereaction time plus 3 times the standard deviation of


the combined latencies of the control mice at all timeperiods. Any reaction time of the test animals whichis greater than the cut-off time is called a positiveresponse indicative of analgesic activity. The lengthof time until response indicates the period of great-est activity after dosing. An ED50 value is calculatedat the peak time of drug activity. ED50 values foundby this method were 1.5 mg/kg s.c. for morphine and7,5 mg/kg for codeine s.c.

CRITICAL ASSESSMENT OF THE TESTThe test does not need any sophisticated equipment buta skilled, preferably “blind”, observer. Peripheral anal-gesics of the salicylate type are not detected by thistest.

MODIFICATIONS OF THE METHODBartoszyk and Wild (1989) described a modification ofthe original Haffner clip test using pressure on the tailof rats instead of mice. Additionally hyperalgesia wasinduced by injection of carrageenan suspension intothe tail. In this case not only an effect of a nonsteroidalanti-inflammatory agent but also a potentiation by B-vitamins could be shown.

Takagi et al. (1966) published a modification ofHAFFNER’s method for testing analgesics.

Ossipov et al. (1988) used the Haffner test to com-pare the antinociceptive effects of intrathecally admin-istered opiates, α2-adrenergic agonists, and local anes-thetics.

Yanagisawa et al. (1984) described a tail pinchmethod in vitro for testing antinociceptive drugs con-sisting of an isolated spinal cord, spinal nerve rootsand the functionally connected tail of a new-born rat.Changes of electric potential in the ventral root areinduced by noxious pressure on the tail. In addition,responses after electric stimulation of the dorsal rootwere recorded. The authors recommend the method forstudying actions of analgesic drugs.

Pinch of the toes of guinea pigs was recommendedas a test for opioid analgesics by Collier (1965).

Tail-pinch feeding in rats after intracerebroventric-ular injection of various opioid antagonists has beenused to differentiate opioid receptor subtypes (Kochand Bodnar 1993).

Person et al. (1985) used three different techniquesof mechanical tail stimulation (reaction threshold de-termined with an Analgesymeter at two different cut-off values and HAFFNER’s tail clip) to study mor-phine-caffeine analgesic interaction in rats.

Arndt et al. (1984) studied pain responses (in-crease of heart rate and arterial pressure, respiratory ef-

fects) to tail clamping in trained unanesthetized spon-taneously breathing dogs after administration of fen-tanyl.

REFERENCES AND FURTHER READINGArndt JO, Mikat M, Parasher C (1984) Fentanyl’s analgesic,

respiratory, and cardiovascular actions in relation to doseand plasma concentrations in unanesthetized dogs. J Anesth61:355–361

Bartoszyk GD, Wild A (1989) B-vitamins potentiate the anti-nociceptive effect of diclofenac in carrageenin-inducedhyperalgesia in the rat tail pressure test. Neurosci Lett101:95–100

Bianchi C, Franceschini J (1954) Experimental observations onHaffner’s method for testing analgesic drugs. Br J Pharma-col 9:280–284

Collier HOJ (1965) Multiple toe-pinch test for potential anal-gesic drugs. In: Keele, Smith (eds) Assessment of Pain inMan and Animals. Livingston, London, pp 262–270

Fleisch A, Dolivo M (1953) Auswertung der Analgetica imTierversuch. Helv Physiol Acta 11:305–322

Haffner F (1929) Experimentelle Prüfung schmerzstillenderMittel. Dtsch Med Wschr 55:731–733

Koch JKE, Bodnar RJ (1993) Involvement of mu1 and mu2 opi-oid receptor subtypes in tail-pinch feeding in rats. PhysiolBehav 53:603–605

Ossipov MH, Suarez LJ, Spaulding TC (1988) A comparisonof the antinociceptive and behavioral effects of intrathe-cally administered opiates, α 2-adrenergic agonists, and lo-cal anesthetics in mice and rats. Anesth Analg 67:616–624

Person DL, Kissin I, Brown PT, Xavier AV, Vinik HR,Bradley EL (1985) Morphine-caffeine analgesic interactionin rats. Anesth Analg 64:851–856

Takagi H, Inukai T, Nakam M (1966) A modification ofHaffner’s method for testing analgesics. Jpn J Pharmacol16:287–295

Vanderwende C, Spoerlein M (1972) Antagonism by DOPA ofmorphine analgesia. A hypothesis for morphine tolerance.Res Comm Chem Pathol Pharmacol 3:37–45

Yanagisawa M, Murakoshi T, Tamai S, Otsuka M (1984) Tail-pinch method in vitro and the effects of some antinocicep-tive compounds. Eur J Pharmacol 106:231–239

H.1.2.3Radiant Heat Method

PURPOSE AND RATIONALEOriginally, the method was developed by Schumacheret al. (1940), Wolff et al. (1940) for quantitative mea-surements of pain threshold in man against thermal ra-diation and for evaluation of analgesic activity of opi-ates. Later on, the procedure has been used by manyauthors to evaluate analgesic activity in animal experi-ments by measuring drug-induced changes in the sen-sitivity of mice or rats to heat stress applied to theirtails. The test is very useful for discriminating be-tween centrally acting morphine-like analgesics andnon-opiate analgesics.

Mice are placed into cages leaving the tail exposed.A light beam is focused to the proximal third of the tail.


Within a few seconds the animal flicks the tail aside ortries to escape. The time until this reaction occurs ismeasured.

PROCEDUREThe method was described by Ther, Lindner andVogel (1963) as a modification of earlier publications(D’Armour and Smith 1941). Groups of 10 mice(NMRI-strain) of both sexes with a weight between 18and 22 g are used for each dose. Before administrationof the test compound or the standard the normalreaction time is determined. The animal is put intoa small cage with an opening for the tail at the rearwall. The tail is held gently by the investigator. Byopening of a shutter, a light beam exerting radiantheat is directed to the proximal third of the tail. Forabout 6 s the reaction of the animal is observed by theinvestigator. The mouse tries to pull the tail away andturns the head. With a switch the shutter is closed assoon as the investigator notices this reaction. Micewith a reaction time of more than 6 s are not used inthe test. The escape reaction which is the endpoint ofthis test can be regarded as a complex phenomenonmediated by the brain. In contrast, the simple tailflick as an endpoint of this test may be mediated asa spinal reflex. Therefore the observation of the escapereaction can be regarded as a true assessment of theinfluence of the drug on the brain.

The test compounds and the standard are adminis-tered either orally or subcutaneously. The animals aresubmitted to the same testing procedure after 30, 60and eventually 120 min. For each individual animal thereaction time is noted. Other time intervals can be usedaccording to the question to be investigated.

EVALUATIONThere are two possibilities for evaluation:

• The average values of reaction time after eachtime interval are calculated and compared with thepretest value by analysis of significance.

• At each time interval only those animals whichshow a reaction time twice as high or higher as thepretest value are regarded as positive. Percentagesof positive animals are counted for each time inter-val and each dose and ED50 values are calculatedaccording to LITCHFIELD and WILCOXON.

As standards codeine, pethidine and morphine canbe used. The ED50 values of these drugs are:

• Codeine 12 mg/kg s.c.• Pethidine 12 mg/kg s.c.• Morphine 2 mg/kg s.c.

CRITICAL ASSESSMENT OF THE TESTThe radiant heat test on the tail of mice is very ef-fective to estimate the efficacy and potency of centralacting analgesic drugs. With pyrazolones ED50 valuesstill can be calculated but these are achieved only withrelatively high doses. Compounds like acetylsalicylicacid and phenyl-acetic acids show only slight effectsmaking it impossible to calculate ED50 values.

MODIFICATIONS OF THE METHODOriginally, the method has been described for testinganalgesic properties in the rat (D’Armour and Smith1941, Winter et al. 1954, Harris and Pierson 1964).Goldstein and Malseed (1979) adapted the procedurefor utilization in cats. The effect of morphine could beantagonized by naloxone in this test. No response tosodium salicylate or pentobarbital was observed. Lutzet al. (1994) used a modification of the rat tail with-drawal test to investigate the structure-activity profileof a series of opioid analgesics. One day before testing,polyethylene tubings were implanted in the femoralvein and externalized behind the neck for intravenousapplication of test substances.

Various instruments have been described for mea-suring tail flick latencies by several authors, e. g.,Davies et al. 1946; Owen et al. (1981), Isabel et al.(1981), Walker and Dixon (1983), Yoburn et al. (1984),Harris et al. (1988).

Tail flick analgesy meters are commercially avail-able (e. g., IITC Life Science, Woodland Hills, CA,USA).

Green and Young (1951) compared the heat andpressure analgesiometric methods in rats. Mohrlandet al. (1983) described an ultrasound-induced tail-flickprocedure.

Hargreaves et al. (1988), Costello and Hargreaves(1989), Hylden et al. (1991) exposed the plantar sur-face of hindpaws of unrestrained rats to a beam of ra-diant heat applied through the glass floor of a testingchamber. Paw withdrawal latency was automaticallyrecorded by a photocell.

This method was also used by Schuligoi et al.(1994).

Taylor et al. (1997) used this method to investigatethe brief (phase 1) and persistent (phase 2) nociceptiveresponses of rats after injection of dilute formalin intothe hindpaw.

Carmon and Frostig (1981) used brief laser inducedheat applied to the rat ear for pharmacological testingof analgesics.

Perkins et al. (1993), Perkins and Kelly (1993) usedultra-violet-induced hyperalgesia in rat paw. Female


Sprague-Dawley rats weighing about 100 g were ex-posed on the plantar surface of one hind paw to UVlight (intensity maximum 365 nm, 69 mW/cm2) for90 s and this was repeated 18 h later. On the followingdays, each group of rats was placed in a transparentPerspex box and the withdrawal threshold to a focusedbeam of radiant heat applied to the underside of eachhind paw was measured.

McCallister et al. (1986) directed radiant heat to theears of rabbits and measured ear-withdrawal time.

REFERENCES AND FURTHER READINGCarmon A, Frostig R (1981) Noxious stimulation of animals

by brief laser induced heat: advantages to pharmacologicaltesting of analgesics. Life Sci 29:11–16

Costello AH, Hargreaves KM (1989) Suppression of car-rageenan-induced hyperalgesia, hyperthermia and edemaby a bradykinin antagonist. Eur J Pharmacol 171:259–263

D’Armour FE, Smith DL (1941) A method for determining lossof pain sensation. J Pharmacol Exp Ther 72:74–79

Davies OL, Raventós J, Walpole AL (1946) A method for theevaluation of analgesic activity using rats. Br J Pharmacol1:255–264

Dewey WL, Harris LS, Howes JF, Nuite JA (1970) The effect ofvarious neurohumoral modulators on the activity of mor-phine and the narcotic antagonists in the tail-flick and thephenylquinone tests. J Pharmacol Exp Ther 175:435–442

Geller I, Axelrod LR (1968) Methods for evaluating anal-gesics in laboratory animals. In: Soulairac A, Cahn J,Charpentier J (eds) Pain. Acad Press, London New York,pp 153–163

Goldstein FJ, Malseed RT (1979) Evaluation of narcotic anal-gesic activity using a cat tail-flick procedure. J PharmacolMeth 2:333–338

Gray WD, Osterberg A, Scuto TJ (1970) Measurement ofthe analgesic efficacy and potency of pentazocine by theD’Armour and Smith method. J Pharmacol Exp Ther172:154–162

Green AF, Young PA (1951) A comparison of heat and pressureanalgesiometric methods in rats. Br J Pharmacol 6:572–585

Hargreaves KM, Dubner R, Brown F, Flores C, Joris J (1988)A new and sensitive method for measuring thermal noci-ception in cutaneous hyperalgesia. Pain 32:77–82

Harris DP, Burton R, Sinclair G (1988) A simple microcomputerinterface for tail-flick determination. J Pharmacol Meth20:103–108

Harris LS, Pierson AK (1964) Some narcotic antagonists in thebenzomorphan series. J Pharmacol Exp Ther 143:141–148

Howes JF, Harris LS, Dewey WL, Voyda CA (1969) Brainacetylcholine levels and inhibition of the tail-flick reflex inmice. J Pharmacol Exp Ther 169:23–28

Hylden JLK, Thomas DA, Iadarola MJ, Nahin RL, Dubner R(1991) Spinal opioid analgesic effects are enhanced ina model of unilateral inflammation/hyperalgesia: possibleinvolvement of noradrenergic mechanisms. Eur J Pharma-col 194:135–143

Isabel G, Wright DM, Henry JL (1981) Design of an inexpensiveunit for measuring tail flick latencies. J Pharmacol Meth5:241–247

Litchfield JT, Wilcoxon F (1949) A simplified method for evalu-ating dose-effect experiments. J Pharmacol Exp Ther 96:99

Lutz MW, Morgan OH, James MK, Feldman OL, Brackeen MF,Lahey AP, James SV, Bilotta JM, Pressley JC (1994)

A pharmacodynamic model to investigate the structure-ac-tivity profile of a series of novel opioid analgesics. J Phar-macol Exp Ther 271:795–803

McCallister LW, Lipton JM, Giesecke AH Jr, Clark WG (1986)The rabbit ear-withdrawal test: A new analgesiometric pro-cedure. Pharmacol Biochem Behav 25:481–482

Mohrland JS, Johnson EE, von Voigtlander PF (1983) An ul-trasound-induced tail-flick procedure: evaluation of non-steroidal antiinflammatory analgesics. J Pharmacol Meth9:297–282

Owen JA, Milne B, Jhamandas K, Nakatsu K (1981) Assemblyof an inexpensive tail flick analgesia meter. J PharmacolMeth 6:33–37

Perkins MN, Kelly D (1993) Induction of bradykinin B1 re-ceptors in vivo in a model of ultra-violet irradiation-in-duced thermal hyperalgesia in the rat. Br J Pharmacol110:1441–1444

Perkins MN, Campell E, Dray A (1993) Antinociceptive activ-ity of the bradykinin B1 and B2 receptor antagonists, des-Arg9,[Leu8]-BK and Hoe 140, in two models of persistenthyperalgesia in rats. Pain 53:191–197

Schuligoi R, Donnerer J, Amann R (1994) Bradykinin-in-duced sensitization of afferent neurons in the rat. Neurosci59:211–215

Schumacher GA, Goodell H, Hardy JD, Wolff HG (1940) Uni-formity of the pain threshold in man. Science 92:110–112

Taylor BK, Peterson MA, Basbaum AI (1997) Early noci-ceptive events influence the temporal profile, but not themagnitude, of the tonic response to subcutaneous for-malin: effects with remifentanil. J Pharmacol Exp Ther280:876–883

Ther L, Lindner E, Vogel G (1963) Zur pharmakodynamischenWirkung der optischen Isomeren des Methadons. DtschApoth Ztg 103:514–520

Tulunay FC, Takemori AE (1974) The increased efficacy of nar-cotic antagonists induced by various narcotic analgesics.J Pharmacol Exp Ther 190:395–400

Walker JM, Dixon WC (1983) A solid state device for measuringsensitivity to thermal pain. Physiol Behav 30:481–483

Winter CA, Orahovats PD, Flataker L, Lehman EG, Leh-man JT (1954) Studies on the pharmacology of N-allylnormorphine. J Pharmacol Exp Ther 112:152–160

Wolff HG, Hardy JD, Goodell H (1940) Studies on pain. Mea-surement of the effect of morphine, codeine, and other opi-ates on the pain threshold and an analysis of their relationto the pain experience. J Clin Invest 19:659–680

Yoburn BC, Morales R, Kelly DD, Inturrisi CE (1984) Con-strains on the tail flick assay: morphine analgesia and toler-ance are dependent upon locus of tail stimulation. Life Sci34:1755–1762

H.1.2.4Hot Plate Method

PURPOSE AND RATIONALEThe paws of mice and rats are very sensitive to heat attemperatures which are not damaging the skin. The re-sponses are jumping, withdrawal of the paws and lick-ing of the paws. The time until these responses occur isprolonged after administration of centrally acting anal-gesics, whereas peripheral analgesics of the acetylsal-icylic acid or phenyl-acetic acid type do not generallyaffect these responses.


PROCEDUREThe method originally described by Woolfe and MacDonald (1944) has been modified by several investiga-tors. The following modification has been proven to besuitable:

Groups of 10 mice of either sex with an initialweight of 18 to 22 g are used for each dose. Thehot plate, which is commercially available, consists ofa electrically heated surface. The temperature is con-trolled for 55° to 56°C. This can be a copper plateor a heated glass surface. The animals are placed onthe hot plate and the time until either licking or jump-ing occurs is recorded by a stop-watch. The latency isrecorded before and after 20, 60 and 90 min followingoral or subcutaneous administration of the standard orthe test compound.

EVALUATIONThe prolongation of the latency times comparing thevalues before and after administration of the test com-pounds or the values of the control with the experi-mental groups can be used for statistical comparisonusing the t-test. Alternatively, the values which exceedthe value before administration for 50% or 100% canbe regarded as positive and ED50 values can be calcu-lated.

Doses of 7.5 mg/kg s.c. morphine hydrochloride,30 mg/kg s.c. codeine hydrochloride, 30 mg/kg s.c.pethidine hydrochloride and 400 mg/kg s.c. phenazonewere found to be effective, whereas aspirin showed noeffect even at high doses.

CRITICAL ASSESSMENT OF THE TESTThe hot plate test has been used by many investigatorsand has been found to be suitable for evaluation of cen-trally but not of peripherally acting analgesics. Mice aswell as rats have been used. The method has the draw-back that sedatives and muscle relaxants (Woolfe andMacDonald 1944) or psychotomimetics (Knoll 1967)cause false positives, while mixed opiate agonists-an-tagonists provide unreliable results. The validity of thetest has been shown even in the presence of substan-tial impairment of motor performance (Plummer et al.1991). Mixed opiate agonists-antagonists can be eval-uated if the temperature of the hot plate is lowered to49.5°C (O’Callaghan and Holtzman 1975; Zimer et al.1986).

MODIFICATIONS OF THE METHODO’Neill et al. (1983) described an automated, high-capacity method for measuring jump latencies on a hotplate. A hot-plate test with increasing temperature wasrecommended by Tjølsen et al. (1991).

Hot plate analgesy meters are commercially avail-able (e. g., IITC Life Science, Woodland Hills, CA,USA).

REFERENCES AND FURTHER READINGEddy NB, Leimbach D (1953) Synthetic analgesics: II. Dithien-

ylbutenyl- and dithienylbutylamines. J Pharmacol Exp Ther107:385–393

Jacob J, Blozovski M (1961) Action des divers analgésiques surle comportement de souris exposées a un stimulus ther-moalgésique. Arch Int Pharmacodyn 138:296–309

Jacob J, Loiseau G, Echinard-Garin P, Barthelemy C, Lafille C(1964) Caractérisation et détection pharmacologiques dessubstances hallucinogènes. II.-antagonismes vis-a-vis de lamorphine chez la souris. Arch Int Pharmacodyn 148:14–30

Kitchen I, Crowder M (1985) Assessment of the hot-plate anti-nociceptive test in mice. A new method for the statisticaltreatment of graded data. J Pharmacol Meth 13:1–7

Knoll J (1967) Screening and grouping of psychopharmacologi-cal agents. In: Siegler PE, Moyer HJ (eds) Animal and Clin-ical Pharmacologic Techniques in Drug Evaluation. Year-book Med Publ. Inc., Chicago, pp 305–321

O’Neill KA, Courtney C, Rankin R, Weissman A (1983) An au-tomated, high-capacity method for measuring jump laten-cies on a hot plate. J Pharmacol Meth 10:13–18

O’Callaghan JP, Holtzman SG (1975) Quantification of the anal-gesic activity of the narcotic antagonists by a modified hotplate procedure. J Pharm Exp Ther 192:497–505

Plummer JL, Cmielewski PL, Gourlay GK, Owen H, CousinsMJ (1991) Assessment of antinociceptive drug effects inthe presence of impaired motor performance. J PharmacolMeth 26:79–87

Tjølsen A, Rosland JH, Berge OG, Hole K (1991) The increas-ing temperature hot-plate test: an improved test of nocicep-tion in mice and rats. J Pharmacol Meth 25:241–250

Witkin LB, Heubner CF, Galgi F, O’Keefe E, Spitaletta P, Plum-mer AJ (1961) Pharmacology of 2-aminino-indane hy-drochloride (SU 8629): a potent non-narcotic analgesic.J Pharmacol Exp Ther 133:400–408

Woolfe G, MacDonald AD (1944) The evaluation of the anal-gesic action of pethidine hydrochloride (DEMEROL) JPharmacol Exper Ther 80:300–307

Zimer PO, Wynn RL, Ford RD, Rudo FG (1986) Effect ofhot plate temperature on the antinociceptive activity ofmixed opioid agonist antagonist compounds. Drug Dev Res7:277–280

H.1.2.5Tail Immersion Test

PURPOSE AND RATIONALEThe method has been developed to be selective formorphine-like compounds. The procedure is based onthe observation that morphine-like drugs are selec-tively capable of prolonging the reaction time of thetypical tail-withdrawal reflex in rats induced by im-mersing the end of the tail in warm water of 55°C.

PROCEDUREYoung female Wistar rats (170–210 g body weight) areused. They are placed into individual restraining cages


leaving the tail hanging out freely. The animals are al-lowed to adapt to the cages for 30 min before testing.The lower 5 cm portion of the tail is marked. This partof the tail is immersed in a cup of freshly filled waterof exactly 55°C. Within a few seconds the rat reactsby withdrawing the tail. The reaction time is recordedin 0.5 s units by a stopwatch. After each determinationthe tail is carefully dried. The reaction time is deter-mined before and periodically after either oral or sub-cutaneous administration of the test substance, e. g., af-ter 0.5, 1, 2, 3, 4 and 6 h. The cut off time of the immer-sion is 15 s. The withdrawal time of untreated animalsis between 1 and 5.5 s. A withdrawal time of more than6 s therefore is regarded as a positive response.

EVALUATIONED50 values can be calculated for each compound andtime response curves (onset, peak and duration of theeffect) be measured. All the morphine-like analgesicshave been shown to be active at doses which do notproduce gross behavioral changes. For example, anED50 of 3.5 mg/kg s.c. for morphine and an ED50 of1.7 mg/kg s.c. methadone was found. Acetylsalicylicacid at a dose of 640 mg/kg p.o., phenylbutazone ata dose of 160 mg/kg s.c. as well as nalorphine at a doseof 40 mg/kg s.c. were inactive.

CRITICAL ASSESSMENT OF THE TESTThe test is useful to differentiate central opioid likeanalgesics from peripheral analgesics.

MODIFICATIONS OF THE METHODBen-Bassat et al. (1959) described the receptaclemethod in mice. Each mouse was inserted in a conoidpaper receptacle with its tail protruding, the cone be-ing closed by a stapler. The protruding tail was entirelyimmersed in a water bath (58°C) and the time untilwithdrawal of the tail was measured by a stop watch.

Pizziketti et al. (1985) modified the tail immer-sion test in rats in this way that they used a 1:1 mix-ture of ethylene-glycol and water cooled to a tem-perature of minus 10°C as noxious stimulus. Lineardose-response curves were found with levo-methadoneand morphine. Low ceiling effects or curvilinear dose-response curves were obtained with narcotic agonist-antagonist analgesics such as pentazocine. Diazepamand aspirin were inactive.

Tiseo et al. (1988) could show that the endogenouskappa agonist dynorphin A was inactive in the rat tailimmersion test at 55°C, but gave dose-response curvesin the cold water version of the test.

Abbott and Melzack (1982) examined the effects ofbrainstem lesions on morphine analgesia using the for-malin test which produced moderate pain that lastedabout 2 h, and the tail-flick hot water-immersion testwhich measured brief threshold-level pain.

Abbott and Franklin (1986) used two forms of therat tail flick test: In the restrained form of the test ratswere placed in wire restraining tubes from 10 min be-fore drug injection till the end of the test. In the un-restrained form of the test rats were left free in theirhome cages and handheld during each test for approx-imately 30 s. Responses to the thermal pain stimuluswere assessed by the latency with which the rat re-moved its tail from 55°C water. Two types of morphineanalgesia have been postulated in animals: One type,exemplified in rats that are restrained during tail flicktesting, is sensitive to an interaction between morphineand brain 5-HT, the level of which is elevated by re-strained stress (Kelly and Franklin 1984).

Luttinger (1985) determined the antinociceptive ac-tivity of drugs using different water temperatures ina tail-immersion test in mice. The results roughly par-alleled the differences in the severity of pain for whichvarious analgesics are effective.

Dykstra et al. (1986, 1987) described a tail with-drawal procedure for assessing analgesic activity inRhesus monkeys by immersion of the tail into waterof 55°C. This procedure was used by Rothman et al.(1989) to determine the pharmacological activities ofoptically pure enantiomers of the κ opioid agonist,U50,488, and its cis diastereomer.

Using this method, Ko et al. (1999) found that ac-tivation of peripheral κ opioid receptors inhibits cap-saicin-induced nociception in Rhesus monkeys.

REFERENCES AND FURTHER READINGAbbott FV, Melzack R (1982) Brainstem lesions dissociate neu-

ral mechanisms of morphine analgesia in different kinds ofpain. Brain Res 251:149–155

Abbott FV, Franklin KBJ (1986) Noncompetitive antagonism ofmorphine analgesia by diazepam in the formalin test. Phar-macol Biochem Behav 24:319–321

Ben-Bassat J, Peretz E, Sulman FG (1959) Analgesimetry andranking of analgesic drugs by the receptacle method. ArchInt Pharmacodyn 122:434–447

Cowan A (1990) Recent approaches in the testing of analgesicsin animals. In: Modern Methods in Pharmacology, Vol. 6,Testing and Evaluation of Drugs of Abuse, pp 33–42,Wiley-Liss Inc

Dykstra LA, Woods JH (1986) A tail withdrawal procedure forassessing analgesic activity in Rhesus monkeys. J Pharma-col Meth 15:263–269

Dykstra LA, Gmerek DE, Winger G, Woods JH (1987) Kappaopioids in rhesus monkeys. Diuresis, sedation, analgesiaand discriminative stimulus effects. J Pharm Exp Ther242:413–420


Evangelista S, Pirisino R, Perretti F, Fantozzi R, Brunelleschi S,Malmberg-Aiello P, Bartolini A (1987) The pharmacologi-cal properties of 1,4-dihydro-1-ethyl-7-phenylpyrrol- (1,2-a)-pyrimidine-4-one, a new antipyretic and analgesic drug.Drugs Exp Clin Res 13:501–510

Grotto M, Sulman FG (1967) Modified receptacle method foranimal analgesimetry. Arch Int Pharmacodyn 165:152–159

Janssen P, Niemegeers CJE, Dony JGH (1963) The inhibitory ef-fect of Fentanyl and other morphine-like analgesics on thewarm water induced tail withdrawal reflex in rats. Arzneim-Forsch 13:502–507

Kelly SJ, Franklin KBJ (1984) Evidence that stress augmentsmorphine analgesia by increasing brain tryptophan. Neu-rosci Lett 44:305–310

Ko M-C, Butelman ER, Woods JH (1999) Activation of pe-ripheral κ opioid receptors inhibits capsaicin-induced no-ciception in Rhesus monkeys. J Pharmacol Exp Ther287:378–385

Luttinger D (1985) Determination of antinociceptive activity ofdrugs in mice using different water temperatures in a tail-immersion test. J Pharmacol Meth 13:351–357

Ono M, Satoh T (1988) Pharmacological studies of Lappa-conitine. Analgesic studies. Arzneim Forsch/Drug Res38:892–895

Pizziketti RJ, Pressman NS, Geller EB, Cowan A, Adler MW(1985) Rat cold water tail-flick: A novel analgesic test thatdistinguishes opioid agonists from mixed agonists-antago-nists. Eur J Pharmacol 119:23–29

Ramabadran K, Bansinath M, Turndorf H, Puig MM (1989) Tailimmersion test for the evaluation of a nociceptive reactionin mice. J Pharmacol Meth 21:21–31

Rothman RB, France CP, Bykov V, de Costa BR, Jacobson AE,Woods JH, Rice KC (1989) Pharmacological activitiesof optically pure enantiomers of the κ opioid agonist,U50,488, and its cis diastereomer: evidence for three κ re-ceptor subtypes. Eur J Pharmacol 167:345–353

Sewell RDE, Spencer PSJ (1976) Antinociceptive activity ofnarcotic agonist and partial agonist analgesics and otheragents in the tail-immersion test in mice and rats. Neu-ropharmacol 15:683–688

Tiseo PJ, Geller EB, Adler MW (1988) Antinociceptive action ofintracerebroventricularly administered dynorphin and otheropioid peptides in the rat. J Pharm Exp Ther 246:449–453

H.1.2.6Electrical Stimulation of the Tail

PURPOSE AND RATIONALESince the tail of mice is known to be sensitive to anystimulus, a method of electrical stimulation has beendescribed as early as 1950 by Burn et al. The stimu-lus can be varied either by the duration of the electricshock or by an increase in the electric current.

PROCEDUREAs described by Kakunaga et al. (1966), male micewith a weight of 20 g are placed into special cages.A pair of alligator clips is attached to the tail wherebythe positive electrode is placed at the proximal end ofthe tail. Rectangular wave pulses from a constant volt-age stimulator at an intensity of 40–50 V are applied.The frequency of the stimulation is 1 shock/s, and the

pulse duration 2.5 ms. The normal response time rangeof the stimuli is 3–4 s. Following administration of thedrug, the response time is registered at 15 min intervalsuntil the reaction time returns to control levels.

EVALUATIONThe data for each animal are plotted with reactiontimes on the ordinate and time intervals following ad-ministration on the abscissa. The area under the timeresponse curve is calculated. In control animals the re-action time remains fairly constant and the area underthe curve is approximately zero. Effects of morphine at5 mg/kg s.c. and meperidine 30 mg/kg s.c. could easilybe demonstrated.

CRITICAL ASSESSMENT OF THE TESTThe effect of central analgesics can be clearly demon-strated, however also the activity of peripheral anal-gesics given at higher doses can be detected.

MODIFICATIONS OF THE METHODVidal et al. (1982) measured the thresholds of 3 noci-ceptive reactions (tail withdrawal, vocalization, vocal-ization afterdischarge) following electrical stimulationof the tail.

A variation of the test has been introduced byYanaura et al. (1976), using ultrasonic stimulation in-stead of electric stimulation. The method is consideredto be fast, simple, and precise. The stimulus can be ap-plied repeatedly without causing injury to the tissue.A vocalization test in rats with electrical stimulationof the tail has been described by Hoffmeister (1968).

Ludbrook et al. (1995) described a method for fre-quent measurement of sedation and analgesia in sheepusing the response to a ramped electrical stimulus.Sheep were placed in a canvas sling in their metaboliccrates to allow their limbs to partially bear weight inorder to minimize spontaneous limb movements. Twoneedles were placed subcutaneously 0.5 cm apart inthe anterior aspect of the lower third of the sheep’shind and connected to the nerve stimulator. The currentramp rate was set at one mA per sec. As soon as limbwithdrawal was observed, the stimulus was switchedoff and the highest current and ramp duration wererecorded.

REFERENCES AND FURTHER READINGBurn JH, Finney DJ, Goodwin LG (1950) Chapter XIV: Anti-

pyretics and analgesics. In: Biological Standardization. Ox-ford University Press, London, New York, pp 312–319

Carroll MN, Lim RKS (1960) Observations on the neurophar-macology of morphine and morphinelike analgesia. ArchInt Pharmacodyn 125:383–403


Charpentier J (1968) Analysis and measurement of pain in an-imals. A new conception of pain. In: Soulairac A, Cahn J,Charpentier J (eds) Pain. Acad Press, London New York,pp 171–200

Hoffmeister F (1968) Tierexperimentelle Untersuchungen überden Schmerz und seine pharmakologische Beeinflussung.Arzneim Forsch 16. Beiheft:5–116

Kakunaga T, Kaneto H, Hano K (1966) Pharmacological stud-ies on analgesics. VII. Significance of the calcium ion inmorphine analgesia. J Pharm Exp Ther 153:134–141

Ludbrook G, Grant C, Upton R, Penhall C (1995) A method forfrequent measurement of sedation and analgesia in sheepusing the response to a ramped electrical stimulus. J Phar-macol Toxicol Meth 33:17–22

Nilsen PL (1961) Studies on algesimetry by electrical stimula-tion of the mouse tail. Acta Pharmacol Toxicol 18:10–22

Paalzow G, Paalzow L (1973) The effect of caffeine and theo-phylline on nociceptive stimulation in the rat. Acta Phar-macol Toxicol 32:22–32

Vidal C, Girault JM, Jacob J (1982) The effect of pituitary re-moval on pain reaction in the rat. Brain Res 233:53–64

Yanaura S, Yamatake Y, Ouchi T (1976) A new analgesic testingmethod using ultrasonic stimulation. Effects of narcotic andnon-narcotic analgesics. Jpn J Pharmacol 26:301–308

H.1.2.7Grid Shock Test

PURPOSE AND RATIONALEThe electric grid shock test in mice has been describedby Blake et al. (1963) as a modification of an earlierapproach (Evans 1962) to measure the analgesic prop-erties by the “Flinch-jump” procedure in rats.

PROCEDUREMale mice with a weight between 18 and 20 g are indi-vidually placed into clear plastic chambers. The floorof the box is wired with tightly strung stainless steelwire, spaced about 1 mm apart. The stimulus is givenin the form of square wave pulses, 30 cycles per sec-ond with a duration of 2 ms per pulse. The output of thestimulator has to be connected to alternate wires of thegrid. A fixed resistance is placed in series with the gridand in parallel to an oscilloscope to allow calibrationin milliamperes. With increasing shock intensities themice flinch, exhibit a startling reaction, increase loco-motion or attempt to jump. The behavior is accuratelyreflected on the oscilloscope by marked fluctuations ofthe displayed pulse and defined as pain threshold re-sponse. Pain thresholds are determined in each individ-ual mouse twice before administration of the test drugand 15, 30, 60, 90 and 120 min after dosing. Groups of10 animals are used for control and for the test drugs.

EVALUATIONThe current as measured in milliamperes is recordedfor each animal before and after administration of

the drug. The average values for each group at eachtime interval are calculated and statistically comparedwith the control values. Placebo treated controls showa slight increase of threshold over time. Morphine sul-fate in a dose of 10 mg/kg p.o. but also acetylsalicylicacid in a dose of 200 mg/kg p.o. definitely increase thethreshold.

CRITICAL EVALUATION OF THE METHODThe modification of the method as described by Blakeet al. (1963) showed an effect not only of morphine butalso of acetylsalicylic acid which is not easily pickedup by other tests based on stimulation by physicalmeans.

MODIFICATIONS OF THE TESTWeiss and Laties (1961) in a “fractional escape” pro-cedure trained animals to press a lever to reduce theintensity of shock delivered continuously through thefloor grids of the experimental chamber. Each time, therat depresses the lever, it reduces the intensity of theshock. An external timer is programmed to increasethe intensity of the shock every few seconds. If the an-imal fails to press the lever, the shock continues to in-crease in intensity until lever-pressing behavior drivesit down. Thus, the level of shock fluctuates dependingon the rat’s lever pressing. The action of an analgesicin altering the level of shock which the rat will “toler-ate” can than be measured by comparing the averagelevel at which the rat maintains the shock under con-trol conditions with the average level at which the ratmaintains the shock during treatment.

Painful stimulation of the paws of mice placed intocages equipped with metal bands for electrical stim-ulation was described by Charlier et al. (1961) as“pododolorimetry”.

A modification of the jump-flinch technique formeasuring pain sensitivity in rats based on four cate-gories of responses was described by Bonnet and Pe-terson (1975).

Eschalier et al. (1988) described an automatedmethod to analyze vocalization of unrestrained ratssubmitted to noxious stimuli.

REFERENCES AND FURTHER READINGBanzinger R (1964) Animal techniques for evaluating nar-

cotic and non-narcotic analgesics. In: Nodine JH andSiegler PE (eds) Animal and Clinical Pharmacologic Tech-niques in Drug Evaluation. Year Book Medical Publ, Inc.,pp 392–396

Blake L, Graeme ML, Sigg EB (1963) Grid shock test for anal-gesic assay in mice. Med exp 9:146–150


Bonnet KA, Peterson KE (1975) A modification of the jump-flinch technique for measuring pain sensitivity in rats. Phar-macol Biochem Behav 3:47–55

Charlier R, Prost M, Binon F, Deltour G (1961) Étude pharma-cologique d’un antitussif, le fumarate acide de phénéthyl-1 (propyne-2 yl)-4-propionoxy-4 pipéridine. Arch Int Phar-macodyn 134:306–327

Eschalier A, Montastruc JL, Devoice JL, Rigal F, Gaillard-Plaza G, Péchadre JC (1981) Influence of naloxone andmethysergide on the analgesic effect of clomipramine inrats. Eur J Pharmacol 74:1–7

Eschalier A, Marty H, Trolese JF, Moncharmont L, Fialip J(1988) An automated method to analyze vocalization of un-restrained rats submitted to noxious stimuli. J PharmacolMeth 19:175–184

Evans WO (1961) A new technique for the investigation of someanalgesic drugs on a reflexive behavior in the rat. Psy-chopharmacologia 2:318–325

Evans WO (1962) A comparison of the analgesic potency ofsome analgesics as measured by the “Flinch-jump” proce-dure. Psychopharmacol 3:51–54

Evans WO, Bergner DP (1964) A comparison of the anal-gesic potencies of morphine, pentazocine, and a mixture ofmethamphetamine and pentazocine in the rat. J New Drugs4:82–85

Jokovlev V, Sofia RD, Achterrath-Tuckermann U, vonSchlichtegroll A, Thiemer K (1985) Untersuchungen zurpharmakologischen Wirkung von Flupirtin, einem struk-turell neuartigen Analgeticum. Arzneim Forsch/Drug Res35:30–43

Weiss B, Laties VG (1961) Changes in pain tolerance andother behavior produced by salicylates. J Pharm Exp Ther131:120

H.1.2.8Tooth Pulp Stimulation

PURPOSE AND RATIONALEThe method has been first described by Kohl and Ref-fert (1938) and by Ruckstuhl and Gordanoff (1939)for testing central analgesic activity in rabbits andhas since applied by several authors to various animalspecies. Stimulation of the tooth pulp induces charac-teristic reactions, such as licking, biting, chewing andhead flick which can be observed easily.

PROCEDURERabbits of either sex with an weight between 2 and3 kg are anaesthetized with 15 mg/kg thiopental or0.2 mg/kg fentanyl-citrate intravenously. Pulp cham-bers are exposed close to the gingival line in thelateral margins of the two front upper incisors witha high-speed dental drill. On the day of the experiment,clamping electrodes are placed into the drilled holes.After an accommodation period of 30 min stimulationis started to determine the threshold value. The stimu-lus is applied by rectangular current with a frequencyof 50 Hz and a duration of the stimulus of 1 s. The elec-trical current is started with 0.2 mA and increased until

the phenomenon of licking occurs. In some cases, thecurrent has to be increased and than to be decreasedagain in order to find the appropriate threshold. Forassessing the basic value, the threshold is determined3 times in each animal. Each animal serves as its owncontrol. For testing analgesic activity of a new drugand determination of an ED50 8 to 10 animals are usedfor each dose of the analgesic. The test substance is ei-ther injected intravenously or given orally by gavage.The threshold as the indicator of the antinociceptive ef-fect is determined again after 15, 30, 60 and 120 min.The animals serve as their own controls. Thresholdcurrent is determined again 5, 15, 30, 45 and 60 min af-ter intravenous application and 15, 30, 60 and 120 minafter oral application.

EVALUATIONFor screening procedures the increase of threshold, ex-pressed in mV, is the indicator of intensity and dura-tion of the analgesic effect. For determination of theED50, 8 to 10 rabbits are used for each dose, using3 doses, which provided effects between 10 and 90%.An antinociceptive effect is defined as an increase ofthe threshold versus the initial control by a factor of 2or more.

CRITICAL ASSESSMENT OF THE METHODCentral analgesics, especially opioid agonists, havebeen found to be very active in this test. Comparedwith other tests for central analgesic activity, like thehot plate test in mice, the tests result in lower ED50-values indicating a high sensitivity of the method. Inaddition, non-opiate analgesics like ketamine and pe-ripheral analgesics like pyrazolone derivatives gavea positive response.

MODIFICATIONS OF THE METHODThe method has been performed primarily in rabbits(Hertle et al. 1957; Hoffmeister 1962, 1968; Pierceyand Schröder 1980), but also dogs (Koll and Fleis-chmann 1941; Skinkle and Tyers 1979) and cats(Mitchell 1964) have been used.

Among several methods in different species, Fleischand Dolivo (1953) found the electrical stimulation ofthe tooth pulp in the rabbit as the only satisfactorymethod to test the efficacy of different analgesic drugs.

The effects of tooth pulp stimulation in the thalamusand hypothalamus of the rat have been investigated byShigena et al. (1973).

The method has been adapted for freely moving rats(Steinfels and Cook 1986). Medium effective dosescould be determined for ì and ê agonists. Non-steroidal


anti-inflammatory drugs were also effective in this testprocedure, but the slopes of the dose-response curvesfor these compounds were lower than for the opioidanalgesics. Microinfusion of bradykinin solution ontothe tooth pulp of unrestrained rats was described byFoong et al. (1982) as a reliable method for evaluatinganalgesic potencies of drugs on trigeminal pain.

Kidder and Wynn (1983) described an automaticelectronic apparatus for generating and recordinga ramp stimulus for analgesia testing.

Thut et al. (1995) used the rabbit tooth-pulp assayto quantify efficacy and duration of antinociception bylocal anesthetics infiltrated into maxillary tissues.

Shyu et al. (1984) studied the role of central sero-toninergic neurons in the development of dental painin the monkey.

REFERENCES AND FURTHER READINGChau TT (1989) Analgesic testing in animal models. In: Phar-

macological Methods in the Control of Inflammation. AlanR Liss, Inc. pp 196–212

Chin JH, Domino EF (1961) Effects of morphine on brain po-tentials evoked by stimulation of the tooth pulp of the dog.J Pharmacol Exp Ther 132:74–86

Fleisch A, Dolivo M (1953) Auswertung der Analgetica imTierversuch. Helv Physiol Acta 11:305–322

Foong FW, Satoh M, Takagi H (1982) A newly devised reli-able method for evaluating analgesic potencies of drugs ontrigeminal pain. J Pharmacol Meth 7:271–278

Hertle F, Schanne O, Staib I (1957) Zur Methodik der Prüfungder Analgesie am Kaninchen. Arzneim Forsch 7:311–314

Hoffmeister F (1962) Über cerebrale polysynaptische Reflexedes Kaninchens und ihre Beeinflussbarkeit durch Phar-maka. Arch Int Pharmacodyn 139:512–527

Hoffmeister F (1968) Tierexperimentelle Untersuchungen überden Schmerz und seine pharmakologische Beeinflussung.Arzneim Forsch 16. Beiheft:5–116

Kidder GW, Wynn RL (1983) An automatic electronic apparatusfor generating and recording a ramp stimulus for analgesiatesting. J Pharmacol Meth 10:137–142

Koll W, Fleischmann G (1941) Messungen der analgetis-chen Wirksamkeit einiger Antipyretica am Hund. Naunyn-Schmiedeberg’s Arch Exp Path Pharmakol 198:390–406

Koll W, Reffert H (1938) Eine neue Methode zur Messung anal-getischer Wirkungen im Tierversuch. Versuche mit Mor-phin und einigen Morphinderivaten am Hund. Arch expPath Pharmakol 190:67–87

Matthews B, Searle BN (1976) Electrical stimulation of teeth.Pain 2:245–251

Mitchell CL (1964) A comparison of drug effects upon the jawjerk response to electrical stimulation of the tooth pulp indogs and cats. J Pharmacol Exp Ther 146:1–6

Piercey MF, Schroeder LA (1980) A quantitative analgesic assayin the rabbit based on response to tooth pulp stimulation.Arch Int Pharmacodyn Ther 248:294–304

Ruckstuhl K (1939) Beitrag zur pharmakodynamischen Prüfungder Analgetica. Inaug.-Dissertation, Bern

Shigena Y, Marao S, Okada K, Sakai A (1973) The effects oftooth pulp stimulation in the thalamus and hypothalamusof the rat. Brain Res 63:402–407

Shyu KW, Lin MT, Wu TC (1984) Possible role of centralserotoninergic neurons in the development of dental pain

and aspirin-induced analgesia in the monkey. Exp Neurol84:179–187

Skingle M, Tyers MB (1979) Evaluation of antinociceptive ac-tivity using electrical stimulation of the tooth pulp in theconscious dog. J Pharmacol Meth 2:71–80

Steinfels GF, Cook L (1986) Antinociceptive profiles of μ andκ opioid agonists in a rat tooth pulp stimulation procedure.J Pharm Exp Ther 236:111–117

Thut PD, Turner MD, Cordes CT, Wynn RL (1995) A rabbittooth-pulp assay to quantify efficacy and duration of anti-nociception by local anesthetics infiltrated into maxillarytissues. J Pharmacol Toxicol Meth 33:231–236

Wilhelmi G (1949) Über die pharmakologischen Eigenschaftenvon Irgapyrin, einem neuen Präparat aus der Pyrazolon-reihe. Schweiz Med Wschr 25:577–582

Wirth W, Hoffmeister F (1967) Zur Wirkung von Kombi-nationen aus Phenothiazin-Derivaten mit Analgetika-An-tipyretika. Wien Med Wschr 117:973–978

Wynn RL, El’Baghdady YM, Ford RD, Thut PD, Rudo FG(1984) A rabbit tooth-pulp assay to determine ED50 val-ues and duration of action of analgesics. J Pharmacol Meth11:109–117

Wynn RL, Ford RD, McCourt PJ, Ramkumar V, Bergman SA,Rudo FG (1986) Rabbit tooth pulp compared to 55°Cmouse hot plate assay for detection of antinociceptive ac-tivity of opiate and nonopiate central analgesics. Drug DevRes 9:233–239

Yim GKW, Keasling HH, Gross EG, Mitchell CW (1955) Simul-taneous respiratory minute volume and tooth pulp thresh-old changes following levorphan, morphine and levorphan-levallorphan mixtures in rabbits. J Pharmacol Exp Ther115:96–105

H.1.2.9Monkey Shock Titration Test

PURPOSE AND RATIONALEGenerally, analgesic tests in rats and mice result in cor-relation with the analgesic activity of a drug in man.To clarify the mode of action in more detail and tofind a suitable dose for therapy in man, experimentsin monkeys may be necessary.

PROCEDUREThis test has been recommended by Weiss and Laties(1958) and later developed further by several authors.The monkeys are seated in restraining chairs. Electri-cal current is delivered by a Coulbourn Instrument Pro-grammable Shocker through electrodes coupled to twotest tube clamps which are attached to a shaved portionof the tail. The current ranges from 0 to 4 mA through29 progressive steps. The monkey presses a bar to in-terrupt the shock. A stable baseline shock level is es-tablished for each monkey on the day prior to drug ad-ministration. After drug administration shock titrationactivity is rated according to the change in maximumlevel of median shock intensity attained for drug ascompared to control levels. Doses of 3.0 mg/kg i.m.


morphine, 1.7 mg/kg i.m. methadone and 10 mg/kgi.m. pentazocine were found to be effective.

CRITICAL ASSESSMENTThe monkey shock titration test may be used for finalevaluation of a new compound before administrationto man. For screening activities the procedure can notbe recommended since the test is too time consum-ing and the apparatus too complicated. Furthermore,higher animals such as monkeys should only be usedif absolutely necessary.

REFERENCES AND FURTHER READINGBloss JL, Hammond DL (1985) Shock titration in the rhesus

monkey: effects of opiate and nonopiate analgesics. J Phar-macol Exp Ther 235:423–430

Campell ND, Geller I (1968) Comparison of analgesic ef-fects of O-(4-methoxy phenyl carbamoyl)-3-diethylamino-propiophenone oxime HCl (USVP E-142), pentazocineand morphine in cynomolgus monkeys. Fed Proc FASEB27:653 (2465)

Dykstra LA (1979) Effects of morphine, pentazocine and cycla-zocine alone and in combination with naloxone on electricshock titration in the squirrel monkey. J Pharm Exp Ther211:722–732

Dykstra LA (1980) Nalorphine’s effect under several schedulesof electric shock titration. Psychopharmacology 70:69–72

Dykstra LA, Macmillan DE (1977) Electric shock titration: Ef-fects of morphine, methadone, pentazocine, nalorphine,naloxone, diazepam and amphetamine. J Pharm Exp Ther202:660–669

Römer D (1968) A sensitive method for measuring analgesic ef-fects in the monkey. In: Soulairac A, Cahn J, Charpentier J(eds) Pain. Acad Press London, New York, pp 165–170

Weiss B, Laties VG (1964) Analgesic effects in monkeys ofmorphine, nalorphine, and a benzomorphan narcotic antag-onist. J Pharm Exp Ther 143:169–173

H.1.2.10Formalin Test in Rats

PURPOSE AND RATIONALEThe formalin test in rats has been proposed as a chronicpain model which is sensitive to centrally active anal-gesic agents by Dubuisson and Dennis (1977).

PROCEDUREMale Wistar rats weighing 180–300 g are administered0.05 ml of 10% formalin into the dorsal portion of thefront paw. The test drug is administered simultane-ously either sc. or orally. Each individual rat is placedinto a clear plastic cage for observation. Readings aretaken at 30 and 60 min and scored according to a painscale. Pain responses are indicated by elevation or fa-voring of the paw or excessive licking and biting ofthe paw. Analgesic response or protection is indicated

if both paws are resting on the floor with no obviousfavoring of the injected paw.

EVALUATIONUsing various doses, ED50 values for protection canbe calculated. Doses of 1.7 mg/kg morphine s.c. and15 mg/kg s.c. pethidine were found to be effective.

CRITICAL ASSESSMENTThe formalin test identifies mainly centrally activedrugs, whereas peripherally acting analgesics are al-most ineffective. Therefore, the formalin test may al-low a dissociation between inflammatory and non-inflammatory pain, a rough classification of analgesicsaccording to their site and their mechanism of action(Chau 1989). Cowan (1990) underlined the aspect thatthe formalin-test is a model of chronic pain whereasmost other methods measure only the effect on acutepain.

MODIFICATIONS OF THE METHODMurray et al. (1988) used mice instead of rats. Theyinjected 0.020 ml of 5% formalin solution into the sub-plantar region of the hind paw. Morphine at a dose of2.1 mg/kg s.c. and pentazocine at a dose of 23.8 mg/kgs.c. were active whereas the cyclooxygenase inhibitorzomepirac was inactive even at a dose of 100 mg/kgs.c.

Hunskaar et al. (1986), Hunskaar and Hole (1987)injected a small amount of formalin (20 µl of 1% so-lution) under the skin of the dorsal surface of the righthind paw of mice. A biphasisic response with an early(0–5 min) and a late (20–30 min) phase with high lick-ing activity was observed. Central acting analgesicswere active in both phases, whereas non-steroidal anti-inflammatory drugs and corticosteroids inhibited onlythe late phase. Acetylsalicylic acid and paracetamolwere antinociceptive in both phases.

Shibata et al. (1989) again used lower concen-trations of formalin (0.025 ml of 0.5% formalin so-lution) and also mice instead of rats. They founda characteristic biphasic pain response. Centrally act-ing drugs such as morphine inhibited both phases,whereas according to their data peripherally actingdrugs such as acetylsalicylic acid, oxyphenylbuta-zone and corticosteroids inhibited only the secondphase.

Abbott et al. (1995) used the formalin test for scor-ing properties of the first and second phases of the painresponse in rats.


Abbadie et al. (1997) determined the pattern of c-fos expression in the rat spinal cord to study the twophases of the formalin test.

Clavelou et al. (1989), Dallel et al. (1995). Gilbertet al. (2001) used a modification of the formalin testfor assessing pain and analgesia in the orofacial re-gion of the rat. After injection into the upper lip, painintensity was evaluated by the animal’s behavior ofrubbing of the injected area. A subcutaneous injectionof 0.05 ml of 0.92% formaldehyde solution was madeinto the upper lip, just lateral to the nose. Following in-jection, the rat was immediately brought back in a testbox equipped with a videocamera for a 45 min ob-servation period. The recording time was divided into15 blocks of 5 min and a pain score was determined foreach block, by measuring the number of seconds thatthe animals spent rubbing the injected area with the ip-silateral fore- or hindpaw. The animals were sacrificedafter the end of the experiment to avoid unnecessarysuffering.

Tjølsen et al. (1992) attributed the early phase toC-fibre activation, whereas the late phase appeared tobe dependent on the combination of an inflammatoryreaction in the peripheral tissue and functional changesin the dorsal horn of the spinal cord.

Alreja et al. (1984) used the formalin test for as-sessing pain in monkeys after volunteering of one ofthe authors to carry out the same procedure on himself.

Corrêa and Calixto (1993) studied the par-ticipation of B1 and B2 kinin receptors in theformalin-induced nociceptive response in the mouse.Pain response was increased after ACE-inhibitionand decreased by bradykinin receptor antago-nists.

Herman and Felinska (1979) proposed a rapid testfor screening of narcotic analgesics in mice by evalua-tion of behavioral symptoms after subcutaneous injec-tion of EDTA.

Legat et al. (1994), Dumas et al. (1997) induced hy-peralgesia in rats by subplantar injection of collage-nase (100 µg in 100 µl saline) and rated the behavioralreactions after treatment with analgesics according toa modified formalin-test.

REFERENCES AND FURTHER READINGAbbadie C, Taylor BK, Peterson MA, Basbaum AI (1997) Dif-

ferential contribution of the two phases of the formalin testto the pattern of c-fos expression in the rat spinal cord: stud-ies with remifentanile and lidocaine. Pain 69:101–110

Abbott FV, Franklin KBJ, Ludwick RJ, Melzack R (1981) Ap-parent lack of tolerance in the Formalin test suggests differ-ent mechanisms for morphine analgesia in different typesof pain. Pharmacol Biochem Behav 15:637–640

Abbott FV, Melzack R, Samuel C (1982) Morphine analgesia inthe tail-flick and Formalin pain tests is mediated by differ-ent neural systems. Exp Neurol 75:644–651

Abbott FV, Franklin KBJ, Westbrook RF (1995) The formalintest: scoring properties of the first and second phases of thepain response in rats. Pain 60:91–102

Alreja M, Mutalik P, Nayar U, Machanda SK (1984) The forma-lin test: a tonic pain model in the primate. Pain 20:97–105

Chau TT (1989) Analgesic testing in animal models. In: Phar-macological Methods in the Control of Inflammation. AlanR. Liss, Inc., pp 195–212

Clavelou P, Pajot J, Dallel R, Raboisson P (1989) Applicationof the formalin test to the study of orofacial pain in the rat.Neurosci Lett 103:349–353

Corrêa CR, Calixto JB (1993) Evidence for participation of B1and B2 kinin receptors in formalin-induced nociceptive re-sponse in the mouse. Br J Pharmacol 110:193–198

Cowan A (1990) Recent approaches in the testing of analgesicsin animals. In: Modern Methods in Pharmacology, Vol 6,Testing and Evaluation of Drugs of Abuse. Wiley-Liss, Inc.pp 33–42

Dallel R, Raboisson P, Clavelou P, Saade M, Woda A (1995)Evidence for a peripheral origin of the tonic nociceptiveresponse to subcutaneous formalin. Pain 61:11–16

Dubuisson D, Dennis SG (1977) The Formalin test: A quantita-tive study of the analgesic effects of morphine, meperidineand brain stem stimulation in rats and cats. Pain 4:161–174

Dumas J, Liégeois JF, Bourdon V (1997) Involvement of 5-hydroxytryptamine and bradykinin in the hyperalgesia in-duced in rats by collagenase from clostridium histolyticum.Naunyn-Schmiedeberg’s Arch Pharmacol 355:566–570

Gilbert SD, Clatk TC, Flores CM (2001) Antihyperalgesic activ-ity of epibatidine in the formalin model of facial pain. Pain89:159–165

Herman ZS, Felinska W (1979) Rapid test for screeningof narcotic analgesics in mice. Pol J Pharmacol Pharm31:605–608

Hunskaar S, Berge OG, Hole K (1986) Dissociation betweenantinociceptive and anti-inflammatory effects of acetylsal-icylic acid and indomethacin in the formalin test. Pain25:125–132

Hunskaar S, Hole K (1987) The formalin test in mice: disso-ciation between inflammatory and non-inflammatory pain.Pain 30:103–114

Legat FJ, Griesbacher T, Lembeck F (1994) Mediation ofbradykinin of the rat paw oedema induced by colla-genase from Clostridium histolyticum. Br J Pharmacol112:453–460

Malmberg AB, Yaksh TL (1992) Antinociceptive actions ofspinal nonsteroidal anti-inflammatory agents on the forma-lin test in the rat. J Pharm Exp Ther 263:136–146

Murray CW, Porreca F, Cowan A (1988) Methodological refine-ments to the mouse paw formalin test. J Pharmacol Meth20:175–186

North MA (1977) Naloxone reversal of morphine analgesia butfailure to alter reactivity to pain in the formalin test. LifeSci 22:295–302

Shibata M, Ohkubo T, Takahashi H, Inoki R (1989) Modifiedformalin test: characteristic biphasic pain response. Pain38:347–352

Theobald W (1955) Vergleichende Untersuchung anti-inflammatorischer Wirkstoffe am Formalinoedem. ArchInt Pharmacodyn 103:17–26

Tjølsen A, Berge OG, Hunskaar S, Rosland JH, Hole K (1992)The formalin test: an evaluation of the method. Pain51:5–17


Wheeler H, Porreca F, Cowan A (1989) Formalin is uniqueamong potential noxious agents for the intensity of its be-havioral response in rats. FASEB J 3:A278 (310)

H.1.2.11Neuropathic Pain

H.1.2.11.1General Considerations

Partial injury to somatosensory nerves sometimescauses causalgia in humans. Causalgia is characterizedby spontaneous burning pain combined with hyper-algesia and allodynia and usually follows an incom-plete peripheral nerve injury. Allodynia, a pain sensa-tion due to normally innocuous stimulation, is a partic-ularly troublesome symptom in patients. Neuropathicpains are classified according to either the etiologicaldiagnosis of the neuropathy (e. g., painful diabetic neu-ropathy, post-herpetic neuralgia, post-traumatic neu-ralgia, etc.), or the anatomical lesion (e. g., centralpain, peripheral neuralgia). See Hansson and Dicken-son (2005). Various animal models are described tostudy neuropathic pain. See below.

REFERENCES AND FURTHER READINGHansson PT, Dickenson AH (2005) Pharmacological treat-

ment of peripheral neuropathic pain conditions based onshared commonalities despites multiple etiologies. Pain113:251–254

H.1.2.11.2Chronic Nerve Constriction Injury

PURPOSE AND RATIONALEBennet and Xie (1988) described a peripheral neuropa-thy due to nerve constriction in the rat that producesdisorders of pain sensation like those seen in man. Thismethod with slight modifications was used by Davaret al. (1991), Mao et al. (1992), Munger et al. (1992),Yamamoto and Yaksh (1992), Tal and Bennet (1993)and reviewed by Bennett (1993).

PROCEDUREAnesthesia is induced in male Sprague-Dawley rats byinhalation with halothane 4% and maintained at a con-centration of 2–3% as needed. After a local incision,the biceps femoralis of each leg is bluntly dissectedat midtigh to expose the sciatic nerve. Each nerve isthen mobilized with care taken to avoid undue stretch-ing. Four 4–0 chromic gut sutures are each tied looselywith a square knot around the right sciatic nerve. Theleft sciatic nerve is only mobilized. Both incisions are

closed layer to layer with silk sutures and the rats al-lowed to recover. During the next days, the animalsshow a mild eversion of the affected paw and a mild-to-moderate degree of foot drop.

The thermal nociceptive threshold is measured ac-cording to the method of Hargreaves et al. (1988), (seeH.1.2.3). The rats are placed beneath a clear plasticcage (10 × 20 × 24 cm) upon an elevated floor of clearglass. A radiant heat source (halogen projector lamp)is placed beneath the glass floor on a movable holderand positioned such that is focuses at the plantar areaof one hind paw. The time interval between the appli-cation of the light beam and the brisk hind paw with-drawal response is measured to the nearest 01. sec.

The maximum hyperesthesia occurs between 7 and14 days after nerve ligation. Before intrathecal injec-tion of the drug or vehicle, the hind paws are tested 3times alternatively with 5-min intervals as the baselinedata. The left and right test sequence is carried out at5, 15, 30, 60 and 90 min after injection.

EVALUATIONThe mean ±SEM of the paw withdrawal latency(PWL) is plotted. To analyze the magnitude of hyper-esthesia, the difference score (DS) is calculated by sub-traction the maximum PWL of the control side (leftside) from the maximum PWL of the affected side(right side). Maximum PWL is defined as the PWL thatwas the maximum during the first 30 min after injec-tion. To analyze the drug effects in hyperesthetic rats,the dose is plotted against the change in DS (post-drugdifference score minus pre-drug difference score).

MODIFICATIONS OF THE METHODThe chronic constriction injury (CCI) model accord-ing to Bennett and Xie (1988) has been used by sev-eral authors: Sotgiu and Biella (1998), Toda et al.(1998), Blackburn-Munro and Jensen (2003), Keayet al. (2004), Bingham et al. (2005), Bomholt et al.(2005), Costa et al. (2005), and Howard et al. (2005).

Sotgiu et al. (1996) performed laminectomy fromL1 to S2 in anesthetized rats with sciatic chronic con-striction injury. For extracellular recording, two tung-sten microelectrodes were positioned under a dissect-ing microscope on the surface of the spinal cord at L2and L5–L6 level ipsilaterally to the injured nerve, andwere advanced at steps of 2 µm. Neuronal activity wasconventionally recorded and then digitized; frequencyhistograms were constructed by computer programs.The search stimulus for dorsal horn neurons at L2and L5–L6 segments was the electrical stimulation ofsaphenous and sciatic nerve peripheral territories. Nat-


ural stimuli (brushing of the skin) and noxious stimuli(calibrated pinching) were defined. After the responsesto saphenous stimuli in the neurons were recorded,a small pad of gel-foam soaked with 0.5 ml lidocainewas placed around the intact epineurium proximally tothe ligatures on the sciatic nerve. The saphenous stim-ulation was repeated during the block and after com-plete recovery of the preblock baseline activity. In thisway, the effect of the local anesthetic on the sponta-neous activity and on the response to a noxious stimu-lus could be evaluated.

The first animal model of painful neuropathy wasreported by Wall et al. (1979a, b). The sciatic nerve ofrats or mice was sectioned and either tied or implantedin a polyethylene tube sealed at its far end. Moreover,in one modification also the saphenous nerve was cut,such that the hind paw was completely denervated.This procedure, which is known as the neuroma model,is believed to replicate the human syndromes seen afteramputation (phantom pain) or after nerve transectionin an intact limb (anesthesia dolorosa). Within severaldays, the animals begin to self-mutilate the hindpawon the side of the nerve transection: a behavior named‘autotomy’.

Seltzer et al. (1990) ligated only one-half of the sci-atic nerve in rats unilaterally. The withdrawal thresh-olds to repetitive von Frey hair stimulation at theplantar side were decreased bilaterally as were thewithdrawal thresholds to CO2 laser heat pulses. Thecontralateral phenomena resemble the “mirror image”pains in humans with causalgia.

This “partial sciatic nerve injury model” has beenused by several authors (Malmberg and Basbaum1998; Lindenlaub and Sommer 2000; Bingham et al.2005). Patel et al. (2001) studied the effects of GABABagonists and gabapentin on mechanical hyperalgesia inmodels of neuropathic (partial sciatic ligation) and in-flammatory (Freund’s complete adjuvant) pain in therat and the inhibitory action on spinal transmission invitro.

Hofmann et al. (2003) described the tibial nerve in-jury model in rats as a surgically uncomplicated modelof neuropathic pain based on unilateral transection(neurotomy) of the tibial branch of the sciatic nerve.

Walczak et al. (2005) characterized the saphenousnerve partial ligation in rats as a model of neuropathicpain.

Kim and Chung (1992) described an experimentalmodel for peripheral neuropathy produced by segmen-tal spinal nerve ligation in the rat. Either both the L5and L6 spinal nerves or the L5 spinal nerve alone onone side of the rat were tightly ligated. A modified ver-

sion of this technique was used by LaBuda and Little(2005) and by Bertorelli et al. (2005).

DeLeo et al. (1994) performed cryoneurolysis ofthe sciatic nerve in the rat using a cryoprobe cooledto –60°C in a 30/5/30 s freeze-thaw-freeze sequence.Autotomy was observed after 4–14 days.

Coderre et al. (2004) produced a neuropathic-likepain syndrome in rats following prolonged hind pawischemia and reperfusion, creating an animal modelof complex regional pain syndrome-type I (CRPS-I;reflex sympathetic dystrophy), called chronic post-ischemia pain. A tourniquet ring was placed on onehindlimb of an anesthetized rat just proximal to the an-kle joint for 3 h, which was removed prior to termina-tion of anesthesia to allow reperfusion. Rats exhibitedhyperemia and edema/plasma extravasation of the is-chemic hind paw for a period of 2–4 h after reperfu-sion. Hyperalgesia to noxious mechanical stimulation(pin prick) and cold (acetone exposure), as well as me-chanical allodynia to innocuous mechanical stimula-tion (von Frey hairs) are evident in the affected hind-paw as early as 8 h after reperfusion, and extend for atleast 4 weeks.

Mice that lack protein kinase C gamma (PKCγ )displayed normal response to acute pain stimuli, butthey almost completely failed to develop a neuropathicpain syndrome after partial sciatic nerve section, andthe neurochemical changes that occurred in the spinalcord after nerve injury were blunted (Malmberg et al.1997).

Shimoyama et al. (2002) developed a mouse modelof neuropathic cancer pain by inoculating Meth Asarcoma cells in the immediate proximity of the sciaticnerve in BALB/c mice. The tumor grows predictablywith time and gradually compresses the nerve, therebycausing nerve injury. Time courses of thermal hyper-sensitivity and mechanical sensitivity to von Frey hairswere determined and signs of spontaneous pain wereevaluated. The authors compared this model with thechronic constriction model.

Panesar et al. (1997) and Campbell et al. (1998)studied mechanical hyperalgesia associated with par-tial peripheral ligation in the guinea pig.

REFERENCES AND FURTHER READINGBennett GJ (1993) An animal model of neuropathic pain: a re-

view. Muscle Nerve 16:1040–1048Bennet GJ, Xie YK (1988) A peripheral mononeuropathy in the

rat that produces disorders of pain sensation like those seenin man. Pain 33:87–108

Bertorelli R, Fredduzzi S, Tarozzo G, Campanella M, Grundy R,Beltramo M, Reggiani A (2005) Endogenous and exoge-nous melanocortin antagonists induce anti-allodynic ef-


fects in a model of rat neuropathic pain. Behav Brain Res157:55–62

Bingham S, Beswick PJ, Bountra C, Brown T, CampbellJP, Chessell IP, Clayton N, Collins SD, Davey PT,Goodland H, Gray N, Haslam C, Hatcher JP, Hunter AJ,Lucas F, Murkitt G, Naylor A, Pickup E, Sargent P, Sum-merfield SG, Stevens A, Stratton SC, Wiseman J (2005)The cyclooxygenase-2 inhibitor GW406381X [2-(4-ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]pyrazol[1,5-b]pyridazine] is effective in animal models of neuropathicpain and central sensitization. J Pharmacol Exp Ther312:1161–1169

Blackburn-Munro G, Jensen BS (2003) The anticonvulsant reti-gabine attenuates nociceptive behavior in rat models of per-sistent and neuropathic pain. Eur J Pharmacol 460:109–116

Bomholt SF, Mikkelsen JD, Blackburn-Munro G (2005) Antino-ciceptive effects of the antidepressants amitriptyline, du-loxetine, mirtazepine and citalopram in animal models ofacute, resistant and neuropathic pain. Neuropharmacology48:252–261

Campbell EA, Gentry CT, Patel S, Panesar MS, Walpole CJS,Urban L (1998) Selective neurokinin-1 receptor antagonistsare anti-hyperalgesic in a model of neuropathic pain in theguinea pig. Neuroscience 87:527–532

Coderre TJ, Xanthos DN, Francis L, Bennett GJ (2004) Chronicpost-ischemia pain (CPIP): a novel animal model of com-plex regional pain syndrome-Type I (CRPS-I; reflex sym-pathetic dystrophy) produced by prolonged hindpaw is-chemia and reperfusion in the rat. Pain 112:94–105

Costa B, Trovato AE, Colleoni M, Giagnoni G, Zarini E, Croci T(2005) Effect of the cannabinoid CB1 receptor antago-nist, SR141716, on nociceptive response and demyelina-tion in rodents with chronic injury of the sciatic nerve. Pain116:52–61

Davar G, Hama A, Deykin A, Vos B, Maciewicz R (1991)MK-801 blocks the development of thermal hyperalgesiain a rat model of experimental painful neuropathy. BrainRes 553:327–330

DeLeo JA, Coombs DW, Willenbring S, Colburn RW, Fromm C,Wagner R, Twitchell BB (1994) Characterization of a neu-ropathic pain model: sciatic cryoneurolysis in the rat. Pain56:9–16

Hofmann HA, de Vry J, Siegling A, Spreyer P, Denzer D (2003)Pharmacological sensitivity and gene expression analysisof the tibial nerve injury model of neuropathic pain. Eur JPharmacol 470:17–23

Howard RF, Walker SM, Mota PM, Fitzgerald M (2005) Theontogeny of neuropathic pain: postnatal onset of mechani-cal allodynia in rat spared nerve injury (SNI) and chronicconstriction injury (CCI) models. Pain 115:382–389

Keay KA, Monassi CR, Levinson DB, Bandler R (2004) Pe-ripheral nerve injury evokes disabilities and sensory dys-function in a subpopulation of rats: a closer model tohuman chronic neuropathy pain? Neurosci Lett 361:188–191

Kim SH, Chung JM (1992) An experimental model for periph-eral neuropathy produced by segmental spinal nerve liga-tion in the rat. Pain 50:355–363

LaBuda CJ, Little PJ (2005) Pharmacological evaluation of theselective nerve ligation model of neuropathic pain in therat. J Neurosci Methods 144:175–181

Lindenlaub T, Sommer C (2000) Partial sciatic nerve transectionas a model of neuropathic pain: a qualitative and quantita-tive neuropathological study. Pain 89:97–106

Malmberg A, Basbaum AI (1998) Partial sciatic nerve injury inthe mouse as a model of neuropathic pain: behavioral andneuroanatomical correlation. Pain 76:215–222

Mao J, Price DD, Mayer DJ, Lu J, Hayes RL (1992) IntrathecalMK-801 and local anesthesia synergistically reduce noci-ceptive behaviors in rats with experimental peripheral neu-ropathy. Brain Res 576:254–262

Munger BL, Bennett GJ, Kajander KC (1992) An experimen-tal painful peripheral neuropathy due to nerve constriction.Exper Neurol 118:204–214

Panesar MS, Patel S, Gentry CT, Campbell EA (1997) A novelmodel of neuropathic pain in the guinea pig. Comparativeanalgesic activity in a model of inflammatory pain. Br JPharmacol 120:230P

Patel S, Naeem S, Kesingland A, Froestl W, Capogna M, Ur-ban L, Fox A (2001) The effects of GABAB agonists andgabapentin on mechanical hyperalgesia in models of neu-ropathic and inflammatory pain. Pain 90:217–226

Seltzer Z, Dubner R, Shir Y (1990) A novel behavioral modelof neuropathic pain disorders produced in rats by partialsciatic nerve injury. Pain 43:205–218

Shimoyama M, Tanaka K, Hasue F, Shimoyama N (2002)A mouse model of neuropathic cancer pain. Pain99:167–174

Sotgiu ML, Biella G (1998) Contralateral inhibitory control ofspinal nociceptive transmission in rats with chronic periph-eral nerve injury. Neurosci Lett 253:21–24

Sotgiu ML, Biella G, Lacerenza M (1996) Injured nerve blockalters adjacent nerves spinal interaction in neuropathic rats.NeuroReport 7:1385–1388

Tal M, Bennet GJ (1993) Dextrorphan relieves neuropathic heat-evoked hyperalgesia in the rat. Neurosci Lett 151:107–110

Toda K, Muneshige H, Ikuta Y (1998) Antinociceptive effects ofneurotropin in a rat model of painful peripheral mononeu-ropathy. Life Sci 62:913–921

Walczak JS, Pichette V, Leblond F, Desbiens K, Beaulieu P(2005) Behavioral, pharmacological and molecular char-acterization of the saphenous nerve partial ligation: a newmodel of neuropathic pain. Neuroscience 132:1093–1102

Wall PD, Devor M, Inbal R, Scadding JW, Schonfeld D,Seltzer Z, Tomkiewicz MM (1979a) Autotomy followingperipheral nerve lesions: experimental anesthesia dolorosa.Pain 7:103–113

Wall PD, Scadding JW, Tomkiewicz MM (1979b) The produc-tion and prevention of experimental anesthesia dolorosa.Pain 6:175–182

Yamamoto T, Yaksh TL (1992) Spinal pharmacology of ther-mal hyperesthesia induced by constriction injury of sciaticnerve. Excitatory amino acid antagonists. Pain 49:121–128

H.1.2.11.3Peripheral Nerve Injury Model

PURPOSE AND RATIONALEIn addition to chronic ligation techniques, nerve dis-section and nerve crush models have been used tostudy neuropathic pain. Nerve crush injury was de-scribed by Decosterd et al. (2002).

PROCEDUREMale Sprague Dawley rats weighing 200–250 g wereanesthetized with halothane (1.5%–3%). The left sci-atic nerve was exposed at the mid-thigh level andcrushed by a pair of hemostat forceps with smoothprotective pads that were placed perpendicularly to the


sciatic trunk for 30 s (Bester et al. 2000). Muscle andskin were closed in two layers.

Animals were habituated to the tester, the environ-ment and the handling procedures prior to commence-ment of the testing. A calibrated von Frey monofila-ment was applied five times until it bent on the lateraldorsal side of the hindpaw that is in crushed nerve ter-ritory or the intact sural nerve, ipsilateral to the nervelesion. A series of monofilaments (1.0–200.0 g) wereapplied and the test started with the lowest force fil-ament. The mechanical withdrawal threshold corre-sponds to the minimum force required to elicit a repro-ducible flexor withdrawal movement after applicationof the von Frey hairs. Once baseline threshold was de-termined, eight light strokes were applied manually at1 Hz to the dorsum of the paw at 5-min intervals, for 2h. The mechanical withdrawal threshold was recordedimmediately after the application of the light touch, atthe same interval of 5 min during the whole test period.

EVALUATIONData are expressed as mean ±SEM of the recorded me-chanical withdrawal threshold in grams, or as a per-centage of the baseline value. The differences be-tween groups were analyzed with analysis of variance(ANOVA) two-way repeated measures.

MODIFICATIONS OF THE METHODVogelaar et al. (2004) described sciatic nerve regen-eration in mice and rats. Under anesthesia, the sciaticnerve was carefully exposed. At a point immediatelydistal from the gluteus maximus muscle, the nerve wascrushed for 30 s using a hemostatic forceps. The ani-mals were followed for 70 (rats) and 32 (mice) daysand functional recovery of sciatic nerve function wasmonitored by the foot reflex withdrawal test, locomo-tor pattern, and mechanical withdrawal thresholds.

Rodrigues-Filho et al. (2003, 2004) published thetechnique of avulsion injury of the rat brachial plexusthat triggers hyperalgesia and allodynia in the hind-paws. Male Wistar rats weighing 250–300 g wereanesthetized by chloralose i.p. The brachial plexus wasapproached through a horizontal incision parallel tothe clavicle, running from the sternum to the axillaryregion. The pectoralis major muscle was displaced,leaving the cephalic vein intact. The subclavian ves-sels were located and the lower trunk dissected andcrushed three times for 5 s using microsurgical forceps.At the end of this procedure, the nerve was completelyflattened and transparent. The tissue layers were thenbrought together and the skin closed with silk sutures.

For assessment of mechanical hyperalgesia, me-chanical thresholds were measured in the hindpawswith an analgesymeter (Ugo Basile, Italy) accordingto the method of Randall and Sellitto (1957). Further-more, thermal hyperalgesia, mechanical allodynia, andcold allodynia were measured in the hindpaws, as wellas grasping force in the forepaws.

Sweitzer et al. (2001) studied prevention of allody-nia induced by transection of the L5 spinal nerve inrats.

Devor et al. (2005) studied heritability of symptomsin the neuroma model of neuropathic pain.

REFERENCES AND FURTHER READINGBester H, Beggs S, Woolf CJ (2000) Changes in tactile stimuli-

induced behavior and c-Fos expression in the superficialdorsal horn and in parabrachial nuclei after sciatic nervecrush. J Comp Neurol 428:45–61

Decosterd I, Allchorne A, Woolf CJ (2002) Progressive tac-tile hypersensitivity after peripheral nerve crush: non-noxious mechanical stimulus-induced neuropathic pain.Pain 100:155–162

Devor M, del Canho S, Raber P (2005) Heritability of symptomsin the neuroma model of neuropathic pain: replication andcomplementation analysis. Pain 116:294–301

Randall LO, Selitto JJ (1957) A method for measurement anal-gesic activity in inflamed tissue. Arch Int Pharmacodyn111:409–419

Rodrigues-Filho R, Santos ARS, Bertelli JA, Calixto JB (2003)Avulsion injury of the rat brachial plexus triggers hyperal-gesia and allodynia in the hindpaws: a new model of neu-ropathic pain. Brain Res 982:186–194

Rodrigues-Filho R, Campos MM, Ferreira J, Santos ARS,Bertelli JA, Calixto JB (2004) Pharmacological character-ization of the rat brachial plexus avulsion model of neuro-pathic pain. Brain Res 1018:159–170

Sweitzer SM, Schubert P, DeLeo JA (2001) Propentofylline,a glial modulating agent, exhibits antiallodynic propertiesin a rat model of neuropathic pain. J Pharmacol Exp Ther297:1210–1217

Vogelaar CF, Vrinten DH, Hoekman MFM, Brakkee JH, Bur-bach JPH, Hamers FPT (2004) Sciatic nerve regenerationin mice and rats: recovery and sensory innervation is fol-lowed by a slowly retreating neuropathic pain-like syn-drome. Brain Res 1027:67–72

H.1.2.11.4Spared Nerve Injury Model

PURPOSE AND RATIONALEIn addition to the above-described models of pe-ripheral neuropathic pain [chronic constriction injurymodel according to Bennett and Xie (1988), par-tial ligation according to Seltzer et al. (1990), seg-mental spinal nerve ligation according to Kim andChung (1992)], Decosterd and Woolf (2000) describedspared nerve injury as an animal model of persistentperipheral neuropathic pain.


PROCEDUREAdult male Sprague Dawley rats were used. Underhalothane (2%) anesthesia the skin on the lateral sur-face of the thigh was incised and a section made di-rectly through the biceps femoris muscle exposing thesciatic nerve and its three terminal branches: the sural,common peroneal and tibial nerves. The procedurecomprises an axotomy and ligation of the tibial andcommon peroneal nerves leaving the sural nerve in-tact. The common peroneal and the tibial nerves weretightly ligated with 5.0 silk and sectioned distal to theligation, removing 2–4 mm of the distal nerve stump.Care was taken to avoid any contact with or stretchingof the intact sural nerve. Muscle and skin were closedin two layers. Behavior testing was performed only af-ter a period of at least 1 week.

For testing mechanical allodynia, animals wereplaced on an elevated wire grid and the plantar surfaceof the paw stimulated with a series of ascending forcevon Frey filaments. The threshold was taken as thelowest force that evoked a brisk withdrawal responseto one of five repetitive stimuli. The lateral and medialplantar surface of the paw as well as its dorsal surfacewere tested.

For testing mechanical hyperalgesia, a pin prick testwas performed using a safety pin. The lateral part ofthe plantar surface of the paw was briefly stimulatedat an intensity to indent but not penetrate the skin. Theduration of paw withdrawal was recorded.

For testing cold allodynia, a drop of acetone so-lution was delicately dropped onto the lateral plantarsurface of the paw, using a blunt needle connected toa syringe without touching the skin. The duration ofthe withdrawal response was recorded.

The lateral plantar surface was exposed to a beamof radiant heat through a transparent Perspex surface(Hargreaves et al. 1988). The withdrawal latency andduration were recorded. The heat stimulation was re-peated 3 times at an interval of 5–10 min for each pawand the mean calculated.

EVALUATIONResults were presented as mean ±SEM. The data wereanalyzed by one-way ANOVA and the non-parametricWilcoxon matched-pairs signed-rank tests.

MODIFICATIONS OF THE METHODThe rat spared nerve injury model has been used byseveral authors: Blackburn-Munro and Jensen (2003),Rode et al. (2005), and Howard et al. (2005).

REFERENCES AND FURTHER READINGBennett GJ, Xie YK (1988) A peripheral mononeuropathy in the

rat that produces disorders of pain sensation like those seenin man. Pain 33:87–108

Blackburn-Munro G, Jensen BS (2003) The anticonvulsant reti-gabine attenuates nociceptive behavior in rat models of per-sistent and neuropathic pain. Eur J Pharmacol 460:109–116

Decosterd I, Woolf CJ (2000) Spared nerve injury: an ani-mal model of persistent peripheral neuropathic pain. Pain87:149–158

Hargreaves K, Dubner R, Brown F, Flores C, Joris J (1988)A new and sensitive method for measuring thermal noci-ception in cutaneous hyperalgesia. Pain 32:77–88

Howard RF, Walker SM, Mota PM, Fitzgerald M (2005) Theontogeny of neuropathic pain: postnatal onset of mechani-cal allodynia in rat spared nerve injury (SNI) and chronicconstriction injury (CCI) models. Pain 115:382–389


Rode F, Jensen DG, Blackburn-Munro G, Bjerrum OJ (2005)Centrally-mediated antinociceptive actions of GABAA re-ceptor agonists in the rat spared nerve injury model of neu-ropathic pain. Eur J Pharmacol 516:131–138

Seltzer Z, Dubner R, Shir Y (1990) A novel behavioral modelof neuropathic pain disorders produced in rats by partialsciatic nerve injury. Pain 43:205–218

H.1.2.11.5Spinal Cord Injury

PURPOSE AND RATIONALESpinal cord injuries result in a devastating loss of func-tion. Chronic central pain syndromes frequently de-velop in the majority of affected patients. Several at-tempts have been made to find animal models of thissituation. Xu et al. (1992), Hao and Xu (1996), Haoet al. (1998a, 1998b, 2000), Wu et al. (2003), and Col-paert et al. (2004) performed studies in rats after is-chemic spinal cord injury photochemically induced bylaser irradiation.

PROCEDUREFemale Sprague Dawley rats were anesthetized with300 mg/kg chloral hydrate i.p. and one jugular veinwas cannulated. Vertebrae T11–L2 were exposed aftera midline incision of the skin on the back. The animalswere positioned beneath a tuneable argon ion laser (In-nova, Model 70, Coherent Laser Production Division)and irradiated with a knife edge beam, which was usedto cover the single T13 vertebra with an average powerof 0.16 W for 10 min. No laminectomy was performed.Immediately before the irradiation, erythrosine B (RedNo. 3, Aldrich-Chemie) was injected intravenously in0.9% saline at a dose of 32.5 mg/kg. Since erythro-sine B is rapidly metabolized, the injection at this dosewas repeated at 5-min intervals during the irradiationin order to maintain an adequate blood concentration.


Erythrosine B has an optimal absorption wavelengthsimilar to that of the laser light. When it receives thislight, a photochemical reaction occurs inside the spinalcord blood vessel where the laser is aimed. One of thereaction products accumulates in the vessel, injuringthe endothelial layer and causing platelet release andcoagulation and, thus, ischemia. After irradiation theincision was closed and the animals were kept warmfor 2 h. The bladders were emptied manually 2–3 timesa day until normal function was regained.

A set of calibrated von Frey hairs was used to testthe vocalization threshold in response to graded me-chanical pressure ranging from 0.021 to 410.0 g. Dur-ing the test the rats were gently restrained in a standingposition by the experimenter and the von Frey hair waspushed onto the skin until the filament became bent.The frequency of the stimulation was about every 3–4 sper stimulus and at each intensity. The stimuli were ap-plied 5–10 times. The pressure which induced consis-tent vocalization (>75% response rate) over a relativelylarge skin area was considered to be the pain threshold.

EVALUATIONRats were randomized for different treatment groups.The data are expressed as medians and variability asmean ±SEM. Data were analyzed with Kruskal-Wallisone-way ANOVA followed by Wilcoxon signed rankstest or by Mann-Whitney U-test.

MODIFICATIONS OF THE METHODChristensen et al. (1996), Christensen and Hulsebosch(1997a, 1007b), and Bennett et al. (2000a, 2000b) useda rodent spinal hemisection model of spinal cord injuryin which mechanical and thermal allodynia develop by24 days after injury.

Male Sprague Dawley rats (175–200 g) were deeplyanesthetized by i.p. injection of 75 mg/kg ketamineand 15 mg/kg xylazine. The spinal cord was hemidis-sected at T13 on the left side by the following proce-dure: after palpation of the dorsal surface to locate thecranial borders of the sacrum and the dorsal spinousprocesses of the lower thoracic and lumbar vertebrae,the T11, T12, and T13 laminae were determined by lo-cating the last rib, which attaches the cranial end of theT13 vertebrae. The surgical field was shaved and pre-pared with povidone-iodine, and a longitudinal inci-sion was made exposing several segments. A laminec-tomy was performed at vertebral level T11, the lum-bar spinal cord was identified with the accompanyingdorsal vessel, and the spinal cord was hemisected atT13, cranial to the L1 dorsal root entry zone, witha scalpel blade without damage to the major dorsal

vessel or vascular branches. The musculature and thefascia were then sutured and the skin was apposed byautoclips.

Siddall et al. (1995) and Drew et al. (2004) de-scribed mechanical allodynia following contusion in-jury of the rat spinal cord. Contusive spinal cord in-jury was produced in anesthetized Wistar rats weigh-ing 200–300 g. Laminectomy was performed at thevertebral thoracic T10 level to expose a 3-mm win-dow over the dorsal spinal cord and adjacent rostraland caudal spinous processes were clamped to stabi-lize the spine. A brass guide tube 15 cm in length waspositioned perpendicularly above the exposed cord anda cylindrical 10 g steel weight (2 mm in diameter) witha rounded tip was suspended within the tube 2 cmabove the cord surface. The weight was held withinthe tube by a metal pin. Removing the pin and allow-ing the weight to drop onto the exposed cord producedspinal cord injury at the T12–T13 segmental level. Thewound was closed in layers and antibiotics were ad-ministered.

Abraham et al. (2001) and Caudle et al. (2003) de-scribed excitotoxic spinal cord injury induced by in-traspinal injection of quisqualic acid.

Zochodne et al. (1994) induced a segmental chronicpain syndrome by lumbar intrathecal NMDA infusion.

Malmberg and Yaksh (1992) reported that hyperal-gesia mediated by spinal glutamate or substance P re-ceptor is blocked by spinal cyclooxygenase inhibition.

REFERENCES AND FURTHER READINGAbraham KE, McGinty JF, Brewer KL (2001) The role of kainic

acid/AMPA and metabotropic glutamate receptors in theregulation of mRNA expression and the onset of pain-related behavior following excitotoxic spinal cord injury.Neuroscience 104:863–874

Bennett AD, Everhart AW, Hulsebosch CE (2000a) Intrathecaladministration of an NMDA or a non-NMDA receptor an-tagonist reduces mechanical but not thermal allodynia ina rodent model of chronic pain after spinal cord injury.Brain Res 859:72–82

Bennett AD, Chastain KM, Hulseboscj CE (2000b) Alleviationof mechanical and thermal allodynia by CGRP8–37 in a ro-dent model of chronic central pain. Pain 86:163–175

Caudle RM, Perez FM, King C, Yu CG, Yezierski RP (2003)N-Methyl-D-aspartate subunit expression and phosphory-lation following excitotoxic spinal cord injury in rats. Neu-rosci Lett 349:37–40

Christensen MD, Hulsebosch CE (1997a) Spinal cord injury andanti-NGF treatment results in changes in CGRP densityand distribution in the dorsal horn in the rat. Exp Neurol147:463–475

Christensen MD, Hulsebosch CE (1997b) Chronic central painafter spinal cord injury. J Neurotrauma 14:517–537

Christensen MD, Everhart AW, Pickelman JT, Hulsebosch CE(1996) Mechanical and thermal allodynia in chronic centralpain following spinal cord injury. Pain 68:97–107


Colpaert FC, Wu WP, Hao JX, Royer I, Sautel F, Wiesenfeld-Hallin Z, Xu XJ (2004) High-efficacy 5-HT1A receptor ac-tivation causes a curative-like action on allodynia in ratswith spinal cord injury. Eur J Pharmacol 497:29–33

Drew GM, Siddall PJ, Duggan AW (2004) Mechanical allodyniafollowing contusion injury of the rat spinal cord is associ-ated with loss of GABAergic inhibition in the dorsal horn.Pain 109:379–388

Hao JX, Xu XJ (1996) Treatment of a chronic allodynia-likeresponse in spinally injured rats: effects of systemically ad-ministered excitatory amino acid receptor antagonists. Pain66:279–285

Hao JX, Yo W, Wiesenfeld-Hallin Z, Xu XJ (1998a) Treat-ment of chronic allodynia in spinally injured rats: ef-fects of intrathecal selective opioid receptor agonists. Pain75:209–217

Hao JX, Xu IS, Wiesenfeld-Hallin Z, Xu XJ (1998b) Anti-hyperalgesic and anti-allodynic effects of intrathecal noci-ceptin/orphanin FQ in rats after spinal cord injury, periph-eral nerve injury and inflammation. Pain 76:385–393

Hao JX, Xu JX, Urban L, Wiesenfeld-Hallin Z (2000) Re-peated administration of systemic gabapentin alleviatesallodynia-like behaviors in spinally injured rats. NeurosciLett 280:211–214

Malmberg AB, Yaksh TL (1992) Hyperalgesia mediated byspinal glutamate or substance P receptor is blockedby spinal cyclooxygenase inhibition. Science 257:1276–1279

Malmberg AB, Chen C, Tonegawa S, Basbaum AI (1997) Pre-served acute pain and reduced neuropathic pain in micelacking PKCγ . Science 278:279–283

Siddall PJ, Xu CL, Cousins MJ (1995) Allodynia follow-ing traumatic spinal cord injury in the rat. NeuroReport6:1241–1244

Wu WP, Hao JX, Xu XJ, Wiesenfeld-Hallin Z, Koek W, Col-paert FC (2003) The very-high-efficacy 5-HT1A receptoragonist, F 13640, preempts the development of allodynia-like behaviors in rats with spinal cord injury. Eur J Pharma-col 478:131–137

Xu XJ, Hao JX, Aldskogius H, Seiger A, Wiesenfeld-Hallin Z(1992) Chronic pain-related syndrome in rats after is-chemic spinal cord lesion: a possible animal model for painin patients with spinal cord injury. Pain 48:279–290

Zochodne DW, Murray M, Nag S, Riopelle RJ (1994) A seg-mental chronic pain syndrome in rats associated with in-trathecal infusion of NMDA: evidence for selective actionin the dorsal horn. Can J Neurol Sci 21:24–28

H.1.2.11.6Chemotherapy-Induced Pain

PURPOSE AND RATIONALEWithin the numerous adverse effects associated withantineoplastic drugs, painful peripheral neuropathy isfrequent. Vincristine, an antineoplastic agent widelyused in cancer therapy, was found to be neurotoxicfor all treated patients and can induce peripheral neu-ropathy (Windebank 1999). Aley et al. (1996), Authieret al. (1999, 2003) and Marchand et al. (2003) devel-oped an animal model of nociceptive neuropathy usingrepeated injections of vincristine.

PROCEDUREMale Sprague Dawley rats weighing 180–200 g re-ceived five intravenous injections of 150 µg/kg vin-cristine every 2 days until a cumulative dose of 750µg/kg was reached. Thresholds to paw pressure weredetermined before and 14 days after vincristine treat-ment. Test drug was then applied and the vocalizationthresholds were determined 15, 30, 45, 60, 90, and 120min after this injection.

The C-fiber-evoked flexor reflex elicited in the righthind limb (Falinower et al. 1994; Mestre et al. 1997)was recorded from halothane-anesthetized rats. Rect-angular electric pulses of 6–7 mA strength and 2 msduration were applied every 10 s to the sural nerve re-ceptive field by means of two stainless steel needlesinserted into the skin of toes 4 and 5. The C-fiber-evoked reflex response (electromyographic responses)was recorded from the ipsilateral biceps muscle byutilizing another pair of stainless steel needles. Oncea stable threshold C reflex response was obtained, thestimulus strength was increased by a factor of three-fold. Test drug was then injected in various doses andthe mean C-fiber reflex (mean of the 12 C-fiber reflexesrecorded during the 2-min period) was calculated ev-ery 2 min between 25 and 35 min.

EVALUATIONThe data analysis was performed by a two-way anal-ysis of variance (ANOVA) followed by a Student-Newman-Keuls test for the time course of the effect ofdrug on mechanical nociceptive thresholds. For the Creflex, results were expressed as mean percentage in-hibition of the integrated C reflex responses obtainedbetween 25 and 35 min after drug injection and plot-ted against log dose, allowing ED50 calculation.

MODIFICATIONS OF THE METHODLynch et al. (2005) tested the effect of a nicotinicacetylcholine agonist against allodynia in rats in thevincristine-induced pain model.

Polomano et al. (2001) described painful peripheralneuropathy in the rat produced by the chemotherapeu-tic drug, paclitaxel.

Dalziel et al. (2004) described allodynia in rats in-fected with varicella zoster virus as an animal modelfor post-herpetic neuralgia.

REFERENCES AND FURTHER READINGAley KO, Reichling DB, Levine JD (1996) Vincristine hyperal-

gesia in the rat: a model of painful vincristine neuropathyin humans. Neuroscience 73:259–265


Authier N, Coudore F, Eschalier A, Fialip J (1999) Pain re-lated behavior during vincristine-induced neuropathy inrats. NeuroReport 10:965–968

Authier N, Gillet JP, Fialip J, Eschalier A, Coudore F (2003)A new animal model of vincristine-induced nociceptive pe-ripheral neuropathy. NeuroToxicology 24:797–805

Dalziel RG, Bingham S, Sutton D, Grant D, Champion JM, Den-nis SA, Quinn JP, Bountra C, Mark MA (2004) Allody-nia in rats infected with varicella zoster virus – a smallanimal model for post-herpetic neuralgia. Brain Res Rev46:234–242

Falinower S, Willer JC, Junien JL, Le Bars D (1994) A C-fiberreflex modulated by heterotopic noxious somatic stimuli inthe rat. J Neurophysiol 72:194–213

Lynch III JJ, Wade CL, Mikusa JP, Decker MW, Honore P(2005) ABT-594 (a nicotinic acetylcholine agonist): anti-allodynia in a rat chemotherapy-induced pain model. Eur JPharmacol 509:43–48

Marchand F, Alloui A, Pelissier T, Hernández A, Authier N, Al-varez P, Eschalier A, Ardid D (2003) Evidence of an an-tihyperalgesic effect of venlafaxine in vincristine-inducedneuropathy in rats. Brain Res 980:117–120

Mestre C, Hernandez A, Eschalier A, Pelissier T (1997) Effectsof clomipramine and desimipramine on a C-fiber reflex inrats. Eur J Pharmacol 335:1–8

Polomano RC, Mannes AJ, Clark US, Bennett GJ (2001)A painful peripheral neuropathy in the rat produced by thechemotherapeutic drug, paclitaxel. Pain 94:293–304

Windebank AJ (1999) Chemotherapeutic neuropathy. Curr OpinNeurol 12:565–571

H.1.2.11.7Trigeminal Neuropathic Pain Model

PURPOSE AND RATIONALETrigeminal neuralgia is an extreme form of neuro-pathic pain. Although the pathophysiology of the dis-order is uncertain, vascular compression of the trigem-inal root resulting in damage to primary afferent neu-rons is thought to play a major role in the generationof pain. Idänpään-Heikkilä and Guilbaud (1999) useda rat model of trigeminal neuropathic pain, devel-oped by Gregg (1973), Jacquin and Zeigler (1983),Vos and Maciewicz (1991), Vos et al. (1994), and Vosand Strassman (1995), where the neuropathy is pro-duced by a chronic constriction injury of the infra-orbital branch of the trigeminal nerve, and studied theeffects of various drugs on this purely sensory modelof neuropathic pain.

PROCEDUREMale Sprague Dawley rats weighing 175–200 g wereused. The head of the rat, which was anesthetizedwith sodium pentobarbital 50 mg/kg i.p. and treatedwith 0.4 mg/kg atropine i.p., was fixed in a stereotaxicframe. A mid-line scalp incision was made exposingskull and nasal bone. To expose the intra-orbital partof the left infra-orbital nerve, the edge of the orbit,formed by the maxillary, frontal, lacrimal and zygo-

matic bones, was dissected free. To give access to theinfra-orbital nerve, the orbital contents were gently de-flected with a cotton-tipped wooden rod. The infra-orbital nerve was dissected free at its most rostral ex-tent of the orbital cavity, just caudal to the infra-orbitalforamen. Two chromic catgut (5–0) ligatures (2 mmapart) were loosely tied around the infra-orbital nerve.The ligatures reduced the diameter of the nerve by justa noticeable amount and retarded, but did not interrupt,the epineural circulation. The scalp incision was closedwith silk sutures.

Before the first actual stimulation session, the ratswere allowed to adapt to the observation cage and tothe testing environment. During this period, the exper-imenter reached slowly into the cage to touch the wallswith a plastic rod, similar to the ones on which the vonFrey filaments were mounted.

For mechanical stimulation, a graded series of tenvon Frey filaments with a bending force of between0.217 and 12.5 g was used. The stimuli were appliedwithin the infra-orbital nerve territory, near the cen-ter of the vibrissal pad, on the hairy skin surround-ing the mystacial vibrissae. The complete series ofvon Frey hair intensities was presented in an ascend-ing series and either a brisk withdrawal of the head oran attack/escape reaction was considered as the me-chanical threshold. Local injection of a local anes-thetic (articaine) into the rostral orbital cavity of thelesioned side, in close proximity to the ligated infra-orbital nerve, increased the mechanical threshold tothe upper level. The duration of the effect was dose-dependent.

EVALUATIONData are expressed as means ±SEM. The non-parametric Kruskal–Wallis one-way ANOVA was usedand comparisons between the groups were performedusing the Mann-Whitney U-test.

MODIFICATIONS OF THE METHODChristensen et al. (2001) studied the effect ofgabapentin and lamotrigine on mechanical allodynia-like behavior in the rat model of trigeminal neuro-pathic pain.

Deseure et al. (2002) studied the effects of acute i.p.injections of 5-HT1A receptor agonists on mechanicalallodynia in a rat model of trigeminal pain.

Cutrer and Moskowitz (1996) studied the actions ofvalproate and neurosteroids in a guinea pig model oftrigeminal pain. Hartley guinea pigs were pretreatedwith valproate or allopregnanolone 30 min prior to ac-tivation of trigeminal afferent fibers via intracisternal


injection of capsaicin. The effects were examined on c-fos expression within the trigeminal nucleus caudalis.

REFERENCES AND FURTHER READINGChristensen D, Gautron M, Guilbaud G, Kayser V (2001) Effect

of gabapentin and lamotrigine on mechanical allodynia-likebehavior in a rat model of trigeminal neuropathic pain. Pain93:147–153

Cutrer FM, Moskowitz MA (1996) The actions of valproateand neurosteroids in a model of trigeminal pain. Headache36:579–585

Deseure K, Koek W, Colpaerte FC, Adriansen H (2002) The 5-HT1A receptor agonist F 13640 attenuates mechanical al-lodynia in a rat model of trigeminal pain. Eur J Pharmacol456:51–57

Gregg JM (1973) A surgical approach to the ophthalmic-maxil-lary nerve trunks in the rat. J Dent Res 52:392–395

Idänpään-Heikkilä JJ, Guilbaud G (1999) Pharmacological stud-ies on a rat model of trigeminal neuropathic pain: ba-clofen, but not carbamazepine, morphine or tricyclic an-tidepressants, attenuates the allodynia-like behaviour. Pain79:281–290

Jacquin MF, Zeigler HP (1983) Trigeminal orosensation and in-gestive behavior in the rat. Behav Neurosci 97:62–97

Vos B, Maciewicz R (1991) Behavioral changes following lig-ation of the infraorbital nerve in rat: an animal model oftrigeminal neuropathic pain. In: JM Besso, Giulbaud G(eds) Lesions of primary afferent fibers as a tool for thestudy of clinical pain. Elsevier, Amsterdam, pp 147–158

Vos BP, Strassman AM (1995) Fos expression in the medullarydorsal horn of the rat after chronic constriction injury to theinfraorbital nerve. J Comp Neurol 357:362–375

Vos BP, Strassman AM, Maciewicz RJ (1994) Behavioral ev-idence of trigeminal neuropathic pain following chronicconstriction injury to the rat’s infraorbital nerve. J Neurosci14:2708–2723

H.1.2.11.8Migraine Model in Cats

PURPOSE AND RATIONALEIn order to simulate pain experienced by humans in mi-graine attacks, Storer and Goadsby (1997) developeda model of craniovascular pain in cats by stimulat-ing the superior sagittal sinus and monitoring trigem-inal neuronal activity using electrophysiological tech-niques.

PROCEDUREAdult cats were anesthetized with α-chloralose(60 mg/kg i.p.), paralyzed (gallamine 6 mg/kg i.v.) andventilated. The superior sagittal sinus was accessedand isolated for electrical stimulation by a mid-line cir-cular craniotomy. The region of the dorsal surface ofC2 spinal cord was exposed by a laminectomy and anelectrode placed for recording evoked activity by si-nus stimulation and spontaneous activity of the samecells. Signals were amplified and monitored on-line.Cells were recorded that were activated by stimula-

tion of the sinus and were also spontaneously acti-vated. Cells fired with latencies consistent with Aδ andC fibers, generally firing three or four times per stim-ulus (0.3 Hz, 250 µs duration, 100 V) delivered to thesinus. Both evoked and spontaneous firing could beinhibited by iontophoretic application of serotonin (5-HT)1B/1D agonists.

EVALUATIONThe suppression or activation of cell firing was deter-mined from both peri-stimulus and post-stimulus his-tograms using the criteria of a shift of >30% from base-line. Drug comparisons were made using the Kruskal-Wallis one-way analysis of variance assessing signifi-cance at the P < 0.05 level.

MODIFICATIONS OF THE METHODUsing the same method, Goadsby et al. (2002) studiedthe inhibition of trigeminovascular nociceptive trans-mission by adenosine A1 receptor agonists.

REFERENCES AND FURTHER READINGGoadsby PJ, Hoskin KL, Storer RJ, Edvinsson L, Connor HE

(2002) Adenosine A1 receptor agonists inhibit trigemino-vascular nociceptive transmission. Brain 125:1392–1401

Storer RJ, Goadsby PJ (1997) Microiontophoretic applicationof serotonin (5-HT)1B/1D agonists inhibits trigeminal cellfiring in the cat. Brain 120:2171–2177

H.1.3Side Effects of Central Analgesic Drugs

See Chap. I.J.

H.2Peripheral Analgesic Activity


The differentiation between central and peripheralanalgesic drugs is nowadays of more or less his-torical value. Most of the so called peripheral anal-gesics possess anti-inflammatory properties and insome cases also antipyretic activity besides analge-sia. For many of them the mode of action has beenelucidated as an inhibition of cyclooxygenase in theprostaglandin pathway. Nevertheless, new peripheralanalgesics have to be tested not only for their in vitroactivity on cyclooxygenase but also for their in vivoactivity.

The most commonly used methods for measur-ing peripheral analgesic activity are the writhing tests

H.2 · Peripheral Analgesic Activity 1031

in mice (various modifications) and the RANDALL-SELITTO-test in rats.

H.2.0.2Writhing Tests

PURPOSE AND RATIONALEPain is induced by injection of irritants into the peri-toneal cavity of mice. The animals react with a char-acteristic stretching behavior which is called writhing.The test is suitable to detect analgesic activity althoughsome psychoactive agents also show activity. An irri-tating agent such as phenylquinone or acetic acid isinjected intraperitoneally to mice and the stretching re-action is evaluated. The reaction is not specific for theirritant.

PROCEDUREMice of either sex with a weight between 20 and 25 gare used. Phenylquinone in a concentration of 0.02%is suspended in a 1% suspension of carboxymethylcel-lulose. An aliquot of 0.25 ml of this suspension is in-jected intraperitoneally. Groups of 6 animals are usedfor controls and treated mice. Preferably, two groupsof 6 mice are used as controls. Test animals are ad-ministered the drug or the standard at various pretreat-ment times prior to phenylquinone administration. Themice are placed individually into glass beakers and fivemin are allowed to elapse. The mice are then observedfor a period of ten min and the number of writhesis recorded for each animal. For scoring purposes,a writhe is indicated by stretching of the abdomen withsimultaneous stretching of at least one hind limb. Theformula for computing percent inhibition is: averagewrithes in the control group minus writhes in the druggroup divided by writhes in the control group times100%. The time period with the greatest percent of in-hibition is considered the peak time. A dose range isreserved for interesting compounds or those which in-hibit writhing more than 70%. Compounds with lessthan 70% inhibition are considered to have minimalactivity.

EVALUATIONA dose range is run in the same fashion as the time re-sponse except 8 animals/ group are tested at the peaktime of drug activity. Four drug groups and a vehiclecontrol group are employed. Animals are dosed andtested in a randomized manner. An estimated ED50is calculated. Doses of 1.0 mg/kg p.o. indomethacin,30 mg/kg p.o. acetylsalicylic acid, 40 mg/kg p.o. ami-dopyrine and 80 mg/kg p.o. phenacetin have beenfound to be ED50 values.

CRITICAL ASSESSMENT OF THE TESTIn this test both central and peripheral analgesics aredetected. The test, therefore, has been used by manyinvestigators and can be recommended as a simplescreening method. However, it has to be mentionedthat other drugs such as clonidine and haloperidol alsoshow a pronounced activity in this test. Because of thelack of specificity, caution is required in interpretingthe results, until other tests have been performed. Nev-ertheless, a good relationship exists between the poten-cies of analgesics in writhing assays and their clinicalpotencies.

MODIFICATIONS OF THE METHODInstead of a phenylquinone suspension, 0.1 ml ofa 0.6% solution of acetic acid is injected intraperi-toneally to mice with an weight between 18 and 25 g(Koster et al. 1959; Taber et al. 1969). The response issimilar to that after phenylquinone. Some authors haveused this method together with observation of changesin capillary permeability in order to distinguish be-tween narcotic and non-narcotic analgesics (Whittle1964).

Eckhardt et al. (1958), Collier et al. (1968), Louxet al. (1978) showed that several substances are able toelicit the writhing response. For example, Amanumaet al. (1984) as well as Nolan et al. (1990) used as irri-tant intraperitoneal injections of acetylcholine.

Emele and Shanaman (1963), Burns et al. (1968)proposed bradykinin being a endogenous transmitterof pain as irritant.

Sancillo et al. (1987) induced abdominal constric-tion in mice by intraperitoneal injection of 31.6 µg/kgof prostaglandin E1.

Bhalla and Bhargava (1980) described a method forassessing aspirin-like activity using aconitine to inducewrithing.

Adachi (1994) described a device for automaticmeasurement of writhing in mice.

Analgesic effects of non-acidic non-steroidal anti-inflammatory drugs in the acetic acid writhing test af-ter intracisternal administration have been found byNakamura et al. (1986).

The writhing phenomenon can also be observedin rats (Fukawa et al. 1980). The writhing re-sponses were induced by intraperitoneal injectionof 4% sodium chloride solution. Narcotic and non-narcotic analgesics, antipyretic and nonsteroidal anti-inflammatory drugs were effectively evaluated at rel-atively low doses. Methamphetamine also showed ananalgesic action. VonVoigtlander and Lewis (1982,1983) induced writhing in rats by injection of


7 ml air or 6% aqueous saline into the peritoneal cav-ity.

Ethacrinic acid-induced writhing response in ratswas used by Björkman et al. (1992).

Schweizer et al. (1988) described a photoelectronicmotility monitoring apparatus to measure automati-cally the writhing movements. A good correlation wasfound between ED50 values after oral administrationin mice and the clinically effective oral doses in man.

Heapy et al. (1993) induced the abdominal constric-tion response in mice by intraperitoneally injecting0.4 ml of either 0.25% acetic acid, 7.5 mg/ml kaolinsuspension, 2.4 mg/ml zymosan solution, or 25 µg/mlbradykinin solution.

REFERENCES AND FURTHER READINGAdachi KI (1994) A device for automatic measurement of

writhing and its application to the assessment of analgesicagents. J Pharmacol Toxicol Meth 32:79–84

Amanuma F, Wakaumi C, Tanaka M, Muramatsu M, Ai-hara H (1984) The analgesic effects of non-steroidal anti-inflammatory drugs on acetylcholine-induced writhing inmice. Folia Pharmacol Japon 84:543–551

Bhalla TN, Bhargava KP (1980) Aconitine-induced writhingas a method for assessing Aspirin-like analgesic activity.J Pharmacol Meth 3:9–14

Björkman RL, Hedner T, Hallman KM, Henning M, Hedner J(1992) Localization of central antinociceptive effects of di-clofenac in the rat. Brain Res 590:66–73

Blumberg H, Wolf PS, Dayton HB (1965) Use of writhing testfor evaluating activity of narcotic antagonists. Proc SocExp Biol Med 118:763–766

Burns RBP, Alioto NJ, Hurley KE (1968) Modification of thebradykinin-induced writhing test for analgesia. Arch IntPharmacodyn 175:41–55

Carey F, Haworth D, Edmonds AE, Forder RA (1988) Sim-ple procedure for measuring the pharmacodynamics andanalgesic potential of lipoxygenase inhibitors. J PharmacolMeth 20:347–356

Chernov HI, Wilson DE, Fowler F, Plummer AJ (1967) Non-specificity of the mouse writhing test. Arch Int Pharmaco-dyn 167:171–178

Collier HOJ, Dinneen LC, Johnson CA, Schneider C (1968)The abdominal constriction response and its suppressionby analgesic drugs in the mouse. Br J Pharmac Chemother32:295–310

Eckhardt ET, Cheplovitz F, Lipo M, Govier WM (1958) Etiol-ogy of chemically induced writhing in mouse and rat. ProcSoc Exp Biol Med 98:186–188

Emele JF, Shanaman J (1963) Bradykinin writhing: A methodfor measuring analgesia. Proc Soc Exp Biol Med114:680–682

Fukawa K, Kawano O, Hibi M, Misaki M, Ohba S, HatanakaY (1980) A method for evaluating analgesic agents in rats.J Pharmacol Meth 4:251–259

Heapy CG, Shaw JS, Farmer SC (1993) Differential sensitivityof antinociceptive assays to the bradykinin antagonist Hoe140. Br J Pharmacol 108:209–213

Hendershot LC, Forsaith J (1959) Antagonism of the fre-quency of phenylquinone-induced writhing in the mouseby weak analgesics and non-analgesics. J Pharmacol ExpTher 125:237–240

Kokka N, Fairhurst AS (1977) Naloxone enhancement of aceticacid-induced writhing in rats. Life Sci 21:975–980

Koster R, Anderson M, de Beer EJ (1959) Acetic acid for anal-gesic screening. Fed Proc 18:412

Loux JJ, Smith S, Salem H (1978) Comparative analgesic testingof various compounds in mice using writhing techniques.Arzneim Forsch/Drug Res 28:1644–1677

Nakamura H, Shimoda A, Ishii K, Kadokawa T (1986) Centraland peripheral analgesic action of non-acidic non-steroidalanti-inflammatory drugs in mice and rats. Arch Int Pharma-codyn 282:16–25

Nolan JC, Osman MA, Cheng LK, Sancilio LF (1990) Brom-fenac, a new nonsteroidal anti-inflammatory drug: Rela-tionship between the anti-inflammatory and analgesic ac-tivity and plasma drug levels in rodents. J Pharm Exp Ther254:104–108

Okun R, Liddon SC, Lasagna L (1963) The effects of aggre-gation, electric shock, and adrenergic blocking drugs oninhibition of the “writhing syndrome”. J Pharm Exp Ther139:107–109

Rae GA, Souza RLN, Takahashi RN (1986) Methylnalor-phinium fails to reverse naloxone-sensitive stress-inducedanalgesia in mice. Pharmacol Biochem Behav 24:829–832

Sancillo LF, Nolan JC, Wagner LE, Ward JW (1987) Theanalgesic and antiinflammatory activity and pharmaco-logic properties of Bromfenac. Arzneim Forsch/Drug Res37:513–519

Schweizer A, Brom R, Scherrer H (1988) Combined automaticwrithing/motility test for testing analgesics. Agents Actions23:29–31

Siegmund E, Cadmus R, Lu G (1957) A method for evaluatingboth non-narcotic and narcotic analgesics. Proc Soc ExpBiol Med 95:729

Taber RI, Greenhouse DD, Rendell JK, Irwin S (1969) Ago-nist and antagonist interactions of opioids on acetic acid-induced abdominal stretching in mice. J Pharm Exp Ther169:29–38

VonVoigtlander PF, Lewis RA (1982) Air-induced writhing:a rapid broad spectrum assay for analgesics. Drug Dev Res2:577–581

VonVoigtlander PF, Lewis RA (1983) A withdrawal hyperalge-sia test for physical dependence: evaluation of μ and mixedpartial opioid agonists. J Pharmacol Meth 10:277–282

Whittle BA (1964) The use of changes in capillary permeabilityin mice to distinguish between narcotic and non narcoticanalgesics. Br J Pharmacol 22:246–253

H.2.0.3Pain in Inflamed Tissue (RANDALL-SELITTO-Test)

PURPOSE AND RATIONALEThis method for measuring analgesic activity is basedon the principle that inflammation increases the sen-sitivity to pain and that this sensitivity is susceptibleto modification by analgesics. Inflammation decreasesthe pain reaction threshold and this low pain reac-tion threshold is readily elevated by non-narcotic anal-gesics of the salicylate-amidopyrine type as well as bythe narcotic analgesics. Brewers yeast has been used asan inducer for inflammation which increases pain afterpressure.


PROCEDUREGroups of male Wistar rats (130 to 175 g) are used.Only for oral testing the animals are starved 18 to24 h prior to administration. Otherwise, the route ofadministration can be intraperitoneal or subcutaneous.To induce inflammation, 0.1 ml of a 20% suspensionof Brewer’s yeast in distilled water is injected subcuta-neously into the plantar surface of the left hind paw ofthe rat. Three hours later, pressure is applied througha tip to the plantar surface of the rat’s foot at a constantrate by a special apparatus to the point at which the an-imal struggles, squeals or attempts to bite. The appa-ratus being used has been modified by various authorssuch as using the Analgy Meter (Ugo Basile, Appara-tus for Biological Research, Milan, Italy). Each animalis tested for its control pain threshold. Any animal witha control pain threshold greater than 80 g is eliminatedand replaced.

For a time response, groups of at least 7 animals areused, four groups for the agent to be tested and one forthe vehicle control. The tests are done at 15 min inter-vals after subcutaneous administration and at 30 minintervals after oral administration for any change inpain threshold. The interval of time which indicatesthe greatest increase in pain threshold is regarded asthe peak time.

A dose range is obtained in the same manner as thetime response. The drug to be tested is administered ina randomized manner. The pain threshold is recordedat time zero and again at the determined peak time.

EVALUATIONThe mean applied force is determined for each time in-terval tested. The percentage increase in pain thresholdis calculated by subtracting the applied force of the ve-hicle control from the applied force of the drug groupwhich is divided by the applied force of the vehiclecontrol in order to give the percentage of increase inpain threshold of the drug group. Doses of 50 mg/kgs.c. Na salicylate, 50 mg/kg amidopyrine, 3 mg/ kg s.c.morphine, 12.5 mg/kg s.c. codeine or pethidine havebeen found to be effective.

CRITICAL ASSESSMENT OF THE METHODThe method originally described by RANDALL andSELITTO has been used by many investigators andhas been proven to detect central analgesics as wellas peripheral analgesics. Peripherally acting analgesicssuch as the nonsteroidal anti-inflammatory drugs in-crease only the threshold of the inflamed paw, whereasopiate analgesics increase also the threshold of the in-tact paw (Dubinsky et al. 1987). In most modifica-

tions, the assay has a shallow dose-response curve.Nevertheless, the ED50 values of nonsteroidal anti-inflammatory drugs in this test showed a good corre-lation with human doses (Romer 1980).

MODIFICATIONS OF THE METHODThe test has been modified by various authors. In someinstances the pressure on the inflamed paw has beenomitted. Instead the animals were allowed to walk ona metal grid. The gait of the animals is assessed by anobserver using a scoring system:

• 0 = three-legged gait• 0.5 = marked limping• 1 = normal gait.

The scores are transformed into percent analgesia.Other noxious stimuli were used to induce inflam-

mation and hyperalgesia, such as carrageenin (Win-ter et al. 1962), Freund’s adjuvant or prostaglandin E2(Ferreira et al. 1978a).

Vinegar et al. (1990) injected 0.1 ml of 0.25% solu-tion of trypsin into the subplantar region and appliedthe load force 60 min later. They found a biphasic hy-peralgesia and relatively low ED50 values for centraland peripheral analgesics.

Technically, the method has been improved by sev-eral authors, such as Takesue et al. (1969).

Chipkin et al. (1983) modified the test by decreas-ing the rate of acceleration of the noxious stimulus(mechanical pressure) on the inflamed paw from 20 to12.5 mm Hg/s and an extension of the cut-off timefrom 15 to 60 s. This modification is claimed to dis-criminate analgesics active against mild to severe clin-ical pain (narcotic-like) from those only useful againstmild to moderate pain (non-narcotic-like).

Randall-Selitto analgesy meters are commerciallyavailable (e. g., IITC Life Science, Woodland Hills,CA, USA).

Central and peripheral analgesic action of aspirin-like drugs has been studied with a modification of theRandall-Selitto method applying constant pressure tothe rat’s paw by Ferreira et al. (1978b).

A modification of an analgesia meter for paw pres-sure antinociceptive testing in neonatal rats was de-scribed by Kitchen (1984).

Learning and retention has been tested in rats byGreindl and Preat (1976) inducing pain by a lightquantifiable pressure applied to the normal hind paw.

Hargreaves et al. (1988) described a sensitivemethod for measuring thermal nociception in cuta-neous hyperalgesia in rats. One paw was injected with


0.1 ml carrageenan solution, the other paw with saline.The rats were placed in chambers with glass floor andradiant heat was directed to the paws. A photoelec-tric cell detected the light reflected from the paw andturned off the radiant heat when paw movement inter-rupted the reflected light.

Perkins et al. (1993) described hyperalgesia after in-jection of 100 µl of Freund’s adjuvant into the kneeof anesthetized rats. After 64–70 h the animal wasplaced with each hind paw on a pressure transducerand a downward force was exerted until the uninjectedleg was bearing 100 g. At this point animals were lesstolerant to a load on the injected leg, indicating a hy-peralgesic response.

Davis et al. (1996) induced mechanical hyperalge-sia by injection of substance P and capsaicin in the ratknee joint and measured the download force toleratedby the injected leg.

Ferreira et al. (1993a, b) induced hyperalgesia byintraplantar injection in the hindpaw of rats of vari-ous agents, e. g., bradykinin, carrageenin, LPS, PGE2,dopamine, TNFα, IL-1β, IL-6 and IL-8. A constantpressure of 20 mm Hg was applied to the hind pawsand discontinued when the rats presented a typicalfreezing reaction.

Subplantar injection of 0.1 µg of serotonin in the ratresults in a brief period (up to 20 min) of increased painsensitivity to an applied force (hyperalgesia) whichprecedes a longer period of decreased pain sensitiv-ity (hypoalgesia). Vinegar et al. (1989) used this phe-nomenon for pharmacologic characterization of the al-gesic response.

Similarly, a biphasic algesic behavior after subplan-tar injection of 250 µg of trypsin was described byVinegar et al. (1990)

Courteix et al. (1994) proposed the Randall-Selittopaw pressure test in rats with streptozocin-induced di-abetes as a model of chronic pain with signs of hyper-algesia and allodynia that may reflect signs observedin diabetic humans.

Amann et al. (1955, 1996) evaluated local edemaand effects on thermal nociceptive threshold after in-traplantar injection of nerve growth factor into the rathind paw and studied the effect of a 5-lipoxygenaseinhibitor in this test.

Zhou et al. (1996) tested the effects of peripheral ad-ministration of NMDA, AMPA or KA on pain behav-ior in rats. A 28-gauge needle was inserted in the skinof rats proximal to the footpads and advanced about1 cm so that the tip reached the base of the third toe.A bolus of 20 µl containing concentrations between 1and 10,000 µM of KA, NMDA or AMPA. For behav-

ioral testing, each animal was placed in a Plexiglasbox on a wire mesh screen. Mechanical stimuli wereapplied using four von Frey filaments with differentbending forces. Each von Frey filament was applied10 times to the skin on the base of the third toe. Thepaw withdrawal was rated as scores allowing dose-response curves for the hyperalgesic effects of excita-tory amino acids. Furthermore, using the highest con-centration of the stimulant, effects of antagonists weretested.

REFERENCES AND FURTHER READINGAmann R, Schuligoi R, Herzeg G, Donnerer J (1955) Intraplan-

tar injection of nerve growth factor into the rat hind paw:local edema and effects on thermal nociceptive threshold.Pain 64:323–329

Amann R, Schuligoi R, Lanz I, Peskar BA (1996) Effect ofa 5-lipoxygenase inhibitor on nerve growth factor-inducedthermal hyperalgesia in the rat. Eur J Pharmacol 306:89–91

Chipkin RE, Latranyi MB, Iorio LC, Barnett A (1983) Determi-nation of analgesic drug efficacies by modification of theRandall and Selitto rat yeast paw test. J Pharmacol Meth10:223–229

Courteix C, Bardin M, Chantelauze C, Lavarenne L, Eschalier A(1994) Study of the sensitivity of the diabetes-induced painmodel in rats to a range of analgesics. Pain 57:153–160

Davis AJ, Perkins MN (1996) Substance P and capsaicin-in-duced mechanical hyperalgesia in the rat knee joint; theinvolvement of bradykinin B1 and B2 receptors. Br J Phar-macol 118:2206–2212

Dubinsky B, Gebre-Mariam S, Capetola RJ, Rosenthale ME(1987) The analgesic drugs: Human therapeutic correlatesof their potency in laboratory animals of hyperalgesia.Agents Actions 20:50–60

Ferreira SH, Nakamura M, DeAbreu Castro MS (1978a) Thehyperalgesic effects of prostacyclin and prostaglandin E2.Prostaglandins 16:31–37

Ferreira SH, Lorenzetti BB, Corrêa FMA (1978b) Central andperipheral antialgesic action of aspirin-like drugs. Eur JPharmacol 53:39–48

Ferreira SH, Lorenzetti BB, Poole S (1993a) Bradykinin initiatescytokine-mediated inflammatory hyperalgesia. Br J Phar-macol 110:1227–1231

Ferreira SH, Lorenzetti BB, Cunha FQ, Poole S (1993b)Bradykinin release of TNF-α plays a key role in the de-velopment of inflammatory hyperalgesia. Agents Actions38:C7–C9

Greindl MG, Preat S (1976) A new model of active avoidanceconditioning adequate for pharmacological studies. ArchInt Pharmacodyn 223:168–170

Hargreaves K, Dubner R, Brown F, Flores C, Joris J (1988)A new and sensitive method for measuring thermal noci-ception in cutaneous hyperalgesia. Pain 32:77–88

Kitchen I (1984) Modification of an analgesy meter for paw-pressure antinociceptive testing in neonatal rats. J Pharma-col Meth 12:255–258

Perkins MN, Campell E, Dray A (1993) Antinociceptive activ-ity of the bradykinin B1 and B2 receptor antagonists, des-Arg9, [Leu8]-BK and Hoe 140, in two models of persistenthyperalgesia in rats. Pain 53:191–197

Randall LO, Selitto JJ (1957) A method for measurement ofanalgesic activity on inflamed tissue. Arch Int Pharmaco-dyn 111:409–419


Rios L, Jacob JJC (1982) Inhibition of inflammatory pain bynaloxone and its N-methyl quaternary analogue. Life Sci31:1209–1212

Romer D (1980) Pharmacological evaluation of mild analgesics.Br J Clin Pharmacol 10:247S–251S

Takesue EI, Schaefer W, Jukniewicz E (1969) Modification ofthe Randall-Selitto analgesic apparatus. J Pharm Pharmacol21:788–789

Tanaka K, Shimotori T, Makino S, Aikawa Y, Inaba T, YoshidaC, Takano S (1992) Pharmacological studies of the newanti-inflammatory agent 3-formylamino-7-methylsulfonyl-amino-6-phenoxy-4H-1-benzopyran-4-one. 1st Communi-cation: anti-inflammatory, analgesic and other related prop-erties. Arzneim Forsch/Drug Res 42:935–944

Vinegar R, Truax JF, Selph JL, Johnston PR (1989) Pharmaco-logic characterization of the algesic response to the sub-plantar injection of serotonin in the rat. Eur J Pharmacol164:497–505

Vinegar R, Truax JF, Selph JL, Johnston PR (1990) New anal-gesic assay utilizing trypsin-induced hyperalgesia in thehind limb of the rat. J Pharmacol Meth 23:51–61

Winter CA, Flakater L (1965) Reaction thresholds to pressurein edematous hindpaws of rats and response to analgesicdrugs. J Pharm Exp Ther 150:165–171

Winter CW, Risley EA, Nuss GW (1962) Carrageenin-inducededema in hind paw of the rat as an assay for antiinflamma-tory drugs. Proc Soc Exp Biol Med 111:544–547

Zhou S, Bonasera L, Carlton SM (1996) Peripheral administra-tion of NMDA, AMPA, or KA results in pain behaviors ofrats. NeuroReport 7:895–900

H.2.0.4Mechanical Visceral Pain Model in the Rat

PURPOSE AND RATIONALEAnimal models designed to test the effectiveness ofanalgesic agents against visceral pain typically relyon noxious chemical irritation of the peritoneum, e. g.,acetic acid and phenylquinone induced writhing testsbased on acute inflammation. Ethical constraints pre-vent repeated assessments in a single animal, therebycompounding the difficulty of assessing developmentof tolerance to analgesic agents. To overcome theseconstraints, a model for mechanical visceral pain wasdeveloped based on repeatable and reversible disten-sion of duodenum in the rat (Coburn et al. 1989; deLeoet al. 1989).

PROCEDUREA one-piece balloon catheter is prepared composedof PE 50 tubing with a terminal latex rubber bal-loon which is 7.5 mm long and distensable to holdmore than 1.5 ml fluid. Male Sprague Dawley ratsweighing 175–200 g are anesthetized with N2O andhalothane. The abdomen is shaved and an 2.5 cm in-cision is made transversely just below the left costalmargin. On the greater curvature of the stomach anincision is made 10 to 20 mm above the pylorus

and a purse string is accomplished with 4–0 silkprior to gastrostomy. Through a 2 mm gastrostomythe catheter is introduced and advanced through thepylorus to the first portion of the duodenum (ap-proximately 15–20 mm from the pylorus). The pursestring is tied snugly closing the gastrostomy aroundthe catheter. The catheter is tunneled to the base ofthe scull, externalized and anchored to the dermis witha silicon sleeve and suture. The animals recover fromanesthesia within 5 min. Following a 4–5 days recov-ery period, the duodenal distension volume is deter-mined by the mean threshold that produces writhing(usually 0.5 to 0.7 ml). For the test, the animals arerandomized and administered either saline, the stan-dard (0.1, 0.25, 1, and 10 mg/kg indomethacin i.p.)or the test drug in various doses prior to challeng-ing. The animals are placed in a polypropylene boxand challenged by inflating the balloon with saline, us-ing a 1 ml calibrated syringe, pulsed 5 times over 30 sand then distended for 1 min. Behavioral responses arescored:

• 0 = Normal behavior defined as exploration, escapeattempts and resting

• 1 = Slightly modified behavior defined as cessationof exploration, focusing, wet-dog shake, excessivefacial grooming, teeth chattering and deep breath-ing

• 2 = Mildly to moderately modified behavior de-fined as retching-like activity, hunching, abdomi-nal grooming or nipping and immobility of the hindlimbs (disappears with removal of the stimulus).

• 3 = Severely modified behavior defined as stretching of the hindlimbs, arching and dorsoflection ofthe hind paws.

• 4 = Intensive visceromotor activity defined as repet-itive stretching of the body, extension of the hindlimbs, and pelvis, frequent rotating sideward, i. e.,writhing.

EVALUATIONThe average scores of the groups are plotted on semilog paper and ED50 values are determined by best linefit.

CRITICAL ASSESSMENT OF THE METHODIn the mechanical visceral pain model in the rat, mor-phine and indomethacin have been found to be activebut not other agents involved in prostaglandin inhi-bition, like acetylsalicylic acid and mefenamic acid.Other mechanisms besides those involving the arachi-donic acid cascade have to be investigated.


MODIFICATIONS OF THE METHODNess and Gebhart (1988) used colorectal distensionas a noxious visceral stimulus in awake, unanes-thetized, unrestrained rats. A 7–8 cm flexible latexballoon was inserted intra-anally under ether anes-thesia and kept in position by taping to the baseof the tail. Opening a solenoid gate to a constantpressure air reservoir initiated a 20 s, constant pres-sure stimulus in the descending colon and rectum.Femoral arterial and venous catheters were tunneledsubcutaneously and exteriorized at the back of theneck. Teflon-coated stain-less steel wire electrodeswere stitched into the external oblique musculatureimmediately superior to the inguinal ligament forelectromyographic recordings. Blood pressure andheart frequency increase were proportional to the de-gree of colorectal distension. These effects could bedose-dependently antagonized by morphine and cloni-dine.

Renal pelvis distension with a pressure of 80 cmH2O causes a decline in mean arterial blood pressurein pentobarbital-anesthetized rats. Brasch and Zetler(1982) used this blood pressure response, which disap-pears rapidly after cessation of the distension, to studythe effects of analgesic drugs known to be effective inrenal colic pain in man.

Moss and Sanger (1990) measured falls in dias-tolic blood pressure and intragastric pressure after dis-tension of the duodenum by rapid application of in-traluminal pressure (10–75 cm H2O) in anesthetizedrats. The distension-induced responses were blockedby pretreatment with morphine, an action reversible byinjection of naloxone. Bilateral cervical vagotomy re-duced the distension-evoked fall in intragastric pres-sure but had no effect on the corresponding fall inblood pressure.

REFERENCES AND FURTHER READINGBrasch H, Zetler G (1982) Caerulein and morphine in a model of

visceral pain. Effects on the hypotensive response to renalpelvis distension in the rat. Naunyn-Schmiedeberg’s ArchPharmacol 319:161–167

Colburn RW, Coombs DW, Degnan CC, Rogers LL (1989) Me-chanical visceral pain model: chronic intermittent intestinaldistension in the rat. Physiol Behav 45:191–197

deLeo JA, Colburn RW, Coombs DW, Ellis MA (1989) Thedifferentiation of NSAIDs and prostaglandin action usinga mechanical visceral pain model in the rat. PharmacolBiochem Behav 33:253–255

Moss HE, Sanger GJ (1990) Effects of granisetron, ICS 205–930and ondansetron on the visceral pain reflex induced by duo-denal extension. Br J Pharmacol 100:497–501

Ness TJ, Gebhart FG (1988) colorectal distension as a noxiousvisceral stimulus: physiologic and pharmacologic charac-terization of pseudoaffective reflexes in the rat. Brain Res450:153–169

H.2.0.5Antagonism Against Local Effects of Bradykinin

PURPOSE AND RATIONALEGuzman et al. (1962) and Lim et al. (1964) de-scribed the responses (vocalization, respiratory andblood pressure changes) to intra-arterial injection ofbradykinin and other algesic agents in cats and dogs.Deffenu et al. (1966) and Blane (1968) used thebradykinin-induced effects after intra-arterial injec-tion in rats as an assay for analgesic drugs. Par-avascular sensory nerves which accompany bloodvessels throughout the body to end in unmyeli-nated free-branching terminals close to the capil-laries and venules most likely carry the chemore-ceptors of pain (Lim 1970). Due to rapid enzy-matic degradation, bradykinin is ineffective as nox-ious stimulus after intravenous or oral administra-tion.

PROCEDUREMale Wistar rats weighing 280–320 g are lightly anes-thetized with ether. A polyethylene catheter with an in-ternal diameter of 0.5 mm is inserted centripetally intothe right carotid artery. The catheter is passed throughthe subcutaneous tissues to protrude from the back ofthe animal. One hour after recovery from anesthesia,the first dose of bradykinin is injected into the catheterproducing dextro-rotation of the head, flexing of theforelimb and occasionally squeaking. For each rat theminimum dose of bradykinin is determined necessaryto provoke these effects. The test compounds are ap-plied subcutaneously or intraperitoneally 15 min priorto injection of the threshold dose of bradykinin. Thebradykinin injections are repeated in 5 min intervalsuntil the bradykinin effect reappears. Each rat receivesone drug at one dose level.

EVALUATIONThe criterion for protection is the disappearance of thebradykinin effect after at least 2 consecutive doses ofbradykinin. Using groups of 10 rats for various doselevels, ED50 values are calculated.

CRITICAL ASSESSMENT OF THE METHODNot only narcotic analgesics, but also pyrazolonesand phenacetin or acetylsalicylic acid are active inthis test. In some animals, the bradykinin-induced re-sponse can be diminished after repeated injections,classified as the noxious-adaptable group (Satoh et al.1979).


MODIFICATIONS OF THE METHODHaubrich et al. (1990) tested analgesic activity by theintracarotid bradykinin-induced head/forepaw flexionin the rat. Male Charles River rats (280–320 g) fastedovernight were prepared surgically under light etheranesthesia by insertion of a capped polyethylene can-nula (PE-60) centripetally into the right carotid arteryand then exteriorizing the cannula to a harness on theback, to permit repeated i.a. injections. The rats wereallowed to recover at least 2 h from the surgery, andthen given single i.a. injections of bradykinin (triac-etate salt in 0.2 ml 0.9% NaCl per injection) at 10-minintervals to determine the threshold dose which pro-duced marked dextrorotation of the head and flexionof the right forepaw of each rat. This response waselicited by threshold doses of bradykinin ranging from0.1 to 0.5 µg/injection. After administration of the testdrugs, the response of the threshold dose of bradykininwas determined at 10-min intervals for 1 h and then at20-min intervals during the second hour. ED50 valueswere determined by probit analysis of the maximumpercentage of rats that failed to respond to bradykininat each dose of test drug any time within the 2-h testperiod.

Collier and Lee (1963) described nociceptive re-sponses of guinea pigs to intradermal injections ofbradykinin and kallidin-10.

Vargaftig (1966) measured the effect of non-narcotic analgesics on the hypotension induced byintra-arterial injection of bradykinin in rabbits.

Adachi and Ishii (1979) used the response to injec-tion of bradykinin into the femoral artery of guineapigs for quantitative assessment of analgesic agents.

Griesbacher and Lembeck (1987) and Lembecket al. (1991) used the reflex hypotensive response as anindicator of nociception after injection of bradykinininto the ear artery of anesthetized rabbits. Rabbitswere anesthetized and the blood pressure was recordedfrom the carotid artery. The central artery of one earwas cannulated and the ear was separated from thehead with the exception of the auricular nerve, whichremained connected to the head. The ear was per-fused with Tyrode solution to which acetylcholine andbradykinin were added. The reflex fall in blood pres-sure induced by bradykinin and acetylcholine weremonitored. The effect could be inhibited by bradykininantagonists.

Heapy et al. (1993) tested the effects of thebradykinin antagonist HOE140 on the abdominal con-striction response after intraperitoneal injection ofbradykinin to mice.

Further Methods Used to Study the Role of Bradykininand Bradykinin Antagonists in Inflammation and AlgesiaTeixeira et al. (1993) investigated the mechanisms ofinflammatory response induced by extracts of Schisto-soma mansoni larvae in guinea pig skin. Biomphalariaglabrata snails with patent Schistosoma mansoni in-fections were induced to shed cercariae by exposure tolight and water with a temperature of 31°C. The cer-cariae were concentrated, homogenized and extractsprepared. Purified eosinophils or neutrophils obtainedfrom peritoneal exudates were radiolabeled by incu-bation with 111In chelated to 2-mercaptopyridine-N-oxide. Radiolabeled leukocyte infiltration and edemaformation were measured simultaneously at injectedskin sites. [125I]Human serum albumin was added tothe labeled leukocytes and these were injected i.v. intoanesthetized guinea pigs. After 15 min the extracts ofcercariae were locally injected with/or without the in-hibitors. After 2 h, the animals were sacrificed and theinjected sites were punched out with a 17-mm punch.Serum exudation and leukocyte infiltration were mea-sured by counting the two isotopes. The bradykinin an-tagonist HOE 140 reduced substantially the extract-in-duced inflammation.

Ahluwalia et al. (1994) induced plasma protein ex-travasation in the rat urinary bladder by i.p. injec-tion of cyclophosphamide mediated by capsaicin-sen-sitive primary afferent neurons which could be signifi-cantly inhibited by the bradykinin B2 receptor antago-nist HOE140 and the tachykinin NK1 receptor antago-nist RP67,580.

Davis and Perkins (1994a, b) described a model ofpersistent inflammatory mechanical hyperalgesia us-ing intra-articular injections of bradykinin or cytokinesinto the knee joint of rats.

Lecci et al. (1995) analyzed the local and reflex re-sponses to bradykinin on rat urinary bladder motilityin vivo.

REFERENCES AND FURTHER READINGAdachi KI, Ishii Y (1979) Vocalization response to close-arterial

injection of bradykinin and other algesic agents in guineapigs and its application to quantitative assessment of anal-gesic agents. J Pharm Exp Ther 209:117–124

Ahluwalia A, Maggi CA, Santicioli P, Lecci A, Giuliani S (1994)Characterization of the capsaicin-sensitive component ofcyclophosphamide-induced inflammation in the rat urinarybladder. Br J Pharmacol 111:1017–1022

Beck PW, Handwerker HO (1974) Bradykinin and serotonin ef-fects on various types of cutaneous nerve fibres. PflügersArch 347:209–222

Blane GF (1968) A new laboratory model for evaluating anal-gesic and analgesic-antagonist drugs. In: Soulairac A,


Cahn J, Charpentier J (eds) Pain. Academic Press, London,New York, pp 218–222

Collier HOJ, Lee IR (1963) Nociceptive responses of guinea-pigs to intradermal injections of bradykinin and kallidin-10. Br J Pharmacol 21:155–164

Davis AJ, Perkins MN (1994a) Induction of B1 receptors in vivoin a model of persistent mechanical hyperalgesia in the rat.Neuropharm 33:127–133

Davis AJ, Perkins MN (1994b) Involvement of bradykinin B1and B2 receptor mechanisms in cytokine-induced mechan-ical hyperalgesia in rats. Br J Pharmacol 113:63–68

Deffenu G, Pegrasso L, Lumachi B (1966) The use ofbradykinin-induced effects in rats as an assay for analgesicdrugs. J Pharm Pharmac 18:135

Griesbacher T, Lembeck F (1987) Effect of bradykinin antag-onists on bradykinin-induced plasma extravasation, veno-constriction, prostaglandin E2 release, nociceptor stimula-tion and contraction of the iris sphincter muscle in the rab-bit. Br J Pharmacol 92:333–340

Guzman F, Braun C, Lim RKS (1962) Visceral pain andthe pseudoaffective response to intra-arterial injection ofbradykinin and other algesic agents. Arch Int Pharmacodyn136:353–384

Heapy CG, Shaw JS, Farmer SC (1993) Differential sensitivityof antinociceptive assays to the bradykinin antagonist Hoe140. Br J Pharmacol 108:209–213

Haubrich DR, Ward SJ, Baizman E, Bell MR, Bradford J,Ferrari R, Miller M, Perrone M, Pierson AK, SaelensJK, Luttinger D (1990) Pharmacology of pravodoline:a new analgesic agent. J Pharmacol Exp Ther 255:511–521

Lecci A, Giuliani S, Meine S, Maggi CA (1995) Pharmacologi-cal analysis of the local and reflex responses to bradykininon rat urinary bladder motility in vivo. Br J Pharmacol114:708–714

Lembeck F, Griesbacher T, Eckhardt M, Henke S, Breipohl G,Knolle J (1991) New, long acting, potent bradykinin antag-onists. Br J Pharmacol 102:297–304

Lim RKS (1970) Pain. Annu Rev Physiol 32:269–288Lim RKS, Guzman F (1968) Manifestations of pain in analgesic

evaluation in animals and man. In: Soulairac A, Cahn J,Charpentier J (eds) Pain. Academic Press, London, NewYork, pp 119–152

Lim RKS, Guzman F, Rodgers DW, Goto K, Braun C, Dicker-son GD, Engle RJ (1964) Site of action of narcotic and non-narcotic analgesics determined by blocking bradykinin-evoked visceral pain. Arch Int Pharmacodyn 152:25–58

Satoh M, Kawajiri SI, Yamamoto M, Makino H, Takagi H(1979) Reversal by naloxone of adaptation of rats to nox-ious stimuli. Life Sci 24:685–690

Teixeira MM, Doenhoff MJ, McNeice C, Williams TJ,Hellewell G (1993) Mechanisms of the inflammatory re-sponse induced by extracts of Schistosoma mansoni larvaein guinea pig skin. J Immunol 151:5525–5534

Vargaftig B (1966) Effet des Analgésiques non narcotiquessur l’hypotension due à la Bradykinine. Experientia22:182–183

H.2.0.6Effect of Analgesics on Spinal Neurons

PURPOSE AND RATIONALEThe mode of action of peripheral analgesics is stilla matter of debate. Besides inhibition of the arachi-donic acid derived pathway, activities on the spinal

and central level have been discussed. Schaible andSchmidt (1983a, b, 1985, 1987, 1988) performed elec-trophysiological experiments in anesthetized cats andrats after local mechanical stimulation and after in-duction of acute arthritis of the knee joint. With thismethod Xe et al. (1990a, b), Neugebauer et al. (1994)found evidence for a spinal antinociceptive action ofantipyretic analgesics, such as dipyrone.

PROCEDUREIn cats weighing 2.0–4.0 kg anesthesia is induced byi.m. injection of 15–30 mg/kg ketamine hydrochloridefollowed by i.v. injection of 60 mg/kg α-chloralose.After immobilization with i.v. pancuronium bromidethe cats are artificially ventilated. The skin of the rightthigh is incised from rostral of the inguinal fossa toa point below the medial condyle of the tibia. The ten-don of the sartorius muscle is cut close to its insertionat the capsule of the knee joint. The muscle is removedto expose the medial aspects of the joint and the me-dial articular nerve (MAN). The thigh is rigidly fixedto the mounting table by a threaded bolt fitted throughthe femur so that the lower leg can be flexed and ex-tended in the horizontal plane. The saphenous nerve iscut in the inguinal fossa for recording. Bipolar elec-trodes are inserted at the MAN near the knee for enpassent stimulation of articular afferents. Extracellu-lar recordings from single MAN units in the saphe-nous nerve are performed using platinum wire elec-trodes. According to their conduction velocities unitsare classed as group IV afferents (<2.5 m/s, unmyeli-nated axons) or group III afferents (2.5–20 m/s, thinlymyelinated axons).

For recordings from spinal cord neurons thespinal segments T12–L7 are exposed by laminectomy.The spinal cord is transected at the lower thoracicregion after injection of 0.1 ml of 1% procaine hy-drochloride solution to prevent mechanical activationof axons in the long spinal tracts. The animals arefixed to a rigid frame with spinal and pelvic clamps.A pool is formed by skin flaps and filled with warmparaffin oil. The upper lumbar spinal cord is mountedon a pair of platinum wire stimulating electrodes sur-rounding the whole cord. Ascending tract neurons areidentified by electrical stimulation (Neugebauer andSchaible 1990). Single spinal neurons that can be ex-cited by mechanical stimulation of the knee joint tis-sue are recorded extracellularly using glass-insulatedcarbon filament electrodes. The neurons are either no-ciceptive specific neurons responding only to noxiousmechanical stimuli or wide dynamic range neurons re-


sponding to innocuous stimuli but showing strongestresponses to stimuli of noxious intensity.

Acute arthritis in the right knee joint is inducedseveral hours before recordings are started by inject-ing 0.3–0.5 ml of 4% kaolin suspension and 15–20 minlater 0.3 ml of 2% carrageenan solution. Acute arthritisdevelops within 1–3 h.

Action potentials are displayed on a storage os-cilloscope, amplified, filtered, fed to a window dis-criminator and processed using an interface and a per-sonal computer for construction of peristimulus timehistograms. After a control period of at least 40 minduring which a stable discharge rate of the afferentor spinal cord unit is obtained, the test substancesare administered i.v. in various doses. Effects of thetest substance on ongoing and mechanically evokedactivity (by movements, pressure stimuli) are deter-mined.

EVALUATIONOngoing activity is counted every min. The means andstandard deviations in 10 min periods are calculatedbefore and after drug application. The values after druginjection are calculated as percentage of control val-ues. To calculate the net effects of the different me-chanical stimuli, the number of impulses in the pre-ceding 30 s is subtracted from the total discharges dur-ing the stimulus. The responses to at least 4 stimulibefore drug application are averaged and set to 100%.The responses to the different mechanical stimuli afterdrug administration are expressed as a percentage ofthe controls. Statistical significance is evaluated usingthe t-test for unpaired samples.

MODIFICATIONS OF THE METHODUsing the model of kaolin-induced arthritis in the kneeof rats, Han and Neugebauer (2005) and Han et al.(2005) developed a computerized analysis of audibleand ultrasonic vocalizations of rats as a standardizedmeasure of pain-related behavior.

Several other electrophysiological methods havebeen applied to elucidate the mode of action of non-opioid analgesic agents. Carlsson et al. (1986, 1988),Jurna and Brune (1990) recorded the activity from sin-gle neurons in the dorsomedial part of the ventral nu-cleus of the thalamus in rats. Activity was elicited bysupramaximal stimulation of nociceptive afferents inthe sural nerve. In addition, activity was recorded inascending axons of the spinal cord.

Chapman and Dickenson (1992) studied the spinaland peripheral roles of bradykinin and prostaglandinsin nociceptive processing in the rat by recording C-

fibre activity in the dorsal spinal horn after injection offormalin into the center of the respective field of thetoe of the hind paw.

Dray et al. (1992) described a preparation of theneonatal rat spinal cord with functionally connectedtail maintained in vitro. The preparation was placed ina chamber and the spinal-cord and tail were separatelysuperfused with a physiological salt solution. Periph-eral nociceptive fibers were activated by superfusion ofthe tail with bradykinin, capsaicin or by a superfusateheated to 48–50°C (noxious heat). The activation ofperipheral fibres was assessed by measuring the depo-larization produced in a spinal ventral root.

Malmberg and Yaksh (1992) described a directanalgesic action of NSAIDs through spinal cyclooxy-genase inhibition by blocking the thermal hyperalgesiain rats induced after intrathecal administration of exci-tatory amino acids or substance P.

A simple technique for intrathecal injections bylumbar puncture in unanesthetized mice was describedby Hylden and Wilcox (1980).

Mestre et al. (1994) described a method for per-forming direct intrathecal injections in rats without in-troducing a spinal catheter.

Bahar et al. (1984) performed chronic implantationsof nylon catheters into the subarachnoid space of Wis-tar rats and marmosets and tested the effects of localanesthetics.

McQueen et al. (1991) investigated the effectsof paracetamol and lysine acetylsalicylate on high-threshold mechano-nociceptors by recording neuralactivity from the inflamed ankle joint in anesthetizedrats with mild adjuvant-induced mono-arthritis.

Yamamoto and Yaksh (1992) studied the ef-fects of excitatory amino acid antagonists adminis-tered through chronically implanted lumbar intrathecalcatheters on the thermal hyperesthetic state induced byunilateral partial ligation of the sciatic nerve in rats.

Hashimoto and Fukuda (1990) described a spinalcord injury model produced by spinal cord compres-sion in the rat.

Aanonsen and Wilcox (1987) tested effects ofspinally administered opioids, phencyclidine andsigma agonists on the action of intrathecally adminis-tered NMDA in the tail-flick, hot-plate and biting andscratching nociceptive tests in mice.

Brambilla et al. (1996) demonstrated that in-trathecal administration of AMPA produced a dose-dependent behavioral syndrome in mice characterizedby caudally directed biting, which could be antago-nized by peripheral administration of AMPA-receptorand NMDA-receptor antagonists.


Aanonsen et al. (1990) tested the effect of ion-tophoretically applied excitatory amino acid agonists,such as NMDA, AMPA, quisqualate and kainate, onthe firing rate of rat spinal neurons after peripheralnoxious stimulation.

Cumberbatch et al. (1994) studied the roles of re-ceptors for AMPA in spinal nociceptive and non-nociceptive transmission on dorsal horn wide dynamicrange neurones in anesthetized spinalized rats. Theeffects of systemically administered competitive andnon-competitive AMPA antagonists were examined onresponses to peripheral noxious heat and non-noxioustap stimuli as well as to iontophoretic AMPA andNMDA.

With this technique, Chizh et al. (1994) studied theeffects of intravenous administration of AMPA antag-onists to iontophoretically applied excitatory aminoacids.

Watkins et al. (1994) induced hyperalgesia in ratsby intraperitoneally administered lipopolysaccharidesas measured by radiation heat tail flick in rats. Intrathe-cal catheters were implanted into the subdural spacesurrounding the spinal cord to test the involvement ofexcitatory amino acids, substance P, CCK and opioidsassessing the effects of antagonists.

Mjellem et al. (1993) produced a behavioural syn-drome of caudally directed biting in mice by intrathe-cal injection of either NMDA or AMPA.

REFERENCES AND FURTHER READINGAanonsen LM, Wilcox GL (1987) Nociceptive action of excita-

tory amino acids in the mouse: effects of spinally adminis-tered opioids, phencyclidine and sigma agonists. J Pharma-col Exp Ther 243:9–19

Aanonsen LM, Lei S, Wilcox GL (1990) Excitatory amino acidreceptors and nociceptive neurotransmission in rat spinalcord. Pain 41:309–321

Bahar M, Nunn JF, Rosen M, Flecknell P (1984) Differentialsensory and motor blockade after spinal cocaine in the ratand marmoset. Eur J Anaesthesiol 1:31–36

Brambilla A, Prudentio A, Grippa N, Borsini F (1996) Pharma-cological characterization of AMPA-induced biting behav-ior in mice. Eur J Pharmacol 305:115–117

Carlsson KH, Helmreich J, Jurna I (1986) Comparison ofcentral antinociceptive and analgesic effects of the pyra-zolone derivatives, metamizol (Dipyrone) and aminophen-zone (“Pyramidon”). Schmerz – Pain – Douleur 3:93–100

Carlsson KH, Monzel W, Jurna I (1988) Depression of morphineand the non-opioid analgesic agents, metamizol (dipyrone),lysine acetyl salicylate, and paracetamol, of activity in ratthalamus neurons evoked by electrical stimulation of noci-ceptive afferents. Pain 32:313–326

Chapman V, Dickenson AH (1992) The spinal and peripheralroles of bradykinin and prostaglandins in nociceptive pro-cessing in the rat. Eur J Pharmacol 219:427–433

Chizh BA, Cumberbatch MJ, Headley PM (1994) A comparisonof intravenous NBQX and GYKI 53655 as AMPA antago-nists in the rat spinal cord. Br J Pharmacol 112:843–846

Cumberbatch MJ, Chizh BA, Headley PM (1994) AMPA re-ceptors have an equal role in spinal nociceptive and non-nociceptive transmission. NeuroReport 5:877–880

Dray A, Patel IA, Perkin MN, Rueff A (1992) Bradykinin-in-duced activation of nociceptors: receptor and mechanisticstudies on the neonatal rat spinal cord-tail preparation invitro. Br J Pharmacol 107:1129–1134

Han JS, Neugebauer V (2005) mGluR1 and mGluR5 antago-nists in the amygdala inhibit different components of audi-ble and ultrasonic vocalization in a model of arthritic pain.Pain 113:211–222

Han JS, Bird GC, Li W, Jones J, Neugebauer V (2005) Com-puterized analysis of audible and ultrasonic vocalizationsof rats as a standardized measure of pain-related behavior.J Neurosci Methods 141:261–269

Hashimoto T, Fukuda N (1990) New spinal cord injury modelproduced by spinal cord compression in the rat. J Pharma-col Meth 23:203–212

He X, Neugebauer V, Schaible HG, Schmidt RF (1990a) Ef-fects of antipyretic analgesics on pain-related neurons ofthe spinal cord. In: Brune K, Santoso B (eds) AntipyreticAnalgesics: New Insights. Birkhäuser Verlag, Basel,pp 13–23

He X, Neugebauer V, Schaible HG, Schmidt RF (1990b) Newaspects of the mode of action of dipyrone. In: Brune K (ed)New Pharmacological and Epidemiological Data in Anal-gesics Research. Birkhäuser Verlag, Basel, pp 9–18

Hylden JLK, Wilcox GL (1980) Intrathecal morphine in mice:a new technique. Eur J Pharmacol 67:313–316

Jurna I, Brune K (1990) Central effect of the non-steroidanti-inflammatory agents, indomethacin, ibuprofen, and di-clofenac, determined in C fibre-evoked activity in singleneurons of rat thalamus. Pain 41:71–80

Malmberg AB, Yaksh TL (1992) Hyperalgesia mediated byspinal glutamate or substance P receptor blocked by spinalcyclo-oxygenase inhibition. Science 257:1276–1279

McQueen DS, Iggo A, Birrell GJ, Grubb BD (1991) Effectsof paracetamol and aspirin on neural activity of jointmechanociceptors in adjuvant arthritis. Br J Pharmacol104:178–182

Mestre C, Pélissier T, Fialip J, Wilcox G, Eschalier A (1994)A method to perform direct transcutaneous intrathecal in-jections in rats. J Pharmacol Toxicol Meth 32:197–200

Mjellem N, Lund A, Hole K (1993) Different functions of spinal5-HT1A and 5-HT2 receptor subtypes in modulating be-haviour induced by excitatory amino acid receptor agonistsin mice. Brain Res 626:78–82

Neugebauer V, Schaible HG (1990) Evidence for a central com-ponent in the sensitization of spinal neurons with joint inputduring development of acute arthritis in cat’s knee. J Neu-rophysiol 64:299–311

Neugebauer V, Schaible HG, He X, Lücke T, Gündlich P,Schmidt RF (1994) Electrophysiological evidence fora spinal anti-nociceptive action of dipyrone. Agents Ac-tions 41:62–70

Schaible HG, Schmidt RF (1983a) Responses of fine medial ar-ticular nerve afferents to passive movements of knee joint.J Neurophysiol 49:1118–1126

Schaible HG, Schmidt RF (1983b) Activation of groups III andIV sensory units in medial articular nerve by local mechan-ical stimulation of knee joint. J Neurophysiol 49:35–44

Schaible HG, Schmidt RF (1985) Effects of an experimentalarthritis on the sensory properties of fine articular afferentunits. J Neurophysiol 54:1109–1122

Schaible HG, Schmidt RF (1988) Time course of mechanosen-sitivity changes in articular afferents during a developingexperimental arthritis. J Neurophysiol 60:2180–2195


Schaible HG, Schmidt RF, Willis WD (1987) Enhancement ofthe responses of ascending tract cells in the cat spinal cordby acute inflammation of the knee joint. Exp Brain Res66:489–499

Szolcsányi J (1996) Capsaicin-sensitive sensory nerve terminalswith local and systemic efferent functions: facts and scopesof an unorthodox neuroregulatory mechanism. Progr BrainRes 113:343–359

Watkins LR, Wiertelak EP, Furness LE, Maier SF (1994) Illness-induced hyperalgesia is mediated by spinal neuropeptidesand excitatory amino acids. Brain Res 664:17–24

Yamamoto T, Yaksh TL (1992) Spinal pharmacology of ther-mal hyperesthesia induced by constriction injury of sciaticnerve. Excitatory amino acid antagonists. Pain 49:121–128

H.2.0.7Antagonism to Nerve Growth Factor

H.2.0.7.1General Considerations on Nerve Growth Factor

Nerve growth factor (NGF) is a member of the neu-rotrophin family of structurally related secreted pro-teins that includes brain-derived neurotrophic factor(BDNF), neurotrophin 3 (NT-3) and NT-4. Matureneurotrophins are homodimers that are derived by pro-teolytic cleavage from precursor proteins encoded byseparate genes. They bind to two types of receptor:a common receptor, p75NTR, which binds all neu-rotrophins with a similar affinity; and members of thetrk family of receptor tyrosine kinases, trkA, trkB andtrkC, which bind different neurotrophins.

NGF and BDNF have a crucial role in the gener-ation of pain and hyperalgesia in several acute andchronic pain states (Woolf et al. 1994; McMahon 1996;Mannion et al. 1999; Thompson et al. 1999; Pezet et al.2002; Allen and Dawbarn 2006). The expression ofNGF is high in injured and inflamed tissues, and ac-tivation of the receptor tyrosine kinase trkA in noci-ceptive neurons triggers and potentiates pain signal-ing by multiple mechanisms (McMahon et al. 1995;Huang and Reichardt 2003; Hefti et al. 2006) (see alsoF.2.0.10).

An effective pain therapeutic needs to prevent theactivation of trkA by NGF. This may be achieved byagents that remove free NGF, by molecules that pre-vent binding to trkA, and by molecules that preventactivation of trkA.

REFERENCES AND FURTHER READINGAllen SJ, Dawbarn D (2006) Clinical relevance of the neu-

rotrophins and their receptors. Clin Sci 110:175–191Hefti FF, Rosenthal A, Walicke PA, Wyatt S, Vergara G, Shelton

DL, Davies AM (2006) Novel class of pain drugs based onantagonism of NGF. Trends Pharmacol Sci 27:85–91

Huang EJ, Reichardt LF (2003) trk receptors: roles in neuronalsignal transmission. Annu Rev Biochem 72:609–642

Mannion RJ, Costigan M, Decosterd I, Amaya F, Ma PQ,Holstege JC, Ji RR, Acheson A, Lindsay RM, Wilkin-son GA, Woolf CJ (1999) Neurotrophins: peripherally andcentrally acting modulators of tactile stimulus-induced in-flammatory pain hypersensitivity. Proc Natl Acad Sci USA96:9385–9390

McMahon SB (1996) NGF as a mediator of inflammatory pain.Philos Trans R Soc Lond B Biol Sci 351:431–440

McMahon SB, Bennett DL, Priestley JV, Shelton DL (1995)The biological effects of endogenous nerve growth factoron adult sensory neurons revealed by a trkA-IgG fusionmolecule. Nat Med 1:774–780

Pezet S, Malcangio M, McMahon SB (2002) BDNF: a neu-romodulator in nociceptive pathways? Brain Res Rev40:240–249

Thompson SWN, Bennett DLH, Kerr BJ, Bradbury EJ, McMa-hon SB (1999) Brain-derived neurotrophic factor is an en-dogenous modulator of nociceptive responses in the spinalcord. Proc Natl Acad Sci USA 96:7714–7718

Woolf CJ, Safieh-Garabedian B, Ma PQ, Crilly P, Winter J(1994) Nerve growth factor contributes to the generation ofinflammatory hypersensitivity. Neuroscience 62:327–331

H.2.0.7.2In Vitro Assays of Nerve Growth Factor

PURPOSE AND RATIONALEThe neurotrophin nerve growth factor (NGF) binds totwo receptor types: the tyrosine kinase receptor TrkAand the common neurotrophin receptor p75NTR. Al-though many of the biological effects of NGF (suchas neuronal growth and survival) are associated withTrkA activation, p75NTR also contributes to these ac-tivities by enhancing the action of TrkA when recep-tors are coexpressed (Verdi et al. 1994; Kaplan andMiller 1997). Colquhoun et al. (2004) studied the NGFantagonist PD90780 (Spiegel et al. 1995), which inter-acts with NGF, preventing its binding to p75NTR. Inthis study, the actions of this compound were furtherexplored, and it was found that PD90780 is not able toinhibit the binding of either brain-derived neurotrophicfactor or neurotrophin-3 to p75NTR, consistent with thedirect interactions of the antagonist with NGF. In addi-tion, it was demonstrated that the ability of PD90780to inhibit NGF-p75NTR interactions is lower when re-ceptors are coexpressed, compared with when p75NTR

is the only neurotrophin receptor expressed.

PROCEDURERadiolabeled Neurotrophin and Receptor PreparationThe iodination of NGF (mouse 2.5s; CedarlaneLabs, Toronto, ON) and rhBDNF (Alomone Labs,Jerusalem, Israel) was performed as described by Sut-ter et al. (1979) with modification (Ross et al. 1997).The ability of PD90780 to block neurotrophin bind-ing to the p75NTR receptor was evaluated under vari-ous receptor conditions. This was accomplished with


the use of PC12 cells (TrkA and p75NTR), PC12nnr5

cells (p75NTR only), and truncated p75NTR. The twocell types were cultured in RPMI 1640 medium with10% fetal calf serum. Recovery of the cells waspermitted with the replacement of the medium withcalcium/magnesium-free balanced salt solution fol-lowed by a 15-min incubation at 37°C. Cells were cen-trifuged, and pellets were suspended in HKR buffer(10 mM HEPES, pH 7.35 containing 125 mM NaCl,4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mMKH2PO4, 1 g/l glucose, and 1 g/l bovine serum al-bumin). In the case of truncated p75NTR, the cul-ture medium used to grow PC12 cells was removedand centrifuged to ensure it was free of cells. Thismedium contained p75NTR extracellular domains pre-viously sloughed by the cells (molecular weight ap-proximately 50 kDa) (DiStefano and Johnson 1988).

Chemical Cross-Linking of 125I-NGF to TrkAand/or p75NTR in the Presence of Antagonistsand Immunoprecipitation125I-NGF (0.1 nM) alone or in combination with NGF(100 nM), BDNF (10 nM), or PD90780 (100 µM)was incubated with PC12 cells at a concentration of106 cells/ml in HKR buffer in a volume of 1 mlfor 2 h at 4°C with rocking. After binding, 20 µl ofthe cross-linker bis-(sulfosuccinimidyl)suberate (BS3)was added (final concentration of 0.4 mM) to eachsample and incubated at room temperature for 30 min.The cells were washed three times with TBS, af-ter which reducing SDS sample buffer was added tothe pelleted cells to dissolve the proteins, or in thecase of immunoprecipitations prepared as describedbelow. Cell samples undergoing immunoprecipitationsfor TrkA or p75NTR were solubilized in lysis buffer(TBS containing 10% glycerol, 1% Triton X-100, 1mM phenylmethylsulfonyl fluoride, 10 µg/ml apro-tinin, and 1 µg/ml leupeptin) and incubated for 40 minat 4°C. After centrifugation, the lysates were removedto a new tube, and either rabbit polyclonal anti-Trk cy-toplasmic domain antibody or rabbit polyclonal anti-p75NTR antibody (9992) (antisera against glutathioneS-transferase-fusion protein containing the cytoplas-mic domain of p75NTR) was added to the soluble pro-teins to isolate the respective receptors. The sampleswere left to incubate at 4°C overnight. Antibody com-plexes were removed through application and incuba-tion with 70 µl of a 50% slurry of immobilized Pro-tein G (Pierce Chemical, Rockford, Ill., USA) for 2 hat 4°C. The solid phase was washed with lysis bufferthree times, with distilled water once, and then theproteins were dissolved in SDS sample buffer. Pro-

teins from cross-linking and immunoprecipitation ex-periments were separated via 6% SDS-polyacrylamidegel electrophoresis (PAGE).

Chemical Cross-Linking of 125I-NGF to p75NTR

and Concentration Effect Assays125I-NGF was incubated at 4°C for 2 h with or with-out PD90780. PC12 or PC12nnr5 cells were added at106 cells/ml, and samples were incubated at 4°C for 2h with rocking. Bound 125I-NGF and p75NTR proteinswere cross-linked with final concentrations of 5 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)and 2 mM sulfo-N-hydroxy-sulfosuccinimide (SNHS)(20 µl of each) and incubated with rocking at roomtemperature for 30 min. Samples were washed withTBS [10 mM Tris(hydroxymethyl)-aminomethane,pH8.0, and 150 mM NaCl] three times before the additionof reducing SDS sample buffer to dissolve the proteins.The proteins were then separated on a 6% SDS-PAGEgel. In the experiments involving truncated p75NTR,125I-NGF or 125I-BDNF was exposed to the same con-centrations of PD90780 and incubated with mediumcontaining truncated p75NTR before cross-linking withEDC/SNHS. The reaction was quenched by adding 15µl of 1 M glycine followed by 10 min of mixing. Thesamples were then immunoprecipitated using 192 IgG,which recognizes the extracellular domain of p75NTR

(Calbiochem, San Diego, Calif., USA).

Neurotrophin Receptor BindingNeurotrophins NGF, BDNF, and NT-3 were iodinated,and PC12 and PC12nnr5 cells were cultivated and re-covered as previously described. Tubes were set upcontaining single data points that held iodinated neu-rotrophin (0.5 nM), PD90780 (10 µM), a final concen-tration of 106 cells/ml and NGF (at 50 nM for non-specific binding) as required, and were then incubatedat 4°C for 2 h. Aliquots (100 µl) were layered on top of200 µl of 10% glycerol in HKR buffer in 0.4-ml tubes.Samples were then centrifuged at 5000 rpm for 2 min,after which the tip containing the cell pellet was cut offand radioactivity present was determined.

TrkA Phosphorylation AssayModification of the methods described permitted de-termination of TrkA phosphorylation (Ross et al.1998). NGF (40 pM) was incubated with varying con-centrations of PD90780 (3, 30, or 300 µM) for 2 h inHKR buffer. PC12 cells used at 106 cells/ml were in-cubated with NGF and PD90780 solutions for 15 minat 37°C. Samples were washed once with cold PBSand once with cold TBS and then lysed with solutions


containing 500 µM orthovanadate and immunoprecip-itated with anti-Trk antibody as previously described.An SDS-PAGE run on 6% gel followed by Westernblot analysis performed with antiphosphotyrosine an-tibody (4G10; UBI, Lake Placid, N.Y., USA) and visu-alized with ECL (Amersham) permitted the resolutionof isolated phosphoproteins. The resulting bands werequantified via densitometry analysis.

NGF Protomer Cross-Linking125I-NGF (0.1 nM) was incubated for 2 h at 4°C withZnCl2 (100 µM), PD90780 (30 µM), or ZnCl2 andPD90780 (100 and 30 µ M, respectively), along withHKR buffer, for a total volume of 0.1 ml. After in-cubation, BS3 was added (final concentration of 0.4mM) in 5 µl volume and set at room temperature for30 min. Proteins were dissolved with the addition of50 µl of SDS sample buffer and heating to 95°C for10 min. Separation of 125I-NGF dimers and 125I-NGFmonomers was completed using a 15% acrylamide gelSDS-PAGE.

EVALUATIONFollowing SDS-PAGE, gels were fixed and dried, andthe radio-iodinated ligands cross-linked to receptorswere detected via autoradiography. Receptor ligandbands within SDS-PAGE gels were excised, and ra-dioactivity within each band was detected with a Beck-man gamma counter. The concentration–effect curves,SEM, IC50 values, and 95% confidence intervals (CIs)described in the concentration–effect studies were de-termined by non-linear regression analyses and car-ried out by the program GraphPad Prism, version 3.00(GraphPad Software, San Diego, Calif., USA).

MODIFICATIONS OF THE METHODDebeir et al. (1999) described a nerve growth factormimetic TrkA antagonist that causes withdrawal ofcortical cholinergic boutons in the adult rat. A smallpeptide, C(92–96), which blocks NGF–TrkA interac-tions, was delivered stereotactically into the rat cor-tex over a 2-week period, and its effect and potencywere compared with those of an anti-NGF monoclonalantibody (mAb NGF30). Two presynaptic antigenicsites were studied by immunoreactivity, and the num-ber of presynaptic sites was counted by using an imageanalysis system. Synaptophysin was used as a markerfor overall cortical synapses, and the vesicular acetyl-choline transporter was used as a marker for corticalcholinergic presynaptic sites.

Owolabi et al. (1999) characterized the antiallo-dynic actions of ALE-0540, a novel nerve growth fac-

tor receptor antagonist that inhibits the binding of NGFto tyrosine kinase (Trk) A or both p75 and TrkA (IC505.88± 1.87 µM, 3.72±1.3 µM, respectively), as well assignal transduction and the biological responses medi-ated by TrkA receptors.

REFERENCES AND FURTHER READINGColquhoun A, Lawrence GM, Shamovsky IL, Riopelle RJ,

Ross GM (2004) Differential activity of the Nerve GrowthFactor (NGF) antagonist PD90780 [7-(Benzolylamino)-4,9-dihydro-4-methyl-9-oxo-pyrazolo[5,1-b]quinazoline-2-carboxylic Acid] suggests altered NGF-p75NTR

interactions in the presence of TrkA. J Pharmacol ExpTher 310:505–511

Debeir T, Sargovi HU, Cuello AC (1999) A nerve growth fac-tor mimetic TrkA antagonist causes withdrawal of corticalcholinergic boutons in the adult rat. Proc Natl Acad SciUSA 96:4067–4072

DiStefano PS, Johnson EM Jr (1988) Identification of a trun-cated form of the nerve growth factor receptor. Proc NatlAcad Sci USA 85:270–274

Kaplan DR, Miller FD (1997) Signal transduction by the neu-rotrophin receptors. Curr Opin Cell Biol 9:213–221

Owolabi JB, Rizkalla G, Tehim A, Ross GM, Riopelle RJ, Kam-boj R, Ossipov M, Bian D, Wegert S, Porreca F, Lee DKH(1999) Characterization of antiallodynic actions of ALE-0540, a novel nerve growth factor receptor antagonist, inthe rat. J Pharmacol Exp Ther 289:1271–1276

Ross GM, Shamovsky IL, Lawrance G, Solc M, Dostaler SM,Jimmo SL, Weaver DF, Riopelle RJ (1997) Zinc alters con-formation and inhibits biological activities of nerve growthfactor and related neurotrophins. Nat Med 3:872–878

Ross GM, Shamovsky IL, Lawrance G, Solc M, Dostaler SM,Weaver DF, Riopelle RJ (1998) Reciprocal modulation ofTrkA and p75NTR affinity states is mediated by direct re-ceptor interactions. Eur J Neurosci 10:890–898

Spiegel K, Agrafiotis D, Caprathe B, Davis RE, Dicker-son MR, Fergus JH, Hepburn TW, Marks JS, Van Dorf M,Wieland DM, Jae JC (1995) PD 90780, a nonpeptide in-hibitor of nerve growth factor’s binding to the p75 NGFreceptor. Biochem Biophys Res Commun 217:488–494

Sutter A, Riopelle RJ, Harris-Warrick RM, Shooter EM (1979)Nerve growth factor receptors. Characterization of two dis-tinct classes of binding sites on chick embryo sensory gan-glia cells. J Biol Chem 254:5972–5982

Verdi JM, Birren SJ, Ibáñez CF, Persson H, Kaplan DR,Benedetti M, Chao MV, Anderson DJ (1994) p75LNGFR

regulates Trk signal transduction and NGF-induced neu-ronal differentiation in MAH cells. Neuron 12:733–745

H.2.0.7.3In Vivo Assays of Nerve Growth Factor Antagonism

PURPOSE AND RATIONALEMany studies indicate the role of NGF in pain percep-tion.

Herzberg et al. (1997) reported NGF involvement inpain induced by chronic constriction injury of the ratsciatic nerve.

Ma and Woolf (1997) reported that the progressivetactile hyperalgesia induced by peripheral inflamma-tion is nerve growth factor dependent. An i.p. injection


of anti-NGF antiserum (5 µl/g) 1 h before induction ofinflammation by intraplantar complete Freund’s adju-vant (CFA) injection and 24 h after reduced the basalinflammatory hypersensitivity and significantly atten-uated the progressive increase of spontaneous activity,touch-, pinch- and Aβ-afferent-evoked responses, aswell as the progressive reduction of the mechanicalthreshold of biceps femoris/semitendinosus alpha mo-toneurons normally evoked by repeated (every 5 min)tactile stimulation of the inflamed hindpaw, in decere-brate-spinal rats.

Ro et al. (1999) described the effect of NGF andanti-NGF on neuropathic pain in rats following chronicconstriction injury of the sciatic nerve.

Theodosiou et al. (1999) studied the role of nervegrowth factor in hyperalgesia due to nerve damage.

Gwak et al. (2003) found attenuation of mechanicalhyperalgesia following spinal cord injury by adminis-tration of antibodies to nerve growth factor in the rat.

PROCEDUREAdult Sprague Dawley rats (200–250 g) were spinallyhemisected at T13 (Christensen et al. 1996). Underenflurane anesthesia (induction 3% and maintenance2%) the T11–12 laminae were determined by count-ing the dorsal spinous processes from the sacrum. Thesurgical field was then shaved, a longitudinal inci-sion made exposing several segments, and a laminec-tomy performed at two vertebral segments, T11–T12.The spinal cord was hemisected just cranial to the L1dorsal root entry zone with a micro-dissecting knifewithout damaging the major dorsal vessel or vascularbranches. An insulin syringe with a 28-gauge needlewas placed dorsal-ventrally at the midline of the cord,and pulled laterally to ensure the completeness of thehemisection. The incised skin was sutured, and postop-erative care was done. After the hemisection, animalswere either treated once a day for 10 days with anti-NGF (anti-nerve growth factor-2.5S, Sigma, i.p., 2 µg,0.2 ml), or with saline (0.2 ml), or were untreated.

In the behavioral experiments, rats were housed inclear plastic boxes (8 × 8 × 24 cm) above a metal mesh(0.5 × 0.5 cm) and acclimatized for 30 min to avoidthe stress associated with environmental change. Me-chanical paw withdrawal threshold to the applicationof a von Frey filament was measured by using the up-down testing paradigm (Ro et al. 1999). An ascendingseries of von Frey filaments of incremental force (0.35,0.53, 0.78, 2.5, 3.7, 5.2, 6.0, and 12.5 g) was appliedfor 3 s to the middle of the plantar surface of the hindpaw, starting with the 2.5 g stimulus (Chaplan et al.1994).

For electrophysiology experiments, rats were anes-thetized with sodium pentobarbital (40–50 mg/kg),with supplementary pancuronium bromide (2–4 mg/kgper h), and artificially ventilated. Extracellular record-ings of neuronal activity were made from the dorsalhorn neurons in the lumbar spinal cord (L4–L5), us-ing a recording glass microelectrode with a carbon fila-ment (3–5 M) while mechanical stimuli were appliedonto the receptive fields. The single unit responsesof the dorsal horn neurons, characterized as wide dy-namic range (WDR) neurons by their graded responsesto increased intensities of mechanical stimuli, wereamplified, filtered, and displayed on an oscilloscope.The output signals were also fed into a data acquisitionsystem (CED 1401 plus) via a window discriminatorfor the construction of real-time recordings of peris-timulus time histograms, which were displayed as thenumber of spikes per second.

The mechanical stimuli were applied for 10 s andincluded: (1) brushing the skin with a camel hair brushin a stereotypic manner (brush); (2) sustained applica-tion of a large clamp that produced a sense of firm pres-sure when placed on human skin (pressure); and (3)sustained application of a small clamp that produceda distinctly painful sensation (pinch).

EVALUATIONStatistical analysis was performed using Mann–Whitney’s unmatched pairs rank-sum test to evaluatethe differences between the scores in two groups. Alldata are displayed as means ±standard error.

MODIFICATIONS OF THE METHODSpinal cord injury often leads to central pain syndromeincluding hyperalgesia to mechanical stimulation. Sev-eral authors studied the influence of nerve growth fac-tor in models of neuropathic pain (Ramer and Bisby1999; Li et al. 2002, 2003; Cahill et al. 2003; Ruiz et al.2004).

Nerve Ligation InjuryNerve ligation injury was performed according tothe method described previously (Kim and Chung1992). This technique produces signs of tactile al-lodynia and thermal hyperalgesia. Rats were anes-thetized with halothane and the L5 and L6 spinalnerves were exposed, carefully isolated, and tightlyligated with 4–0 silk suture distal to the dorsal rootganglion (DRG). After ensuring homeostatic stability,the wounds were sutured, and the animals were al-lowed to recover in individual cages. Sham-operated


rats were prepared in an identical fashion except thatthe L5 and L6 spinal nerves were not ligated.

Intrathecal Catheter PlacementTwo routes of administration, a systemic i.p. anda spinal intrathecal (i.th.) route, were used explorethe activity of compounds. For the spinal route, testcompounds were injected through indwelling i.th.catheters in the manner described by Yaksh and Rudy(1976). While under anesthesia, polyethylene tubing10 tubing (8 cm) was inserted through an incisionmade in the atlanto-occipital membrane to the level ofthe lumbar enlargement of the rat and secured. Druginjections were made in a volume of 5 µl followed bya 9-µl saline flush.

Thermal SensitizationRats were lightly anesthetized with ether. The lefthindpaw was placed in a water bath maintained at 50°Cfor 1 min. Inflammation suggested by rubor of the pawdeveloped immediately. The rats were allowed to re-cover from anesthesia and tactile testing was begun2 h after thermal sensitization. This procedure has pro-duced signs of thermal hyperalgesia and tactile allody-nia that persisted for over 12 h.

Evaluation of Tactile AllodyniaMechanical allodynia was determined in the mannerdescribed previously (Chaplan et al. 1994). The pawwithdrawal threshold was determined in response toprobing with calibrated von Frey filaments. The ratswere kept in suspended cages with wire mesh floorsand the von Frey filaments were applied perpendicu-larly to the planar surface of the paw of the rat un-til it bent slightly, and was held for 3–6 s, or untilthe paw was withdrawn. A positive response was in-dicated by a sharp withdrawal of the paw. The 50%paw withdrawal threshold was determined by the non-parametric method (Dixon 1980).

Shelton et al. (2005) found that nerve growth fac-tor mediates hyperalgesia and cachexia in auto-immune arthritis. Function-blocking antibodies toNGF completely reverse established pain in rats withfully developed arthritis despite continuing joint de-struction and inflammation. Likewise, these antibod-ies reverse weight loss while not having any effect onlevels of the pro-cachectic agent tumor necrosis factor(TNF).

Banik et al. (2005) reported that increased nervegrowth factor after rat plantar incision contributes toguarding behavior and heat hyperalgesia. The ther-apeutic effect of a monoclonal antibody against en-

dogenous NGF was evaluated by intraperitoneal ad-ministration of a single preoperative dose of anti-NGF.

Adult male, 225–275 g, Sprague-Dawley rats wereused in a plantar incision animal model. The animalswere anesthetized with 1.5%–2% halothane and thesurgical field was prepared in a sterile manner. A 1-cmlongitudinal incision was made in the plantar aspect ofthe hind paw beginning 0.5 cm from the end of heel;skin, fascia and muscle were incised and the skin wasclosed with 5–0 nylon suture. Topical antibiotics wereadministered. On the second postoperative day, sutureswere removed under brief anesthesia.

For measuring guarding behaviors, unrestrainedrats were placed on a small plastic mesh floor (grid 8 ×8 mm). Using an angled magnifying mirror, the incisedand non-incised paws were viewed. Both paws of eachanimal were closely observed over a 1-min period re-peated every 5 min for 1 h. Depending on the positionin which each paw was found during the majority ofthe 1-min scoring period, a 0, 1 or 2 was given. A scoreof 0 was given for full weight bearing with the area ofthe wound blanched or distorted by the mesh; 1 for thewound area just touching the mesh without blanchingor distortion; and 2 for the wound area completely offthe mesh. The sum of the 12 scores (0–24) obtainedduring 1-h session for each paw was obtained.

For measuring heat sensitivity rats were placed in-dividually on a glass floor covered with a clear plasticcage and allowed to acclimate. Withdrawal latencies toradiant heat were assessed by applying a focused radi-ant heat source underneath a glass floor on the middleof the incision. The latency time to evoke a withdrawalwas determined with a cut-off value of 30 s. The inten-sity of the heat was adjusted to produce a withdrawallatency in normal rats of 25–30 s. Each rat was testedat least three times, at an interval of 10 min. The av-erage of at least three trials was used to obtain pawwithdrawal latency.

For measuring mechanosensitivity, rats were placedindividually on a plastic mesh floor covered witha clear plastic cage and allowed to acclimate. The with-drawal response to punctate mechanical stimulationwas determined using calibrated von Frey hairs ap-plied underneath the cage to an area adjacent to theincision. Each filament was applied once starting with10 mN and continuing until a withdrawal response oc-curred or 250 mN was reached. If a rat did not respondto the 250-mN filaments (522 mN), the next filamentwas recorded. This was repeated a total of three timeswith at least a 5- to 10-min test-free period betweenwithdrawal responses. The lowest force from the three


tests producing a response was considered the with-drawal threshold.

Zahn et al. (2004) described the effect of blockadeof nerve growth factor and tumor necrosis factor onpain behaviors after plantar incision.

Obata et al. (2002) described the expression ofneurotrophic factors in the dorsal root ganglion ina rat model of lumbar disc herniation. The left L4/5nerve roots were exposed after hemilaminectomies andautologous intervertebral discs, which were obtainedfrom coccygeal intervertebral discs, were implanted oneach of the exposed nerve roots without mechanicalcompression.

Lamb et al. (2003) studied nerve growth factor(NGF) and gastric hyperalgesia in the rat. MaleSprague Dawley rats (300–400 g) were anesthetizedand the stomach exposed and placed in a circularclamp. Acetic acid (60%) or saline was injected intothis area and aspirated 45 s later, resulting in kissingulcers. A balloon was surgically placed into the stom-ach and electromyographic responses to gastric disten-sion recorded from the acromiotrapezius muscle. Ani-mals received a daily injection of neutralizing NGF an-tibody or control serum for 5 days; NGF in the stomachwas measured with an ELISA. The severity of gastricinjury was assessed microscopically and by determi-nation of myeloperoxidase activity.

Winston et al. (2003) investigated molecular and be-havioral changes in nociception in a novel rat modelof chronic pancreatitis induced by pancreatic infu-sion of trinitrobenzene sulfonic acid as a model ofpainful pancreatitis. Nociception was assessed by mea-suring mechanical sensitivity of the abdomen and byrecording the number of nocifensive behaviors in re-sponse to electrical stimulation of the pancreas. Ex-pression of neuropeptides calcitonin gene-related pep-tide (CGRP) and substance P (SP) in the thoracicdorsal root ganglia receiving input from the pancreasand nerve growth factor in the pancreas were mea-sured.

Guerios et al. (2006) reported that nerve growth fac-tor (NGF) mediates peripheral mechanical hypersensi-tivity that accompanies experimental cystitis in mice.Cystitis was induced by intraperitoneal injection of cy-clophosphamide (CYP) in female mice. Sensitivity ofhind paws to mechanical stimuli was determined priorto and 4, 9, and 24 h after CYP, and the sensitivity ofthe tail to thermal stimuli was determined prior to, and4 and 24 h after CYP treatment. To investigate the roleof NGF in these processes, other groups of mice re-ceived NGF antiserum or normal serum intravenously30 min after CYP administration. CYP induced blad-

der inflammation that was not ablated by treatmentwith NGF antiserum. Sensitivity to mechanical stim-uli was increased 4 and 9 h after CYP administration.This was reversed by NGF antiserum but not by nor-mal serum.

Cyclophosphamide-induced cystitis was also usedas a model for visceral pain by Lanteri-Minet et al.(1995), Boucher et al. (2000), and Bon et al. (2003).

Jaggar et al. (1999) studied hyperalgesia to thermalstimulation of the hind limb of rats after inflammationof the urinary bladder by instillation of 0.5 ml of 50%turpentine in olive oil.

Dmitrieva and McMahon (1996) and Dmitrievaet al. (1997) reported sensitization of visceral afferentsby NGF in the adult rat.

Delafoy et al. (2003) studied the role of NGF intrinitrobenzene sulfonic acid-induced colonic hy-persensitivity. The function of NGF as a mediatorof persistent pain states was tested in a model ofcolonic hypersensitivity measured by isobaric disten-sion in conscious rats. The effects of exogenous NGFon colonic pain threshold, the involvement of NGF intrinitrobenzene sulfonic acid-induced colonic hyper-sensitivity, and the involvement of sensory nerves inthe effects of NGF and trinitrobenzene sulfonic acidusing rats treated neonatally with capsaicin were stud-ied.

Sevcik et al. (2005) found that anti-NGF therapyprofoundly reduced bone cancer pain and the accom-panying increase of markers of peripheral and centralsensitization. Osteolytic murine sarcoma cells were in-jected into the intramedullary space of the mouse fe-mur. Administrations of a NGF-sequestering antibodyproduced a profound reduction in cancer pain-relatedbehavior that was greater than that achieved with ad-ministration of morphine.

REFERENCES AND FURTHER READINGBanik RK, Subieta AR, Wu C, Brennan TJ (2005) Increased

nerve growth factor after rat plantar incision contributes toguarding behavior and heat hyperalgesia. Pain 117:68–76

Bon K, Lichtensteiger CA, Wilson SG, Mogli JS (2003) Charac-terization of cycophosphamide cystitis, a model of visceraland referred pain, in the mouse: species and strain differ-ences. J Urol 170:1008–1012

Boucher M, Meen M, Codron JP, Coudore F, Kemeny JL, Es-chalier A (2000) Cyclophosphamide-induced cystitis infreely moving conscious rats: behavioural approach toa new model of visceral pain. J Urol 164:203–208

Cahill CM, Dray A, Coderre TJ (2003) Intrathecal nerve growthfactor restores opioid effectiveness in an animal model ofneuropathic pain. Neuropharmacology 45:543–552

Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL (1994)Quantitative measurement of tactile allodynia in the ratpaw. J Neurosci Methods 53:55–63

H.3 · Anti-Inflammatory Activity 1047

Christensen MD, Evertart AW, Pickelman JT, Hulsebosch CE(1996) Mechanical and thermal allodynia in chronic centralpain following spinal cord injury. Pain 68:97–107

Delafoy L, Raymond F, Doherty AM, Eschalier A, Diop L(2003) Role of nerve growth factor in the trinitroben-zene sulfonic acid-induced colonic hypersensitivity. Pain105:489–497

Dixon WJ (1980) Efficient analysis of experimental observa-tions. Annu Rev Pharmacol Toxicol 20:441–462

Dmitrieva N, McMahon SB (1996) Sensitization of visceralafferents by nerve growth factor in the adult rat. Pain66:87–97

Dmitrieva N, Shelton D, Rice ASC, McMahon SB (1997) Therole of nerve growth factor in a model of visceral inflam-mation. Neuroscience 78:449–459

Guerios S, Wang ZY, Bjorling DE (2006) Nerve growth fac-tor mediates peripheral mechanical hypersensitivity thataccompanies experimental cystitis in mice. Neurosci Lett392:193–197

Gwak YS, Nam TS, Paik KS, Hulsebosch CE, Leem JW (2003)Attenuation of mechanical hyperalgesia following spinalcord injury by administration of antibodies to nerve growthfactor in the rat. Neurosci Lett 336:117–120

Herzberg U, Eliav E, Dorsey JM, Gracely RH, Kopin IJ (1997)NGF involvement in pain induced by chronic constric-tion injury of the rat sciatic nerve. NeuroReport 8:1613–1618

Jaggar SI, Scott HCF, Rice ASC (1999) Inflammation of the raturinary bladder is associated with a referred thermal hy-peralgesia which is nerve growth factor dependent. Br JAnaesth 83:442–448


Lamb K, Kang YM, Gebhart GF, Bielefeldt K (2003) Nervegrowth factor and gastric hyperalgesia in the rat. Neuro-gastroenterol Mot 15:355–361

Lanteri-Minet M, Bon K, de Pommery J, Michiels JF, Mene-trey D (1995) Cyclophosphamide cystitis as a model of vis-ceral pain in rats: model elaboration and spinal structuresinvolved as revealed by the expression of c-Fos and Krox-24 proteins. Exp Brain Res 105:220–232

Li L, Xian CJ, Zhong JH, Zhou XF (2002) Effect of lumbar 5ventral root transection on pain behaviours: a novel modelfor neuropathic pain without axotomy of primary sensoryneurons. Exp Neurol 175:23–34

Li L, Xian CJ, Zhong JH, Zhou XF (2003) Lumbar 5 ventral rootdissection-induced upregulation of nerve growth factor insensory neurons and their target tissues: a mechanism ofneuropathic pain. Mol Cell Neurosci 23:232–250

Ma QP, Woolf CJ (1997) The progressive tactile hyperalgesiainduced by peripheral inflammation is nerve growth factordependent. NeuroReport 8:807–810

Obata K, Tsujino H, Yamanka H, Yi D, Fukuoka T, HashimotoN, Yomenobu K, Yoshikawa H, Noguchi K (2002) Expres-sion of neurotrophic factors in the dorsal root ganglion ina rat model of lumbar disc herniation. Pain 99:121–132

Ramer MS, Bisby MA (1999) Adrenergic innervation of ratsensory ganglia following proximal or distal painful sci-atic neuropathy: distinct mechanisms revealed by anti-NGFtreatment. Eur J Neurosci 11:837–846

Ro LS, Chen ST, Tang LM, Jacobs JM (1999) Effect of NGFand anti-NGF on neuropathic pain in rats following chronicconstriction injury of the sciatic nerve. Pain 79:265–274

Ruiz G, Ceballos D, Baños JE (2004) Behavioral and histolog-ical effects of endoneurial administration of nerve growth

factor: possible implications in neuropathic pain. Brain Res1011:1–6

Sevcik MA, Ghilardi JR, Peters CM, Lindsay TH, HalvorsonKG, Jonas BM, Kubota K, Kuskowski MA, Boustany L,Shelton DL, Mantyh PW (2005) Anti-NGF therapy pro-foundly reduced bone cancer pain and the accompanyingincrease of markers of peripheral and central sensitization.Pain 115:128–141

Shelton DL, Zeller J, Ho WH, Pons J, Rosenthal A (2005) Nervegrowth factor mediates hyperalgesia and cachexia in auto-immune arthritis. Pain 116:8–16

Theodosiou M, Rush RA, Zhou XF, Hu D, Walker JS, TraceyDJ (1999) Hyperalgesia due to nerve damage: role of nervegrowth factor. Pain 81:245–255

Winston JH, He ZJ, Shenoy M, Xiao SY, Pasricha PJ (2003)Molecular and behavioural changes in nociception ina novel rat model of chronic pancreatitis for the study ofpain. Pain 117:214–222

Yaksh TL, Rudy TA (1976) Chronic catheterization of the spinalsubarachnoid space. Physiol Behav 17:1031–1036

Zahn PK, Subieta A, Park SS, Brennan TJ (2004) Effect ofblockade of nerve growth factor and tumor necrosis factoron pain behaviours after plantar incision. J Pain 5:157–163

H.3Anti-Inflammatory Activity


Inflammation was characterized two thousand yearsago by Celsus by the four Latin words: Rubor, calor,tumor and dolor. Inflammation has different phases:the first phase is caused by an increase of vascular per-meability resulting in exudation of fluid from the bloodinto the interstitial space, the second one by infiltrationof leukocytes from the blood into the tissues and thethird one by granuloma formation. Accordingly, anti-inflammatory tests have to be divided into those mea-suring acute inflammation, subacute inflammation andchronic repair processes. In some cases, the screeningis directed to test compounds for local application. Pre-dominantly, however, these studies are aimed to findnew drugs against polyarthritis and other rheumaticdiseases. Since the etiology of polyarthritis is consid-ered to be largely immunologically, special tests havebeen developed to investigate various immunologicaland allergic factors (see Chapter I).

H.3.1In Vitro Methods for Anti-Inflammatory Activity


An array of physiological substances, sometimescalled autacoids, are involved in the process of inflam-mation and repair. These include histamine, serotonin,


bradykinin, substance P, and the group of eicosanoids(prostaglandins, thromboxanes and leucotrienes), theplatelet-activating factor (PAF) as well as cytokinesand lymphokines. Their discovery makes the use of invitro studies possible. The influence of non-steroidalanti-inflammatory agents on the eicosanoid pathwaygave rise to numerous studies.

H.3.1.23H-Bradykinin Receptor Binding

PURPOSE AND RATIONALETissue injury or trauma initiates a cascade of reac-tions which results in the proteolytic generation ofbradykinin and kallidin from high-molecular-weightprecursors, kininogens, found in blood and tissue. Therapid enzymatic cleavage of kininogens is accom-plished by the kallikreins, a group of proteolytic en-zymes which are present in most tissues and body flu-ids. Bradykinin produces pain by stimulating A andC fibers in the peripheral nerves, participates in the in-flammatory reaction and lowers blood pressure by va-sodilatation. Since its breakdown occurs via the sameenzyme responsible for converting angiotensin I intoangiotensin II some of the effects of converting en-zyme inhibitors may be due to presence of bradykinin.The 3H-bradykinin receptor binding is used to de-tect compounds that inhibit binding of 3H-bradykininin membrane preparations obtained from guinea-pigileum. Two types of bradykinin receptors (B1 andB2 receptors) are known (Feres et al. 1992; Bascandset al. 1993; Tropea et al. 1994; Marceau et al. 1998;Calixto et al. 2004; Leeb-Lundberg et al. 2005). Theexistence of a pulmonary BK3 receptor has been pro-posed by Farmer et al. (1989) and Meini et al. (2004).Evidence was obtained for the existence of three sub-types of B2 receptors, B2a, B2b, and B2c (Seguin et al.1992; Seguin and Widdowson 1993).

PROCEDUREIleum from guinea pigs is cleaned from its contentand cut into pieces of 2 cm length. They are homog-enized for 30 s in ice-cold TES buffer, pH 6.8, con-taining 1 mM 1,10-phenanthroline, in a Potter homog-enizer. The homogenates are filtered through 3 layersof gauze and centrifuged twice at 50,000 g for 10 minwith an intermediate rehomogenization in buffer.

For routine studies the final pellets are resuspendedin 40 vol of incubation buffer (25 mM TES buffer,pH 6.8, containing 1 mM 1,10-phenanthroline, 0.1%bovine serum albumin, 140 µg/ml bacitracin, 1 mMdithiothreitol, 0.1 µM captopril). In the competition

experiment, 50 µl 3H-bradykinin (one constant con-centration of 0.5–2 × 10−9 M), 50 µl test compound(6 concentrations, 10−5–10−10 M) and 150 µl mem-brane suspension from guinea pig ileum (approx.6.6 mg wet weight/ml) per sample are incubated ina shaker bath at 25°C for 90 min.

Saturation experiments are performed with 12 con-centrations of 3H-bradykinin (14.2–0.007× 10−9 M).Total binding is determined in the presence of incuba-tion buffer, non-specific binding is determined in thepresence of non-labeled bradykinin (10−6 M).

The reaction is stopped by rapid vacuum filtra-tion through glass fibre filters. Thereby the membrane-bound radioactivity is separated from the free one. Theretained membrane-bound radioactivity on the filteris measured after addition of 3 ml liquid scintillationcocktail per sample in a liquid scintillation counter.

EVALUATIONThe following parameters are calculated:

• total binding of 3H-bradykinin• non-specific binding in the presence of 10 µM

bradykinin• specific binding = total binding–non-specific bind-

ing• % inhibition: 100–specific binding as percentage of

control value

Compounds are first tested at a single high concen-tration (10,000 nM) in triplicate. For those showingmore than 50% inhibition a displacement curve is con-structed using 7 different concentrations of test com-pound. Binding potency of compounds is expressed ei-ther as a relative binding affinity (RBA) with respect tothe standard compound (bradykinin) which is tested inparallel or as an IC50.

RBA = IC50 standard compound

IC50 compound× 100%

The dissociation constant (Ki) and the IC50 value ofthe test drug are determined from the competition ex-periment of 3H-bradykinin versus non-labeled drugby a computer-supported analysis of the binding data(McPherson 1985).

Tests for Bradykinin Receptor Types and SubtypesPrado et al. (2002) described mechanisms regulatingthe expression, self-maintenance, and signaling func-tion of the bradykinin B2 and B1 receptors.


Cloning and pharmacological characterization ofa human bradykinin (BK-2) receptor was reported byHess et al. (1992).

Menke et al. (1994) reported the expression cloningof a human bradykinin B1 receptor.

Bradykinin B1 receptors have been studied in theisolated rabbit aorta (Bouthillier et al. 1987), in theisolated rabbit carotid artery (Pruneau and Bélichard1993) and in the rabbit urinary bladder (Butt et al.1955).

A potent bradykinin B1 receptor antagonist hasbeen described by Wirth et al. (1991).

Heterogeneity of B1 receptors has been suggestedby Wirth et al. (1992).

Heitsch (2002) reviewed non-peptide antagonistsand agonists of the bradykinin B2 receptor.

Bradykinin receptor ligands were described byMarceau and Regoli (2004) and Fortin and Marceau(2006).

Drummond and Cocks (1995) used rings of bovineleft anterior descending coronary artery to studyendothelium-dependent relaxations mediated by in-ducible B1 and constitutive B2 kinin receptors.

The production of cyclic GMP via activation of B1and B2 kinin receptors in cultured bovine aortic en-dothelial cells was described by Wiemer and Wirth(1992).

Pharmacological characterization of bradykinin re-ceptors in canine cultured tracheal smooth muscle cellshas been reported by Yang et al. (1995).

Bradykinin B2 receptors and their antagonists havebeen studied in human fibroblasts by Alla et al. (1993),with the high affinity radioligand [125I]PIP HOE 140by Brenner et al. (1993), in guinea pig gall bladder byFalcone et al. (1993), in the smooth muscle of guinea-pig taenia caeci by Field et al. (1994), in guinea pigileum membranes by Graneß and Liebmann (1994),Liebmann et al. (1994a), in isolated blood vessels fromdifferent species by Félétou et al. (1994), in endothe-lial cells by Wirth et al. (1994).

Hallé et al. (2000) described in vitro and in vivo ef-fects of kinin B1 and B2 receptor agonists and antago-nists in inbred control and cardiomyopathic hamsters.

The role of B1 and B2 receptors and of nitric ox-ide in bradykinin-induced relaxation and contractionof isolated rat duodenum was studied by Rhaleb andCarretero (1994).

Campos et al. (1996) investigated the effect of pre-treatment with bacterial endotoxin on the bradykininB1 and B2 receptor-induced edema in the rat paw andthe interaction of B1-mediated responses with other in-flammatory mediators.

Characterization of kinin receptors by bioassayswas described by Gobeil and Regoli (1994). Molec-ular cloning, functional expression and pharmacologi-cal characterization of a human bradykinin B2 receptorgene was performed by Eggerickx et al. (1992).

Simpson et al. (2000) characterized bradykinin ana-logues on recombinant human bradykinin B1 and B2receptors using a high throughput functional assaywhich measures intracellular Ca2+ responses.

Bradykinin B2 receptor subtypes were discussed byLiebmann et al. (1994b) and Regoli et al. (1994).

Evidence for a pulmonary B3 bradykinin receptorhas been given by Farmer et al., (1989) and Meini et al.(2004).

Bradykinin B3 receptors have been described byField et al. (1992) in the smooth muscle of the guinea-pig taenia caeci and trachea.

REFERENCES AND FURTHER READINGAlla SA, Buschko J, Quitterer U, Maidhof A, Haasemann M,

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Bascands JL, Pecher C, Rounaud S, Emond C, Tack JL, BastieMJ, Burch R, Regoli D, Girolami JP (1993) Evidence forexistence of two distinct bradykinin receptors on rat mesan-gial cells. Am J Physiol 264:F548–F556

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Burch RM, Kyle DJ (1992) Minireview: Recent developmentsin the understanding of bradykinin receptors. Life Sci50:829–838

Burch RM, Farmer SG, Steranka LR (1990) Bradykinin receptorantagonists. Medicin Res Rev 10:237–239

Burch RM, Kyle DJ, Stormann TM (1993) Molecular Biol-ogy and Pharmacology of Bradykinin Receptors: The Phar-macological Classification of Kinins. RG Landes Comp.,Austin, pp 6–18

Butt SK, Dawson LG, Hall JM (1995) Bradykinin B1 recep-tors in the rabbit urinary bladder: induction of responses,smooth muscle contraction, and phopshatidylinositol hy-drolysis. Br J Pharmacol 114:612–617

Calixto JB, Medeiros R, Fernandes ES, Ferreira J, Caprini DA,Campos MM (2004) Kinin B1 receptors: key G-protein-coupled receptors and their role in inflammatory andpainful processes. Br J Pharmacol 143:803–818

Campos MM, Souza GEP, Calixto JB (1996) Upregulation of B1receptor mediating des-Arg9-BK-induced rat paw edemaby systemic treatment with bacterial endotoxin. Br J Phar-macol 117:793–798

Drummond GR, Cocks TM (1995) Endothelium-dependent re-laxations mediated by inducible B1 and constitutive B2kinin receptors in the bovine coronary artery. Br J Phar-macol 116:2473–2481

Eggerickx D, Raspe E, Bertrand D, Vassart G, Parmen-tier M (1992) Molecular cloning, functional expres-


sion and pharmacological characterization of a humanbradykinin B2 receptor gene. Biochem Biophys Res Com-mun 187:1306–1313

Emond C, Bascands JL, Pecher C, Cabos-Boutot G, Pradelles P,Regoli D, Girolami JP (1990) Characterization of a B2-bradykinin receptor in rat mesangial cells. Eur J Pharmacol190:381–392

Falcone RC, Hubbs SJ, Vanderloo JD, Prosser JC, Little J,Gomes B, Aharony D, Krell RD (1993) Characterizationof bradykinin receptors in guinea pig gall bladder. J PharmExp Ther 266:1291–1299

Farmer SG, Burch RM, Meeker SA, Wilkins DE (1989) Evi-dence for a pulmonary B3 bradykinin receptor. Mol Phar-macol 36:1–8

Félétou M, Germain M, Thurieau C, Fauchère JL, Canet E(1994) Agonistic and antagonistic properties of thebradykinin B2 receptor antagonist, Hoe 140, in isolatedblood vessels from different species. Br J Pharmacol112:683–689

Feres T, Paiva ACM, Paiva TB (1992) BK1 and BK2 bradykininreceptors in the rat duodenum smooth muscle. Br J Phar-macol 107:991–995

Field JL, Hall JM, Morton IKM (1992) Putative novelbradykinin B3 receptors in the smooth muscle of theguinea-pig taenia caeci and trachea. Recent Progress onKinins, Birkhäuser Basel, pp 540–545

Field JL, Butt SK, Morton IKM, Hall JM (1994) Bradykinin B2receptors and coupling mechanisms in the smooth muscleof guinea-pig taenia caeci. Br J Pharmacol 113:607–613

Fortin JP, Marceau F (2006) Advances in the developmentof bradykinin receptor ligands. Curr Topics Med Chem6:1353–1363

Galizzi JP, Bodinier MC, Chapelain B, Ly SM, Coussy L, Gi-raud S, Neliat G, Jean T (1994) Up-regulation of [3H]-des-arg10-kallidin binding to the bradykinin B1 receptor byinterleukin-1β in isolated smooth muscle cells: correlationwith B1 agonist-induced PGI2 production. Br J Pharmacol113:389–394

Gobeil F, Regoli D (1994) Characterization of kinin receptors bybioassays. Braz J Med Biol Res 27:1781–1791

Graneß A, Liebmann C (1994) Affinity cross-linking ofbradykinin B2 receptors in guinea pig ileum membranes.Eur J Pharmacol 268:271–274

Hallé S, Gobeil F Jr, Ouelette J, Lambert C, Regoli D (2000) Invitro and in vivo effects of kinin B1 and B2 receptor ago-nists and antagonists in inbred control and cardiomyopathichamsters. Br J Pharmacol 129:1641–2648

Heitsch H (2002) Non-peptide antagonists and agonists of thebradykinin B2 receptor. Curr Med Chem 9:913–928

Hess JKF, Borkowski JA, Young GS, Strader CD, Ramson RW(1992) Cloning and pharmacological characterization ofa human bradykinin (BK-2) receptor. Biochem BiophysRes Commun 184:260–268

Hock FJ, Wirth K, Albus U, Linz W, Gerhards HJ, Wiemer G,Henke S, Breipohl G, König W, Knolle J, Schölkens BA(1991) Hoe 140 a new potent and long acting bradykininantagonist: in vitro studies. Br J Pharmacol 102:769–773

Innis RB, Manning DC, Stewart JM, Snyder SH (1981)[3H]Bradykinin receptor binding in mammalian tis-sue membranes. Proc Natl Acad Sci USA., 78:2630–2634

Kachur JF, Allbee W, Danjo W, Gaginella TS (1987) Bradykininreceptors: functional similarities in guinea pig gut muscleand mucosa. Regul Peptides 17:63–70

Leeb-Lundberg LMF, Marceau F, Müller-Esterl W, Petti-bone DJ, Zuraw BL (2005) International Union of Phar-macology. XLV. Classification of the kinin receptor family:

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Liebmann C, Bossé R, Escher E (1994b) Discrimination be-tween putative bradykinin B2 receptor subtypes in guineapig ileum smooth muscle membranes with a selec-tive, iodinatable, bradykinin analogue. Molec Pharmacol46:949–956

Liebmann C, Mammery K, Graneß A (1994a) Bradykinin in-hibits adenylate cyclase activity in guinea pig membranesvia a separate high-affinity bradykinin B2 receptor. Eur JPharmacol 288:35–43

Manning DC, Vavrek R, Stewart JM, Snyder SH (1986) Twobradykinin binding sites with picomolar affinities. J Phar-macol Exp Ther 237:504–512

Marceau F, Regoli D (2004) Bradykinin receptor ligands: thera-peutic perspectives. Nature Rev Drug Disc 3:845–852

Marceau F, Hess JF, Bachvarov DR (1998) The B1 receptors forkinins. Pharmacol Rev 50:357–386

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Menke JG, Borkowski JA, Bierilo KK, MacNeill T, Derrick AW,Schneck KA, Ransom RW, Strader CD, Linemeyer DL,Hess JF (1994) Expression cloning of a human bradykininB1 receptor. J Biol Chem 269:21583–21586

Prado GN, Taylor L, Zhou X, Ricupero D, Mierke DF, Pol-gar P (2002) Mechanisms regulating the expression, self-maintenance, and signaling-function of the bradykinin B2and B1 receptors. J Cell Physiol 193:275–286

Pruneau D, Bélichard P (1993) Induction of bradykinin B1 re-ceptor-mediated relaxation in the isolated rabbit carotidartery. Eur J Pharmacol 239:63–67

Regoli D, Gobeil F, Nguyen QT, Jukic D, Seoane PR, Sal-vino JM, Sawutz DG (1994) Bradykinin receptor types andB2 subtypes. Life Sci 55:735–749

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Rhaleb NE, Rouissi N, Jukic D, Regoli D, Henke S, Breipohl G,Knolle J (1992) Pharmacological characterization of a newhighly potent B2 receptor antagonist (HOE 140: D-arg-[hyp3,thi5,D-tic7,oic8]bradykinin. Eur J Pharmacol210:115–120

Schneck KA, Hess JF, Stonisifer GY, Ransom RW (1994)Bradykinin B1 receptors in rabbit aorta smooth mus-cle in culture. Eur J Pharmacol, Mol Pharmacol Sect266:277–282

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H.3.1.3Substance P and the Tachykinin Family


Substance P belongs to the tachykinin family ofpeptides that share a common carboxy-terminal se-quence (Phe-X-Gly-Leu-Met-NH2). It was first de-scribed by von Euler and Gaddum (1931) as a brainand gut extract that stimulates smooth muscle contrac-tion. Bioassay extracts from spinal dorsal roots impli-cated substance P as a pain neurotransmitter (Lem-beck 1953; Lembeck and Holzer 1979). After deter-mination of the amino acid sequence (Chang et al.1971) the distribution of substance P in the CNS couldbe studied (Hökfelt et al. 1975). Neurokinins belonglike substance P to a group of neuropeptides namedtachykinins. Following the discovery of neurokinin Aand neurokinin B, three distinct G protein-coupled re-ceptors, NK1, NK2 and NK3, were described (Maggiet al. 1993; Mussap et al. 1993; Patacchini and Maggi1995). Neurokinin A and substance P are preferredagonists of the tachykinin NK1 and NK2 receptors,whereas neurokinin B preferentially interacts with thetachykinin NK3 receptor. The receptor sensitivity ofthese peptides is relatively poor, and it is possible thattheir actions could be mediated by interactions withtheir less preferred receptors.

Nomenclature of tachykinins and tachykinin re-ceptors has been discussed repeatedly (Henry 1987;Maggi 2000).

Tachykinin NK1 antagonists are potent antiemet-ics, however other possible therapeutic uses, includ-ing rheumatoid arthritis, asthma, migraine, pain andpsychiatric disorders, were suggested (Longmore et al.1995). The P-preferring NK1 receptor has attractedmost interest as a CNS target because it is the pre-dominant tachykinin receptor expressed in the humanbrain, while NK2 and NK3 receptor expression is inextremely low abundance or absent. Several NK1 re-ceptor agonists antagonists were synthesized and eval-uated (Snider et al. 1991; Emonds-Alt et al. 1993; Ca-scieri et al. 1992; Sakurada et al. 1993; Bristow andYoung 1994; Jung et al. 1994; Rupniak and Williams1994; Smith et al. 1994; Vassout et al. 1994; Patacchiniand Maggi 1995; Bonnet et al. 1996; Chapman et al.1996; Herbert and Bernat 1996; Palframan et al. 1996;Ren et al. 1996). Moreover, agonists and antagonists atthe NK2 receptor (Hagan et al. 1991, 1993; Beresfordet al. 1995; Robineau et al. 1995; Kudlacz et al. 1997;Lecci et al. 1997) and at the NK3 receptor (Guard et al.1990; Edmonds-Alt et al. 1995; Patacchini et al. 1995;Nguyen-Le et al. 1996; Beaujouan et al. 1997; Sarauet al. 1997) were reported (Longmore et al. 1995; Rup-niak 1999).

Understanding the role of substance P in the brainhas been complicated by marked species differencesin the distribution of the tachykinin receptor types.There appears to be a relative increase in NK1 recep-tor density during evolution, such that NK3 receptorsara abundant in lower vertebrates and mammals but,like NK2 receptors, are apparently absent in humanbrain. Preclinical studies with substance P receptor an-tagonists have been hindered not only by phylogeneticdifferences in tachykinin receptor expression, but alsoby pharmacological heterogeneity of the NK1 receptorand the NK2 receptor. An other confounding feature ofneurokinin receptor antagonist is the blockade of Na+

and Ca2+ channels at high doses which produces ef-fects in various assays that are independent of recep-tor antagonism (Patacchini and Maggi 1995; Rupniak1999). Most developments were guided by the effectsof substance P as a pain neurotransmitter. Surprisingly,most clinical studies of analgesic activity of NK1 re-ceptor antagonists were negative (Rupniak and Kramer1999). However, clinical findings indicated that sub-stance P receptor antagonists ara able to alleviate de-pression and anxiety in patients suffering from majordepressive disorder (Kramer et al. 1998).

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Snider RM, Constantine JW, Lowe JA III, Longo KP, Lebel WS,Woody HA, Drozda SE, Desai MC, Vinick FJ, Spencer RW,Hess HJ (1991) A potent nonpeptide antagonist of the sub-stance P (NK1) receptor. Science 251:435–437

Vassout A, Schaub M, Gentsch C, Ofner S, Schilling W, Veen-stra S (1994) P7/CGP 49823, a novel NK1 receptor antag-onist: behavioural effects. Neuropeptides 26, Suppl 1:38

Von Euler US, Gaddum JH (1931) An unidentified depressorsubstance in certain tissue extracts. J Physiol 72:74–89

H.3.1.3.23H-Substance P Receptor Binding

PURPOSE AND RATIONALESubstance P is an undekapeptide which is widely dis-tributed in the central and peripheral nervous systemsand functions as a neurotransmitter/neuromodulator ina variety of physiological processes. Substance P isreleased from neurons in the midbrain in response tostress where it facilitates dopaminergic neurotransmis-sion and from sensory neurons in the spinal cord inthe response to noxious stimuli, where it excites dorsalneurons. In the periphery, release of substance P fromsensory neurons causes vasodilatation and plasma ex-travasation, suggesting a role in neurogenic inflamma-tion. Selective antagonists to substance P found in re-ceptor binding studies may elucidate the physiologicalrole of substance P and may be candidates for anti-inflammatory and analgesic drugs.

PROCEDUREFresh porcine brains are obtained from the slaugh-terhouse. Striata are dissected and homogenized(Ultraturrax) in 50 mM ice-cold Tris-HCl buffer,pH 7.4) containing 150 mM NaCl, 150 mM KCl,12 mM EDTA, 200 µM phenylmethylsulfonylfluoride,40 µg/ml bacitracin, 4 µ g/ml leupeptin, and 2 µg/mlchymostatin. These homogenates are then incubatedfor 30 min at 4°C before being centrifuged at 30,000 gfor 20 min at 4°C and washed twice with 50 mM Tris-HCl (pH 7.4) buffer. Pellets are resuspended in 0.32 Msucrose containing 200 µM phenylmethyl sulfonylflu-

oride and 40 µg/ml bacitracin before storage at –80°Cuntil use.

Sixty-minute incubations are carried out at roomtemperature in 50 mM Tris-HCl buffer, pH 7.4, con-taining various concentrations of [3H]substance P([3H]SP) (0.05–20 nM), 5 mM MgSO4, 40 mg/ml bac-itracin, 4 mg/ml leupeptin, and 2 mg/ml chymostatinin the presence of 0.8–1 mg of membrane protein ina final volume of 1 ml. Total binding and nonspe-cific binding are determined in triplicate in the ab-sence or presence of 1 mM unlabeled substance P. In-cubations are terminated by adding 4 ml of ice-coldTris-HCl buffer (pH 7.4) and membranes are filteredon Whatman glass fiber filters that are presoaked in0.5% polyethylenimine for a minimum of 3 h to reduceabsorption. Filters are then washed three times (5 mleach) using ice-cold Tris-HCl buffer (pH 7.4). Boundradioactivities are determined using a liquid scintilla-tion counter.

EVALUATIONSaturation and competition data are analyzed us-ing a computer program as described by McPherson(1985).

REFERENCES AND FURTHER READINGIversen LL, Jessell T, Kanazawa I (1976) Release and

metabolism of substance P in rat hypothalamus. Nature264:81–83

Lee CM, Javitch JA, Snyder SH (1983) 3H-Substance P bindingto salivary gland membranes. Mol Pharmacol 23:563–569

Liu YF, Quirion R (1991) Presence of various carbohydratemoieties including β-galactose and N-acetylglucosamineresidues on solubilized porcine brain neurokinin-1/substance P receptors. J Neurochem 57:1944–1950

McLean S, Ganong AH, Seeger TF, Bryce DK, Pratt KG,Reynolds LS, Siok CJ, Lowe III JA, Heym J (1991) Ac-tivity of binding sites in brain of a nonpeptide substance P(NK1) receptor antagonist. Science 251:437–439


Mizrahi J, D’Orléans-Juste P, Drapeau G, Escher E, Regoli D(1983) Partial agonists and antagonists for substance P. EurJ Pharmacol 91:139–140

Perrone MH, Diehl RE, Haubrich DR (1983) Binding of[3H]substance P to putative substance P receptors in ratbrain membranes. Eur J Pharmacol 95:131–133

H.3.1.3.3Neurokinin Receptor Binding

PURPOSE AND RATIONALEThe actions of tachykinins are mediated through threesubtypes of neurokinin receptors belonging to theG protein-linked receptor family, namely, NK1, NK2and NK3. Substance P displays highest affinity to NK1


receptors, whereas neurokinin A and neurokinin Bpreferably bind to NK2 and NK3 receptors, respec-tively. NK1 receptors are expressed in a wide variety ofperipheral tissues and in the CNS. NK2 receptors areexpressed primarily in the periphery, while NK3 recep-tors are primarily expressed in the CNS.

PROCEDURETachykinin NK1receptor binding assay is performedin intact Chinese hamster ovary (CHO) cells express-ing the human tachykinin NK1 receptor (Cascieriet al. 1992). The receptor is expressed at a level of3 × 105 receptors per cell. Cells are grown in a mono-layer culture, detached from the plate with enzyme-free cell dissociation solution (Specialty Media), andwashed prior to use in the assay. 125I[Tyr8]substance P(0.1 nM, 2000 Ci/mmol; New England Nuclear) is in-cubated in the presence or absence of test compounds(dissolved on 5 µl DMSO) with 5 × 104 CHO cells.Ligand binding is performed in 0.25 ml of 50 mM Tris-HCl, pH 7.5, containing 5 mM MnCl2, 150 mM NaCl,0.02% bovine serum albumin, 40 µg/ml bacitracin,0.01 mM phosphoramidon and 4 µg/ml leupeptin. Theincubation proceeds at room temperature until equi-librium is achieved (>40 min) and the receptor ligandcomplex is harvested by filtration over GF/C filterspresoaked in 0.1% polyethylenimine using a Tomtek96-well harvester. Nonspecific binding is determinedusing excess substance P (1 µM) representing <10% oftotal binding.

For NK2receptor binding assays membranes ofCHO cells transfected with human ileum NK2 receptorare used (Hagan et al. 1993; Beresford et al. 1995). Themembrane suspensions (5 µg protein) in assay buffer(Tris base (50 mM), MnCl2 (3 mM), bovine serum al-bumin (0.05%), chymostatin (2 µg/ml) and leupeptin4 µg/ml, pH 7.4) are incubated for 90 min at roomtemperature with wash buffer (Tris base (50 mM),MnCl2 (3 mM), lauryl sulphate (0.01%), pH 7.4) ortest compound, and [3H]-GR100679 (0.5 nM final con-centration). Non-specific binding is defined by use ofGR159897 (1 µM).

For NK3 receptor binding assays guinea pig cor-tical membranes (Guard et al. 1990) are incubated atroom temperature for 60 min with HEPES wash bufferor test compound and [3H]-senktide (final concentra-tion 0.8–1.0 nM). Non-specific binding is defined byaddition of eledoisin (10 µM).

EVALUATIONInhibition curves are analyzed and pIC50 values calcu-lated by use of a curve fitting program. pIC50 values

are converted to inhibition constants (pKi values) us-ing the Cheng Prussoff equation

Ki = IC50/(1 + L/KD)

where L is the ligand concentration and KD is the dis-sociation constant. The KD and Bmax (maximum num-ber of binding site per mg of tissue) are determinedfrom saturation curves and analyzed by a curve fittingprogram. Values are expressed as means ±SEM.

MODIFICATIONS OF THE METHODWatson et al. (1955) performed substance P bind-ing assays in ferret brain membranes and assessedneurokinin NK1 receptor binding using human lym-phoblasts (IM-9 cells).

Rupniak et al. (1997) studied displacement of 125I-[Tyr8]substance P binding to cloned human tachykininNK1 receptors and to ferret brain membranes in vitro.

Beattie et al. (1995) used U373 MG cell membranesand cerebral cortical membranes from rat, ferret andgerbil and [3H]substance P for NK1 receptor bindingassays.

Bonnet et al. (1996), McLean et al. (1996) used theIM9 lymphoblastoma cell line expressing the humanNK1 receptor.

Emonds-Alt et al. (1995) studied binding of [125I]Bolton-Hunter labelled substance P to NK1receptorsof rat brain cortex, human lymphoblast cells (IM9),and human astrocytoma cells (U373MG, STTG1),binding of [125I]iodohistidyl-NKA (or [125I]neuro-peptide γ ) to NK2receptors of rat or hamsterurinary bladder or guinea pig ileum, binding of[125I]iodohistidyl-[Me-Phe7]NKB (or [125I]Eledoisin)to tachykinin NK3 receptors of rat, guinea pig andgerbil brain cortex., and of [125I]iodohistidyl-[Me-Phe7]NKB to the human NK3 receptor, cloned and ex-pressed in CHO cells (Buell et al. 1992).

Cascieri et al. (1992) described the binding of a po-tent, selective, radioiodinated antagonist to the humanneurokinin-1 receptor.

A radioligand of the tachykinin NK2 receptor wasdescribed by Renzetti et al. (1998).

Jordan et al. (1998) evaluated the Cytosensor micro-physiometer, a system that measures the extracellularacidification rate as an index of the integrated func-tional response to receptor activation, as a method tostudy NK3 receptor pharmacology and used this sys-tem to assess the functional activity of novel com-pounds at this receptor.

Appell et al. (1998) reported biological character-ization of neurokinin antagonists discovered through


screening of a combinatorial library. Using stablytransfected CHO-K1 cell lines expressing human NK-1, NK-2 and NK-3 receptor subtypes and europiumtime-resolved fluorescence, primary receptor bindingassays were designed to define active compounds. Inaddition, a secondary, functional assay measuring in-tracellular calcium flux with the calcium-sensitive flu-orophore, fluo-3, in CHO cells transfected with the hu-man NK-1 or NK-2 receptor was used to determine ag-onist or antagonist activities.

REFERENCES AND FURTHER READINGAppell KC, Chung TDY, Solly KJ, Chelsky D (1998) Bio-

logical characterization of neurokinin antagonists discov-ered through screening of a combinatorial library. J BiomolScreening 3:19–27

Beattie DT, Beresford IJM, Connor HE, Marshall FH, HawcockAB, Hagen RM, Bowers J, Birch PJ, Ward P (1995) Thepharmacology of GR203040, a novel, potent and selec-tive tachykinin NK1 receptor antagonist. Br J Pharmacol116:3149–3157

Beresford IJM, Ball DI, Sheldrick RGL, Turpin MP, Walsh DM,Hawcock AB, Coleman RM, Tyers MB (1995) GR 159897,a potent, non-peptide antagonist at NK2 receptors. Eur JPharmacol 272:241–248


Buell G, Schulz MF, Arkinstall SJ, Maury K, Missotten M,Adami N, Talabot F, Kawashima E (1992) Molecularcharacterization, expression and localization of humanneurokinin-3 receptor. FEBS Lett 299:90–95

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Emonds-Alt X, Bichon D, Ducoux JP, Heaulme M, Miloux B,Poncelet M, Proietto V, Van Broeck D, Vilain P, Neliat G,Soubrié P, Le Fur G, Brelière JC (1995) SR 142801, thefirst potent non-peptide antagonist of the tachykinin NK3receptor. Life Sci 56:27–32

Guard S, Watson SP, Maggio JE, Phon Too H, Watling KJ (1990)Pharmacological analysis of [3H]-senktide binding to NK3tachykinin receptors in guinea pig ileum longitudinal mus-cle-myenteric plexus and cerebral cortex membranes. Br JPharmacol 99:767–773

Hagan RM, Beresford IJ, Stables J, Dupere J, Stubbs CM, El-liott PJ, Sheldrick RL, Chollet A, Kawashima E, McEl-roy AB, Ward P (1993) Characterization, CNS distribution

and function of NK2 receptors studied using potent NK2receptor antagonists. Regul Peptides 46:9–19

Jordan RE, Smart D, Grimson P, Suman-Chauhan N, McK-night AT (1998) Activation of the cloned human NK3 re-ceptor in Chinese hamster ovary cells characterized by thecellular acidification using the Cytosensor microphysiome-ter. Br J Pharmacol 125:761–766

Longmore J, Swain CJ, Hill RG (1995) Neurokinin receptors.Drug News Perspect 8:5–12

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Matuszek MA, Zeng XP, Strigas J, Burcher E (1998) An investi-gation of tachykinin NK2 receptor subtypes in the rat. EurJ Pharmacol 352:103–109

McLean S, Ganong A, Seymour PA, Bryce DK, Crawford RT,Morrone J, Reynolds LS, Schmidt AW, Zorn S, Watson J,Fossa A, DePasquale M, Rosen T, Nagahisa A, TsuchiyaM, Heym J (1996) Characterization of CP-122,721, a non-peptide antagonist of the neurokinin NK-1 receptor. J Phar-macol Exp Ther 277:900–908

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Nakanishi S (1991) Mammalian tachykinin receptors. Annu RevNeurosci 14:123–136

Otsuka M, Yoshioka K (1939) Neurotransmitter functions ofmammalian tachykinins. Phys Rev 73:229–308

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Regoli D, Boudon A, Fachere JL (1994) Receptors and antago-nists for substance P and related peptides. Pharmacol Rev46:551–559

Renzetti AR, Catalioto RM, Criscuoli M, Cucchi P, Lippi AS,Guelfi M, Quartara L, Maggi CA (1998) Characterizationof [3H]MEN 11420, a novel glycosylated peptide antago-nist of the tachykinin NK2 receptor. Biochem Biophys ResCommun 248:78–82

Rupniak NMJ, Tattersall FD, Williams AR, Rycroft W, CarlsonEJ, Cascieri MA, Sadowski S, Ber E, Hale JJ, Mills SG,McCoss M, Seward E, Huscroft I, Owen S, Swain CJ, HillRG, Hargreaves RJ (1997) In vitro and in vivo predictors ofthe anti-emetic activity of tachykinin NK1 receptor antag-onists. Eur J Pharmacol 326:201–209

Snider RM, Constantine SJW, Lowe JA III, Longo KP, LebelWS, Woody HA, Drozda SE, Desai MC, Vinick FJ,Spencer RW, Hess HJ (1991) A potent nonpeptide an-tagonist of the substance P (NK1) receptor. Science251:435–437

Watson JW, Gonsalves SF, Fossa AA, McLean S, Seeger T,Obach S, Andrews PLR (1995) The anti-emetic effects ofCP-99,994 in the ferret and the dog: Role of the NK1 re-ceptor. Br J Pharmacol 115:84–94

H.3.1.3.4Characterization of Neurokinin Agonistsand Antagonists by Biological Assays

PURPOSE AND RATIONALESeveral biological assays have been used to character-ize neurokinin agonists and antagonists on their recep-tors (Review by Regoli et al. 1994).

The following functional assays are recommendedfor evaluation of antagonists:


For NK1Receptors

• Inhibition of [Sar9,Met(O2)11]substance P-inducedendothelium-dependent relaxation of rabbit pul-monary artery, previously contracted with 0.1 µMnoradrenaline (D’Orléans-Juste et al. 1986; Rubinoet al. 1992; Emonds-Alt et al. 1993),

• inhibition of [Sar9,Met(O2)11]substance P or[Sar9]substance P sulfone-induced contractions ofguinea pig ileum in the presence of 3 µM atropineand 3 µM mepyramine and indomethacin (Dion etal. 1987; Emonds-Alt et al. 1993; Patacchini et al.1995; Hosoki et al. 1998; Walpole et al. 1998),

• rabbit vena cava stimulated by substance P or[Sar9,Met(O2)11]substance P (Nantel et al. 1991;Regoli et al. 1994; Gitter et al. 1995; Robineau et al.1995; Bonnet et al. 1996; Nguyen-Le et al. 1996),

• inhibition of substance P-induced relaxation of theisolated dog carotid artery previously contractedwith norepinephrine (Snider et al. 1991),

• rat urinary bladder, stimulated by the selectiveagonist [Sar9,Met(O2)11]substance P and treatedwith SR 48986 (1.7 × 10−7 mol/l) to eliminate NK-2 functional sites (Rouissi et al. 1993; Nguyen-Leet al. 1996),

• Ca2+ mobilization in rat vas deferens (Nagata et al.1991),

• inhibition of substance P-induced plasma ex-travasation in the bladder and bronchi of theguinea pig (Bonnet et al. 1996),

• inhibition of substance P-induced vasodilation inthe nasal mucosa of pigs using an acoustic rhi-nometer (Rinder and Lundberg 1996),

• inhibition of [Sar9,Met(O2)11]substance P-induced plasma extravasation in guinea-pigbronchi (Cirillo et al. 1998),

• inhibition of methacholine-induced contractionsof isolated rat tracheal strips (Tian et al. 1997),

• inhibition of cyclophosphamide- and radiation-induced damage in the rat and ferret organs (Al-fieri and Gardner 1997, 1998),

• inhibition of edema formation induced by sub-stance P and antigen in rat skin (Herbert andBernat 1996),

• inhibition of reciprocal hindlimb scratching afterintracerebroventricular injection of substance P,[Sar9, Met(O2)11]substance P or septide in mice(Jung et al. 1994) or gerbils (Smith et al. 1994),

• inhibition of turning behavior after intrac-erebroventricular injection of substance P,[Sar9,Met(O2)11] substance P or septide in mice(Jung et al. 1994),

• inhibition of hind paw tapping and chromodacry-orrhea after intracerebroventricular injectionof tachykinin agonists in gerbils (Graham et al.1993; Bristow and Young 1994; Rupniak andWilliams 1994; Rupniak et al. 1995, 1997; Vassoutet al. 1994),

• inhibition of cis-platin-induced emesis in ferrets(Rupniak et al. 1997; Singh et al. 1997; Minamiet al. 1998).

For NK2Receptors

• Inhibition of neurokinin A-induced contraction ofisolated rabbit aorta (Snider et al. 1991),

• inhibition of neurokinin A-induced contractionof isolated endothelium-deprived rabbit pul-monary artery or hamster trachea (D’Orléans-Juste et al. 1986; Emonds-Alt et al. 1993; Patacchiniet al. 1995),

• the hamster urinary bladder (Dion et al. 1987;Maggi et al. 1990; Regoli et al. 1994; Emonds-Altet al. 1997; Tramontana et al. 1998),

• inhibition of motor responses induced by intrav-esival administration of capsaicin in rats in vivo(Lecci et al. 1997),

• rat esophageal tunica muscularis (Croci et al.1995),

• inhibition of turning behavior induced by intras-triatal injection of Nle10-neurokinin A in mice(Emonds-Alt et al. 1997).

For NK3Receptors

• Inhibition of senktide- or neurokinin B-inducedcontractions of the rat portal vein (Mastrangeloet al. 1987; Snider et al. 1991; Emonds-Alt et al.1993; Patacchini et al. 1995),

• antagonism against senktide-induced contrac-tions in the isolated rabbit iris sphincter muscle(Medhurst et al. 1997; Sarau et al. 1997),

• inhibition ofcolonic propulsion in rats (Broccardoet al. 1999)

• inhibition of turning behavior induced by intras-triatal injection of senktide in gerbils (Emonds-Alt et al. 1994),

• inhibition of citric acid-induced cough in guineapigs (Daoui et al. 1998).

• The failure of NK1 receptor antagonists in mostclinical tests for analgesia in spite of clear preclin-ical data is a matter of discussion (Hill R 2000a, b;Urban and Fox 2000).


REFERENCES AND FURTHER READINGAlfieri A, Gardner C (1997) The NK-1 antagonist GR203040

inhibits cyclophosphamide-induced damage in the rat andferret bladder. Gen Pharmacol 29:245–250

Alfieri A, Gardner C (1998) Effects of GR203940, an NK-1 an-tagonist, on radiation- and cisplatin-induced tissue damagein the ferret. Gen Pharmacol 31:741–746


Bristow LJ, Young L (1994) Chromodacryorrhea and repeti-tive hind paw tapping: models of peripheral and centraltachykinin NK1 receptor activation in gerbils. Eur J Phar-macol 253:245–252

Broccardo M, Improta G, Tabacco A (1999) Central tachykininNK3 receptors in the inhibitory action on rat colonicpropulsion of a new tachykinin, PG-KII. Eur J Pharmacol376:67–71

Cirillo R, Astolfi M, Conte B, Lopez G, Parlani M, Terracci-ano R, Fincham CI, Manzini S (1998) Pharmacology of thepeptidomimetic, MEN 11149, a new potent, selective andorally effective tachykinin NK1 receptor antagonist. Eur JPharmacol 341:201–209

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Daoui S, Cognon C, Naline E, Emonds-Alt X, Advenier C(1998) Involvement of tachykinin NK3 receptors in citric-acid-induced cough and bronchial responses in guinea pigs.Am J Respir Crit Care Med 158:42–48

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Emonds-Alt X, Advenier C, Cognon C, Croci T, Daoul S,Ducoux JP, Landl M, Maline E, Nellat G, Poncelet M,Proletto V, Von Broeck D, Vilain P, Soubrié P, Le Fur G,Maffrand JP, Brelière JC (1997) Biochemical and phar-macological activities of SR 144190, a new potent non-peptide tachykinin NK2 receptor antagonist. Neuropeptides31:449–458

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Hill R (2000b) Reply: will changing the testing paradigms showthat NK1 receptor antagonists are analgesic in humans?Trends Pharmacol Sci 21:265

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Rupniak NMJ, Webb JK, Williams AR, Carlson E, Boyce S,Hill HG (1995) Antinociceptive activity of the tachykininNK1 receptor antagonist, CP-99994, in conscious gerbils.Br J Pharmacol 116:1937–1943

Rupniak NMJ, Tattersall FD, Williams AR, Rycroft W, Carl-son EJ, Cascieri MA, Sadowski S, Ber E, Hale JJ, MillsSG, MacCoss M, Seward E, Huscroft I, Owen S, Swain CJ,Hill RG, Hargreaves RJ (1997) In vitro and in vivo predic-tors of the anti-emetic activity of tachykinin NK1 receptorantagonists. Eur J Pharmacol 326:201–209

Sarau HM, Griswold DE, Potts W, Foley JJ, Schmidt DB,Webb EF, Martin LD, Brawner ME, Elshourbagy NA,Medhurst AD, Giardina GAM, Hay DWP (1997) Non-peptide tachykinin receptor antagonists: I. Pharmacolog-ical and pharmacokinetic characterization of SB 223412,a novel, potent and selective neurokinin-3 receptor antago-nist. J Pharmacol Exp Ther 281:1303–1311

Singh L, Field MJ, Hughes J, Kuo BS, Suman-Chauhan N, Tu-ladhar BR, Wright DS, Naylor RJ (1997) The tachykininNK1 receptor antagonist PD 154075 blocks cisplatin-in-duced delayed emesis in the ferret. Eur J Pharmacol321:209–216

Snider RM, Constantine JW, Lowe JA III, Longo KP, Lebel WS,Woody HA, Drozda SE, Desai MC, Vinick FJ, Spencer RW,Hess HJ (1991) A potent nonpeptide antagonist of the sub-stance P (NK1) receptor. Science 251:435–437

Smith G, Harrison S, Bowers J, Wiseman J, Birch P (1994) Non-specific effects of the tachykinin NK1 receptor antagonist,CP-99,994, in antinociceptive tests in rat, mouse and gerbil.Eur J Pharmacol 271:481–487

Tian J, Wei EQ, Chen JS, Zhang WP (1997) Effect of SR140333, a neurokinin NK1 receptor antagonist, on airwayreactivity to methacholine in sedated rats. Acta PharmacolSin 18:485–488

Tramontana M, Patacchini R, Lecci A, Giuliani S, Maggi CA(1998) Tachykinin NK2 receptors in the hamster urinarybladder: In vitro and in vivo characterization. Naunyn-Schmiedeberg’s Arch Pharmacol 358:293–300

Urban LA, Fox A (2000) NK1 receptor antagonists – are theyreally without effects in the pain clinic? Trends PharmacolSci 21:462–464

Vassout A, Schaub M, Gentsch C, Ofner S, Schilling W,Veenstra S (1994) P7/CGP 49823, a novel NK1 re-ceptor antagonist: behavioural effects. Neuropeptides 26,Suppl 1:38

Walpole CSJ, Brown MCS, James IF, Campbell EA, McIntyreP, Docherty R, Ko S, Hedley L, Ewan S, Buchheit KH,Urban LA (1998) Comparative, general pharmacology ofSDZ NKT 343, a novel, selective NK1 receptor antagonist.Br J Pharmacol 124:83–92

H.3.1.4Assay of Polymorphonuclear LeukocyteChemotaxis In Vitro

PURPOSE AND RATIONALELeukocyte accumulation is an important aspect ofhost defense mechanisms. Chemotactic factors attractleukocytes to an infected or inflamed site. The methodof Boyden (1962) has been widely employed to mea-sure the chemotactic effects on polymorphonuclearleukocytes. Watanabe et al. (1989) described a rapidassay of polymorphonuclear leukocyte chemotaxis invitro.

PROCEDURETwo 96-well tissue-culture plates are utilized as oneset of multiple Boyden chambers. One plate as mul-tiple lower compartments and the other plate upsidedown as multiple upper compartments can be tightlysandwiched with the aid of 12 sets of bolts. The holes,into which bolts are set and polymorphonuclear leuko-cytes (PMN) suspensions are introduced, are openedby a heated stick. Eight sets of 6 holes are made onthe bottom of the upper plate to serve as multiple up-per compartments for introducing PMN suspensions.Eight sets of polycarbonate filter (approximately 3.2 ×2.2 cm), cut from a round filter (Nuclepore Co.) withpores of 2 µm in diameter, are sandwiched betweenthe upper and lower plates. One sheet of the filter canseparate six sets of the upper compartments from thelower ones. Silicon grease is spread on all the platesurfaces that attach to the filters. The lower compart-ments are filled with a chemoattractant (400 µ l/well)diluted in RPMI-1640 medium. The eight sheets offilter paper are carefully placed on each set of thelower compartments to avoid air bubbles. The upperplate is positioned over the lower plate and fastenedwith bolts. The upper compartments are filled with0.3 ml of PMN suspension (at 107 cells/ml). The as-sembly is incubated at 37°C for 60 min in a humid-ified atmosphere containing 5% CO2. Then the fluidin the upper compartment is decanted and the up-per compartments are completely washed with a jetof water. The lower plate is centrifuged at 2400 rpmfor 5 min at room temperature and the supernatantin the well is decanted. The pellet of PMNs is dis-persed in 200 µl of phosphate buffered saline contain-ing 0.1% EDTA. Absorbance at 660 nm of each wellcontaining PMN suspension is determined with a 96-well microplate reader. The number of PMNs in thelower compartments is further determined by a Coul-ter counter.


EVALUATIONThe migration rate is calculated as percentage from thenumber of PMNs in the lower compartment/numberof PMNs applied in the upper compartment. Themigration rate is dependent on the concentration ofthe chemoattractant (e. g., zymosan-activated serum).Moreover, a dose dependent decrease of migration rateis achieved by chemotaxis inhibitors.

MODIFICATIONS OF THE METHODNelson et al. (1975) described chemotaxis underagarose as a simple method for measuring chemotaxisand spontaneous migration of human polymorphonu-clear leukocytes and monocytes.

Migration of PMNs in agarose gel was measuredafter fixation with glutaraldehyde and staining withGiemsa by Shalaby et al. (1987).

Optimal conditions for simultaneous purification ofmononuclear and polymorphonuclear leukocytes fromhuman blood were described by Ferrante and Thong(1980).

PMN chemotaxis was measured in multiwell mi-crochemotaxis chambers separated by 5-µm pore sizepolyvinylpyrrolidone-free polycarbonate membranesby Harvath et al. (1980), Figari et al. (1987).

REFERENCES AND FURTHER READINGAtkins PC, Norman ME, Zweiman B (1978) Antigen-induced

neutrophil chemotactic activity in man. J Allergy Clin Im-munol 62:149–155

Boyden S (1962) The chemotactic effects of mixtures of anti-body and antigen on polymorphonuclear leukocytes. J ExpMed 115:453–466

Bray MA, Ford-Hutchinson AW, Shipley ME, Smith MJH(1980) Calcium ionophore A23187 induces release ofchemokinetic and aggregating factors from polymorphonu-clear leukocytes. Br J Pharmacol 71:507–512

Camussi G, Tetta C, Bussolino F, Baglioni C (1990) An-tiinflammatory peptides (antiflammins) inhibit synthesisof platelet-activating factor, neutrophil aggregation andchemotaxis, and intradermal inflammatory reactions. J ExpMed 171:913–927

Ferrante A, Thong YH (1980) Optimal conditions for simul-taneous purification of mononuclear and polymorphonu-clear leukocytes from human blood by the Hypaque-Ficollmethod. J Immunol Meth 36:109–117

Figari IS, Mori NA, Palladino MA (1987) Regulation ofneutrophil migration and superoxide production by re-combinant tumor necrosis factors-α and -β: Comparisonto recombinant interferon-γ and interleukin-1α. Blood70:979–984

Harvath L, Falk W, Leonard EJ (1980) Rapid quantitation ofneutrophil chemotaxis: Use of a polyvinylpyrrolidone-freepolycarbonate membrane in a multiwell assembly. J Im-munol Meth 37:39–45

Issekutz AC, Issekutz TB (1989) Quantitation of blood cell ac-cumulation and vascular responses in inflammatory reac-tions. In: Pharmacological Methods in the Control of In-flammation. Alan R. Liss, Inc., pp 129–150

Matzner Y, Drexler R, Levy M (1984) Effect of dipyrone, acetyl-salicylic acid and acetaminophen on human neutrophilchemotaxis. Eur J Clin Invest 14:440–443

Nelson RD, Quie PG, Simmons RL (1975) Chemotaxis un-der agarose: a new and simple method for measuringchemotaxis and spontaneous migration of human poly-morphonuclear leukocytes and monocytes. J Immunol115:1650–1656

Roch-Arveiller M, Roblin G, Allain M, Giroud JP (1985) A vi-sual technique of chemotactic assessment for pharmacolog-ical studies. J Pharmacol Meth 14:313–321

Shalaby MR, Palladino MA, Hirabayashi SE, Eessalu TE,Lewis GT, Shepard HM, Aggarwal BB (1987) Receptorbinding and activation of polymorphonuclear neutrophilsby tumor necrosis factor-alpha. J Leukoc Biol 41:196–204

Watanabe K, Kinoshita S, Nakagawa H (1989) Very rapid as-say of polymorphonuclear leukocyte chemotaxis in vitro.J Pharmacol Meth 22:13–18

H.3.1.5Polymorphonuclear Leukocytes AggregationInduced by FMLP

PURPOSE AND RATIONALEAggregation of polymorphonuclear leukocytes(PMNs) can be induced by FMLP (formyl-L-methi-onyl-L-leucyl-L-phenylalanine). The aggregation canbe inhibited by xanthine derivatives.

PROCEDUREPMNs cell suspensions are prepared from peritonealexudates obtained 17 h after intraperitoneal injec-tion of 10 ml 6% sodium caseinate into SpragueDawley rats. The cells are washed twice in Geys-balanced-salt-solution (Gibco GBSS) and resuspendedto a final concentration of 15 × 106 cells/ml. Thetest compounds and the standard (pentoxiphylline)are dissolved in GBSS. FMLP (formyl-L-methionyl-L-leucyl-L-phenylalanine) is dissolved in DMSO. Thefurther dilutions are made up to a final concentration of10−7 mol FMLP in GBSS. Before addition of FMLP,the cell suspensions are pre-incubated for 10 min withthe drugs. PMNs aggregation is carried out in a Bornaggregometer.

EVALUATIONThe results are expressed as change in transmittance,measured in mm on the recorder. The mean peak ofthe untreated cells is set 100%.

MODIFICATIONS OF THE METHODMoqbel et al. (1986) measured the activation of humanleukocytes after FMLP in a rosette assay by the changein the expression of complement (C3b) and IgG (Fc)


receptors and in a cytotoxic assay by the in vitro capac-ity to adhere to and kill the complement-coated larvae(schistosomula) of Schistosoma mansoni.

Bradford and Rubin (1986) determined the effect ofvarious drugs on IP3 accumulation evoked by FMLPin neutrophils from New Zealand white rabbits.

Bourgoin et al. (1991) studied the influence of gran-ulocyte-macrophage colony-stimulating factor (GM-CSF) on phosphatidylcholine breakdown by phospho-lipase D in human neutrophils.

REFERENCES AND FURTHER READINGBradford PG, Rubin RP (1986) The differential effects of ne-

docromil sodium and sodium cromoglycate on the secre-tory response of rabbit peritoneal neutrophils. Eur J RespirDis 69 (Suppl 147):238–240

Bray MA, Ford-Hutchinson AW, Shipley ME, Smith MJH(1980) Calcium ionophore A23187 induces release ofchemokinetic and aggregating factors from polymorphonu-clear leucocytes. Br J Pharmacol 71:507–512

Bourgoin S, Borgeat P, Poubelle PE (1991) Granulocyte-macrophage colony-stimulating factor (GM-CSF) primeshuman neutrophils for enhanced phosphatidylcholinebreakdown by phospholipase D. Agents Actions 34:32–34

Moqbel R, Walsh GM, Kay AB (1986) Inhibition of humangranulocyte activation by nedocromil sodium. Eur J RespirDis 69 (Suppl 147):227–229

H.3.1.6Constitutive and Inducible Cellular Arachidonic AcidMetabolism In Vitro

PURPOSE AND RATIONALEThe various metabolites of arachidonic acid are in-volved in many inflammatory processes. Arachidonicacid is released from the cellular phospholipid fractionby the action of phospholipase A2, and subsequentlymetabolized via two major routes: the cyclooxyge-nase pathway yielding the primary prostaglandins andthromboxane, and the 5-lipoxygenase pathway yield-ing the leukotrienes. Thromboxanes, prostaglandins,and leukotrienes play a pathophysiological role inmany diseases. Receptor assays for these autocoidswere developed (Haluska et al. 1989). Murata et al.(1997) produced mice lacking the prostaglandin recep-tor which showed increased susceptibility to throm-bosis and altered pain reception and inflammatory re-sponse.

The therapeutic mode of action of the classicalnon-steroidal anti-inflammatory drugs (NSAID), suchas aspirin or indomethacin, is primarily explained bytheir inhibitory effect on cyclooxygenase, the key en-zyme of the prostaglandin pathway. Inhibitors of the5-lipoxygenase pathway have attracted considerable

attention as potential anti-inflammatories with highpotency. Appropriate assay systems for the determi-nation of the different eicosanoids allow to studythe influence of drugs towards the specific path-ways of the arachidonic acid cascade in various cel-lular systems (Samuelsson 1986; Vane and Botting1987).

According to recent discoveries there are two formsof cyclooxygenase (Xie et al. 1992; Lee et al. 1992;Gierse et al. 1996). Cyclooxygenase-1 (COX-1) isfound as a constitutive enzyme in most tissues in-cluding blood platelets. Prostaglandins generated byconstitutive pathways may exert cytoprotective effects,and are involved in maintaining vital functions in vas-cular hemostasis, gastric mucosa and kidney.

Chandrasekharan et al. (2002) described the clo-ning, structure and expression COX-3, a cyclo-oxygenase-1 variant, which is selectively inhibited byanalgesic/antipyretic drugs, such as acetaminophen,phenacetin, antipyrine, and dipyrone.

The inhibition of these prostaglandins by the clas-sical cyclooxygenase inhibitors is now generally ac-cepted as an explanation of their adverse side effects.

COX-2 which shares about 62% amino acid homol-ogy with COX-1, is only expressed after cell activa-tion, especially by mitogenic or inflammatory stim-uli (Herrmann et al. 1990; Funk et al. 1991; Crof-ford 1997). Thus, specific suppression of the COX-2-pathway may represent a superior target for the eval-uation of new antiinflammatory drugs. Drugs whichhave a high potency on COX-2 and a favorable COX-2/COX-1 ratio have potent anti-inflammatory activitywith fewer side effects (Riendeau et al. 1997; Vane1998; Hawkey 1999; Song et al. 1999; Chan et al.1999). Shigeta et al. (1998) described the role ofcyclooxygenase-2 (COX-2) in the healing of gastriculcers in rats. Hull et al. (2005) investigated the ex-pression of cyclooxygenase-1 and -2 by human gastricendothelial cells.

The cardiovascular value of selective COX-2 in-hibitors has been questioned because they selectivelyreduce prostacyclin production, thus disrupting thehormonal balance and promoting a prothrombotic state(Hankey and Eikelboom 2003).

These theoretical concerns were supported bythe results of clinical trials demonstrating an in-creased risk of myocardial infarction with COX-2inhibitors compared with conventional non-steroidalanti-inflammatory drugs (NSAIDs) (Bombadier et al.2000). The debate on benefit-risk assessment of COX-2 inhibitors is ongoing (Bing 2003; Schmidt et al.2004).


H.3.1.6.1Formation of Leukotriene B4in Human White Blood Cells In Vitro

PROCEDUREHuman white blood cells are prepared according tothe standard procedure published by Salari et al.(1984): 40 ml freshly drawn citrated blood are ad-mixed with 8 ml of PM16-buffer, containing 6% (v/v)dextran (MW = 480,000), and incubated at room tem-perature for one hour. The supernatant containing thewhite blood cell fraction is removed, diluted 1:1 (v/v)with PM16 and centrifuged for 15 min at 300 g. Theprecipitate is resuspended in PM16 and adjusted to1010 cells/l (Counter HT, Coulter Electronics, Krefeld,FRG).

The metabolism of endogenously bound arachi-donic acid to LTB4 is measured in a total volume of0.3 ml of the cell suspension at 37°C. The reactiontube contains 2 mmol/l CaCl2, 0.5 mmol/l MgCl2 andthe investigational drug. After 15 min pre-incubationthe reaction is started by addition of 12.5 µg of the Ca-ionophore A 23187 and 2 µg glutathione. After 5 minthe reaction is stopped with 30 µl 0.1 M HCl at 0°C.After centrifugation for 2 min at 0°C, aliquots of thesupernatant are subjected to HPLC, similarly as de-scribed by Veenstra et al. (1988), using a C-18 Nu-cleosil column (5 µm, 100 × 3 mm, Chrompack GmbH,Frankfurt, FRG) and, at a flow rate of 0.7 ml/min, a sol-vent mixture consisting of 725 ml methanol, 275 mlwater and 0.1 ml acetic acid. Authentic standard drugsare used to identify cis-, trans- and epi-LTB4. The sep-aration of the three isomers can be followed photomet-rically at the UV-maximum of 278 nm.

EVALUATIONThe peak areas of cis-, trans- and epi-LTB4 are mea-sured as a function of drug concentration, and relatedto a control experiment without drug.

CRITICAL ASSESSMENTThe tested pathway involves the enzymatic steps ofphospholipase A2 and the 5-lipoxygenase. Thus in-hibitors of these enzymes are to decrease formation ofthe three isomers of LTB4. The step of phospholipaseA2 can be circumvented by the addition of exogenousarachidonic acid (1 µmol/l).

Inhibitors of LTA4-hydrolase which catalyzes theintermediary conversion of LTA4 to the biologicallyactive cis-LTB4, can also be identified by this assay.Such drugs are to exhibit an increased formation ofthe non-enzymatic hydrolysis products trans- and epi-LTB4 at the expense of decreased cis-LTB4.

MODIFICATIONS OF THE METHODWinkler et al. (1988) used differentiated U-937 cellsexpressing LTB4 receptors to study Ca2+ mobilizationin response to LTB4.

Jones et al. (1995) tested inhibition of [3H]leu-kotriene D4 specific binding in guinea pig lung, sheeplung, and dimethylsulfoxide-differentiated U837 cellplasma membrane preparations.

H.3.1.6.2Formation of Lipoxygenase Products from14C-Arachidonic Acid in Human PolymorphonuclearNeutrophils (PMN) In Vitro

PROCEDUREPMN are prepared by the standard procedure ofBöyum (1976). The first steps are carried out at roomtemperature: 50 ml citrated human blood are cen-trifuged at 130 g for 15 min. The pellet is resuspendedin 20 ml Dulbecco’s minimal essential medium, andsubsequently underlayered with 15 ml lymphoprep.After centrifugation at 400 g for 25 min the pellet isresuspended in 28 ml Dulbecco’s HBSS containing3% dextran. After incubation at 0°C for 120 minthe decanted supernatant is centrifuged at 400 g for15 min. The pellet is resuspended in 1 ml Dulbecco’sPBS, containing 11 µmol/l glucose, to a leukocytecount of 2 × 1010/l.

The method of HPLC determination of cellularmetabolites of exogenous 1-C-14-arachidonic acid, aspublished by Borgeat and Samuelsson (1979), is mod-ified as briefly described:

0.1 ml of the leukocyte suspension are incubatedin Dulbecco’s phosphate buffered saline (DPBS) at37°C with the test drugs for 15 min. The incubationis then interrupted by cooling in an ice bath. Cal-cium ionophore A 23187 (final concentration 7 × 10−5

mol/l) and 1-C-14-arachidonic acid (final concentra-tion 8.4 × 10−5 mol/l, 0.5 µCi) are added, and, aftera second incubation period of 15 min at 37°C, ter-minated by the addition of 0.4 ml methanol. The as-say mixture is then extracted with chloroform. Thechloroform is evaporated, and, after redissolving theresidue in a minor amount of methanol/water, ana-lyzed by HPLC and radiomonitoring for the C-14-eicosanoids.

HPLC-conditions are as follows: Column: Nucle-osil C-18, 5 µm; organic phase: 700 ml methanol,300 ml water, 0.1 ml acetic acid; after 35 min change topure methanol. Flow: 1 ml/min, 2000 psi. Radiomoni-tor: LB 507 (Berthold, Wildbad, FRG).


A viability assay (trypan blue exclusion) ascertainsthat cells remain intact during incubation periods.

EVALUATIONThe radioactivity of the separated 5-HETE and LTB4is measured as a function of drug concentration, andrelated to a control experiment without drug. Two fur-ther lipoxygenase products, 12-HETE and 15-HETE,which are additionally generated under the test condi-tions, can be quantified in a similar way.

CRITICAL ASSESSMENTThe measured reaction sequence starts with arachi-donic acid, and involves its transformation to 5-HETEby 5-lipoxygenase, as well as the subsequent enzy-matic hydrolysis to LTB4. Inhibitors of 5-lipoxygenaseexhibit a decreased formation of 5-HETE and LTB4.Effects of drugs on the side products 12-HETE and 15-HETE can also be studied in this test system.

H.3.1.6.3Formation of Eicosanoids from 14C-Arachidonic Acidin Human Platelets In Vitro

PROCEDUREBlood is drawn from the vena brachialis of healthyvolunteers and collected into plastic tubes containingsodium citrate (0.38% final concentration (w/v)). Af-ter centrifugation at 100 g for 15 min the platelet richplasma (PRP) containing about 2,5 × 1011 platelets/l(Counter HT, Coulter Electronics, Krefeld, FRG) issaved, and kept at 20°C no longer than one houruntil the experiment is started. Metabolism of 14C-arachidonic acid is followed by the HPLC procedurespublished by Weithmann et al. (1994), modifying themethod of Powell (1985). PRP is mixed with thesame volume of a citrate/D-glucose solution (27.35 gtrisodium citrate· 2H2O, 1.47 g citric acid and 27.74 gD(+)-glucose· H2O ad 1 l water) and centrifuged at1000 g for 20 min (4°C). The saved precipitate is re-suspended in Dulbecco’s solution (DPBS, Serva, Hei-delberg, FRG) in the original volume. 0.495 ml of thisplatelet suspension is incubated at 37°C for 15 minwith the test compound. Subsequently eicosanoid for-mation is started by the addition of 5 µl of 1-14C-arachidonic acid solution (50 Ci/mol, 9 × 10−4 Ci/l,NEN, Dreieich, FRG). After 5 min (37°C) the re-action is stopped by adding 0.5 ml of chilled ace-tone/0.1 ml 1n HCl, cooled to 0°C, and extractedtwo times with 3 ml ethylacetate. The combined or-ganic extracts are evaporated and the residue redis-solved in 0.2 ml methanol. Aliquots are separated by

HPLC at a flow rate of 1.5 ml/min, using a C-18 Nu-cleosil column (5 µm, 125 × 4.6 mm, Bischoff, Leon-gang, FRG) connected with a pre-column C-18 Nu-cleosil (5 µm, 20 × 4.6 mm) of the same type. Theformed 14C-eicosanoids are analyzed using a liquidscintillation flow detector LB 507 (Berthold, Wild-bad, FRG). The radiochromatogram is analyzed bycomparison with tritiated authentic eicosanoids (NEN,Dreieich, FRG). The elution system consists of thefollowing solvent mixtures (elution time in parenthe-sis): I 725 ml water/275 ml acetonitrile/1 ml acetic acid(40 min), II 700 ml methanol/300 ml H2O/1 ml aceticacid (40 min), III pure methanol (20 min).

EVALUATIONThe radioactivity of the separated TXB2 and PGE2 ismeasured as a function of drug concentration, and re-lated to a control experiment without drug addition.

A further lipoxygenase product, 12-HETE, which isadditionally generated under the test conditions, can bequantified in a similar way.

Lasché and Larson determined PGI2 by a bioassaybased on its generation by aortic rings and assay by itsability to inhibit platelet aggregation.

CRITICAL ASSESSMENTThe measured reaction sequence starts with arachi-donic acid, and involves its transformation toprostaglandin endoperoxides, which is catalyzed bycyclooxygenase. The unstable and short-living en-doperoxides transform immediately into thromboxaneA2 by the action of thromboxane isomerase. TXA2 isunstable, too, and yields the stable non-enzymatic hy-drolysis product TXB2.

Inhibitors of cyclooxygenase exhibit a decreasedformation of TXB2. Specific inhibition of the throm-boxane isomerase step results in an accumulation ofthe mentioned endoperoxide intermediates, which dueto their chemical instability are non-enzymaticallytransformed to the primary PGE2. Thus, inhibitors ofthe TXA2-isomerase lead to a significant increase ofthe PGE2-peak at the expense of the TXB2-peak. Ef-fects of drugs on the side product 12-HETE can alsobe studied in this test system.

H.3.1.6.4Stimulation of Inducible Prostaglandin Pathwayin Human PMNL

PROCEDUREThe procedure of Herrmann et al. (1990) with the mod-ification of Weithmann et al. (1994), is used to stimu-


late cyclohexamide-inhibitable generation of PGE2 inhuman PMNL by LPS.

H.2.5 × 109 PMNL/l culture medium (RPMI 1640,completed with 1 mmol/l sodium pyruvate, 5% FCS(w/v), 2 mmol/l glutamine and each 100 U/ml peni-cillin/streptomycin) are incubated with 100 µmol/lacetylsalicylic acid for 60 min (37°C, 5% CO2),and the latter subsequently removed by four timeswashing with medium. 0.25 ml-aliquots (contain-ing 2.5 × 109 PMNL/l medium) are incubated with0.1 mg/ml LPS (lipopolysaccharide from Salmonellaabortus equii, Sigma GmbH, Deisenhofen, FRG) alongwith the test compound for 18 h (96-well plates, 37°C,5% CO2). The generation of eicosanoids is inducedby the administration of 7 × 10−5 mol/l of the calciumionophore A 23187. After incubation for 30 min at37°C the plates are centrifuged at 400 g for 15 min.PGE2 is determined in the supernatant, using anELISA-kit commercially available from several dis-tributors. Alternatively the test compound is addedalong with the calcium ionophore. At least 90% ofthe cells remain intact during incubation times (trypanblue exclusion assay).

EVALUATIONThe PGE2-concentration in the sample is deter-mined from appropriate calibration curves. The PGE2-concentration is measured as a function of drug con-centration, and related to a control experiment withoutdrug.

CRITICAL ASSESSMENTLong term activation with LPS or other inflamma-tory effectors leads in human polymorphonuclear neu-trophils to the stimulation of a prostaglandin synthe-sizing capacity, which under normal conditions is notpresent in this system. The described assay system de-tects compounds which interfere with this activationprocess.

Incubation of drugs with already activated cells al-lows to search for drugs that directly influence the en-zyme activity.

H.3.1.6.5COX-1 and COX-2 Inhibition

PURPOSE AND RATIONALESeveral assays were described to characterize COX-1 and COX-2-inhibitors, such as in vitro COX en-zyme assay (Seibert et al. 1994), COX-2 protein ex-traction and analysis (Anderson et al. 1996), a humanwhole blood assay using LPS-induced PGE2 produc-tion as an index for cellular COX-2 activity (Riendeau

et al. 1997) or whole-cell assays with transfected Chi-nese hamster ovary cells expressing COX-1 and COX-2 or COX-2 specific (osteosarcoma cells) and COX-1 specific (U937 cells) making use of PGE2 produc-tion after arachidonic challenge as an index of cellularpotency and selectivity of cyclooxygenase inhibitors(Chan et al. 1999; FitzGerald and Loll 2001; Rao et al.2003).

PROCEDUREIn Vitro Cyclooxygenase InhibitionThe ability of test compounds to inhibit COX-1 andCOX-2 (IC50 values, µM) is determined using an en-zyme immunoassay kit (Cyman Chemical, Ann Arbor,Mich., USA, no. 560101) (Uddin et al. 2004). ThisCOX (ovine) inhibitor screening assay directly mea-sures the amount of the prostaglandin PGF2α producedin the cyclooxygenase reaction. The prostanoid prod-uct is quantified via enzyme immunoassay (EIA) usinga broadly specific antibody that binds to all the ma-jor prostaglandin compounds. Thus, this COX assay ismore accurate and reliable than an assay based on per-oxidase inhibition. The COX (ovine) inhibitor screen-ing assay includes both ovine COX-1 and COX-2 en-zymes in order to screen isozyme-specific inhibitors.This assay is an excellent tool, which can be usedfor general inhibitor screening, or to eliminate false-positive leads generated by less specific methods.

Cyclooxygenase catalyzes the first step in thebiosynthesis of arachidonic acid to PGH2. PGF2α pro-duced from PGH2 by reduction with stannous chlo-ride is measured by enzyme immunoassay. This assayis based on the competition between PGs and a PG-acetylcholinesterase conjugate (PG tracer) for a lim-ited amount of PG antiserum. The amount of PG tracerthat is able to bind to the PG antiserum is inversely pro-portional to the concentration of PGs in the wells sincethe concentration of PG tracer is held constant whilethe concentration of PGs varies. This antibody–PGcomplex binds to a mouse anti-rabbit monoclonal an-tibody that was previously attached to the well. Theplate is washed to remove any unbound reagents andthen Ellman’s reagent, which contains the substrate foracetylcholinesterase, is added to the well. The prod-uct of this enzymatic reaction produces a distinct yel-low color that absorbs at 405 nm. The intensity ofthis color, determined spectrophotometrically, is pro-portional to the tracer bound to the well, which is in-versely proportional to the amount of PGs present inthe well during the incubation. Percent inhibition iscalculated by the comparison of compound treated tovarious control incubations. The concentration of the


test compound causing 50% inhibition (IC50, µM) iscalculated from the concentration–inhibition responsecurve.

Inhibition Studies with Recombinant HumanCOX-1 and COX-2Microsomal preparations of recombinant human COX-1 and COX-2 are prepared from a vaccine virus-COS-7 cell expression system (O’Neill et al. 1994). Re-combinant human COX-1 and COX-2 are expressed inbaculovirus-Sf9 cells, and enzymes are purified (Ouel-let and Percival 1995; Cromlish and Kennedy 1996).Enzymatic activity is monitored continuously by ei-ther a fluorescence assay measuring the appearance ofthe oxidized form of the reducing agent cosubstratehomovanillic acid or by oxygen consumption (Ouelletand Percival 1995).

Classical non-steroidal anti-inflammatory drugs(NSAIDs) and COX-2 inhibitors are time-dependent,irreversible inhibitors of hCOX-2, which is consis-tent with a two-step process involving an initial rapidequilibrium binding of enzyme and inhibitor, fol-lowed by the slow formation of a tightly boundenzyme–inhibitor complex. COX-2 inhibitors showa time-independent inhibition of hCOX-1, consistentwith the formation of a reversible enzyme–inhibitorcomplex (Ouellet and Percival 1995; Riendeau et al.2001).

HPLC Assay for Oxygenationof Radiolabeled Arachidonic Acid by COX-1Purified recombinant human COX-1 (50 µl of 1 µg/mlin 100 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM phe-nol, 1 µM hematin) is preincubated with 2 µl of the in-hibitor solution (50 fold concentrated stock in DMSO,0–2.5 mM) for 15 min. The reaction is then initiatedby the addition of 5 µl of 1 µM [14C]-arachidonic acid(0.005 µCi) to obtain a final concentration of 0.1 µM.After 7 min incubation at room temperature, the re-action is stopped by the addition of 5 µl 1 M HCland 50 µl acetonitrile. Aliquots of 50 µl of each re-action mixture are analyzed for substrate conversionby reverse phase HPLC onto a C-18 Nova-Pak col-umn (3.9 × 150 mm) which is developed with acetoni-trile/water/acetic acid (85:15:0.1) at 2 ml/min. Arachi-donic acid metabolites and arachidonic acid eluted at0.6–1 min and 2.2–2.5 min, respectively, are quanti-tated by a Packard radiochromatography detector. Per-centages of inhibition are calculated from the differ-ence in conversion of arachidonic acid to prostaglandinmetabolites between inhibitor-treated samples andcontrols exposed to DMSO vehicle.

Determination of the Stoichiometry of Inhibitor BindingAliquots of purified COX-2 (0.25 mg/ml, concentra-tion of subunit of 3.4 mM) are incubated in buffer(100 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM phe-nol) in the presence of various inhibitors (0–8 µM)for 15 or 30 min. An aliquot (20 µl) is then re-moved for determination of the cyclo-oxygenase ac-tivity which is monitored continuously by oxygen con-sumption by a Clark-type polarographic oxygen probe.The oxygen chamber is filled with 0.6 ml of reac-tion buffer (100 mM Tris-HCl, pH 8.0, 5 mM EDTA,1 µM hematin,1 mM phenol, 100 µM arachidonic acidat 30° or 37°C) and the reaction is initiated bythe addition of 20 µl of a solution of 4 mM hydro-gen peroxide and 0.5 mM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) in assay buffer. Enzymeconcentration is determined by amino acid concentra-tion following acid hydrolysis.

Determination of the Dissociation Rate Constantof the Enzyme-Inhibitor ComplexPurified COX-2 (2.0 nmol, 2 ml) in 20 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 2 µM hematin, 0.1% β-octylglucoside is treated with 2.0 nmol [14C]-DFU(18 Ci/mol) and incubated at 20°C for 3 h. A control(0.7 ml) is removed and 13 nmol unlabelled DFU isadded to the remaining 1.3 ml of the mixture contain-ing COX-2 and [14C]-DFU. At timed intervals, 0.1 ml(in duplicate) is transferred to a Microcon-30 microconcentration device (Amicon) and the free inhibitoris separated from enzyme-bound inhibitor by centrifu-gation at 14,000 g for 6 min at 4°C. Buffer (0.1 ml) isadded to the retentate and the centrifugation repeated.The filtrate and retentate are then removed and mixedwith 10 ml scintillation fluid and counted in a liquidscintillation counter.

An aliquot of purified COX-2 (1.0 nmol) is treatedwith 1.25 mol equivalents of inhibitor or with DMSOvehicle control and incubated at 20°C for 1 h. The en-zyme-inhibitor mixture is then transferred to a PierceMicrodialyzer 100 apparatus and dialyzed continu-ously against 2 l of buffer (20 mM Tris-HCl, pH 8.0,0.1 mM EDTA, 0.1 mM phenol, 0.1% octylglucoside)for 5 h at 22°C during which aliquots are moved andfrozen at –70°C until assayed for cyclyo-oxygenaseactivity by oxygen uptake as described above.

Recovery of Inhibitor from the COX-2-Inhibitor ComplexPurified COX-2 (0.79 nmol) is treated with 1.0 molequivalent of inhibitor and the mixture is incubatedfor 60 min at room temperature. The remaining activ-ity at this time is 4% that of a vehicle-treated con-


trol. The sample is then divided in two and the pro-tein denaturated by treatment with four volumes ofethyl acetate/methanol/1 M citric acid (30:4:1). Afterextraction and centrifugation (10,000 g for 5 min), theorganic layer is removed and the extraction repeated.The two organic layers are combined and dried un-der N2. The extract is dissolved in 10 µl of HPLCsolvent mixture consisting of water/acetonitrile/aceticacid (50:41:0.1) and 50 µl are injected onto a Nova-pak C-18 column (3.9 × 150 mm) and developed at1 ml/min. The inhibitor is detected by absorption at260 nm and eluted with a retention time of 6.6 min inthis system. Control experiments for inhibitor recov-ery are performed with incubation of the inhibitor inthe absence of enzyme and processing of the samplesin an identical fashion before quantitation by HPLC.

Spectrophotometric Assay of Recombinant Human COX-2Enzymatic activity of the purified COX-2 is mea-sured using a chromogenic assay based on the oxi-dation of N,N,N′,N′-tetramethyl-p-phenylenediamine(TMPD) during the reduction of PGG2 to PGH2(Copeland et al. 1994). The assay mixture (180 µl)contains 100 mM sodium phosphate, pH 6.5, 1 µMhematin, 1 mg/ml gelatin, 2 to 5 µg/ml of purifiedCOX-2, and 4 µl of the test compound in DMSO. Theassay is also performed in the presence of the detergentGenapol X-100 at a final concentration of 2 mM. Themixture is preincubated at room temperature (22°C)for 15 min before the initiation of the enzymatic re-action by the addition of 20 µl of a solution of 1 mMarachidonic acid and 1 mM TMPD in assay buffer(without enzyme or hematin). For assays in the pres-ence of Genapol, the arachidonic acid and TMPD so-lution is prepared in 50% aqueous ethanol. The en-zyme activity is measured by estimation of the initialvelocity of TMPD oxidation over the first 36 s of thereaction as followed from the increase in absorbancyat 610 nm. A low rate of non-enzymatic oxidation isobserved in the absence of COX-2 and is subtractedbefore the calculation of the percentage of inhibition.

Whole-Cell Assays with Transfected Chinese Hamster(CHO) Cells Expressing COX-1 and COX-2Stably transfected CHO cells expressing human COX-1 and COX-2 are cultured and assayed for the produc-tion of PGE2 after stimulation with arachidonic acid(Kargman et al. 1996). Cells (0.3 × 106 cells in 200 µ l)are pre-incubated in HBSS containing 15 mM HEPES,pH 7.4, with 3 µl of the test drug or DMSO vehiclefor 15 min at 37°C before challenge with arachidonicacid. Cells are challenged for 15 min with an arachi-

donic acid solution (10% ethanol in HBSS) to yieldfinal concentrations of 10 µM arachidonic acid in theCHO[COX-2] assay and 0.5 µM arachidonic acid inthe CHO[COX-1] assay. In the absence of addition ofexogenous arachidonic acid, levels of PGE2 in sam-ples from CHO[COX-1] are <30 pg PGE2/106 cells.In the presence of 0.5 µM exogenous arachidonic acid,levels of PGE2 in samples from CHO[COX-1] cellsincrease to 260 to 1500 pg PGE2/106 cells. After stim-ulation with 10 µM exogenous arachidonic acid, lev-els of PGE2 in samples from CHO[COX-2] cells in-crease from <120 to 700 to 1600 pg PGE2/106 cells.Compounds are tested in eight concentrations in dupli-cate using 3-fold serial dilutions in DMSO. COX activ-ity in the absence of test compounds is determined asthe difference in PGE2 levels of cells challenged witharachidonic acid versus PGE2 levels in cells mock-challenged with ethanol vehicle.

Arachidonic acid-dependent production of PGE2is measured in both cell lines after addition of testdrugs. Indomethacin shows similar IC50 values in bothCHO[COX-1] and CHO[COX-2] cells, whereas spe-cific COX-2 inhibitors show a 1,000-fold specificity.

Assays with Murine MacrophagesMitchell et al. (1994), Hu et al. (2003), and Joo et al.(2004) used mouse peritoneal macrophages for evalu-ation of COX-2 inhibitors.

Adherent peritoneal macrophages were harvestedfrom the peritoneal cells of male C5BL-6J mice af-ter intraperitoneal injection of brewer thioglycollatemedium (5 ml/100 g body weight) for 3 days. Theperitoneal cells obtained from three to four mice weremixed and seeded in 48-well culture cluster at a celldensity of 1 × 109 cell/l in RPMI-1640 supplementedwith 5% newborn calf serum, penicillin, and strep-tomycin. After settlement for 2–3 h, non-adherentcells were washed by D-Hanks’ balanced salt solu-tion. Then macrophages were cultured in RPMI-1640without serum. Almost all of the adherent cells weremacrophages as assessed by Giemsa staining. Cell via-bility was examined by trypan blue dye exclusion. Allincubation procedures were performed with 5% CO2in humidified air at 37°C.

COX-1 AssayMacrophages were incubated with test compound atdifferent concentrations or solvent (Me2SO) for 1 hand were stimulated with calcimycin 1 µmol/l for 1 h.The amount of 6-keto-PGF1α (a stable metabolite ofPGI2) in supernatants was measured by RIA accord-ing to manufacturer’s guide. The inhibitory ratio was


calculated as

IR = (Cs − Ct)

(Cs − Cc).

Cs, Ct, Cc refer to 6-keto-PGFα concentration in su-pernatants of calcimycin, test compound, and controlgroups, respectively.

COX-2 AssayMacrophages were incubated with test compound atdifferent concentrations or solvent (Me2SO) for 1 hand were stimulated with lipopolysaccharide (LPS)1 mg/l for 9 h. The amount of PGE2 in supernatantswas measured by RIA. The inhibitory ratio was calcu-lated using the same formula as in COX-1 assay sec-tion. Cs, Ct, Cc refer to PGE2 concentration in super-natants of LPS, test compound, and control groups, re-spectively.

Statistical analysis data were expressed as the mean±SD of more than three independent experiments.Dose–inhibitory effect curves were fit through “up-hill dose–response curves, variable slope” using Prism,GraphPad version3.00:

Y = 1

1 + 10[(log IC50−X)×Hillslope].

Whole-Cell Assays with Osteosarcoma Cells (COX-2)and U937 Cells (COX-1)The human osteosarcoma cell line has been shownto selectively express COX-2 by reverse transcription-polymerase chain reaction and immunoblot analysis,whereas undifferentiated human lymphoma U937 cellsselectively express COX-1. The production of PGE2by these cells after arachidonic acid challenge is usedas an index of cellular COX-2 and COX-1 activ-ity, respectively. Test substances are preincubated for5 to 15 min with the cells under serum-free condi-tions (HBSS) before a 10-min stimulation with 10 µMarachidonic acid and measurement of PGE2 produc-tion (Wong et al. 1997). COX activity in each cellline is defined as the difference in PGE2 concentra-tions in samples incubated in the presence or absenceof arachidonic acid.

Human Whole Blood AssayFor the COX-2 assay, fresh heparinized human wholeblood is incubated with lipopolysaccharide fromE. coli at 100 µg/ml and with 2 µl of vehicle or a testcompound for 24 h at 37°C (Brideau et al. 1996).PGE2 levels in the plasma are measured using radioim-munoassay after deproteination. For the COX-1 assay,

an aliquot of fresh blood is mixed with either DMSO ortest compound and is allowed to clot for 1 h at 37°C.TBX2 levels in the serum are measured using an en-zyme immunoassay after deproteination.

MODIFICATIONS OF THE METHODYoung et al. (1996) and Khanapure et al. (2003) useda similar assay to determine COX-1 and COX-2 en-zyme activity in human whole blood. Human bloodfrom non-fasted donors, who had not taken any as-pirin or NSAIDs for 14 days, was collected in sodiumheparin and distributed in 1-ml aliquots per well ina 24-well tissue culture plate. The plate was placed ona gently rotating platform shaker in a 5% CO2 incuba-tor at 37°C for 15 min. Test compounds were dissolvedand diluted in dimethylsulfoxide (DMSO) and 1 µl ofeach dilution of test compound was added per well induplicate wells. To induce COX-2, lipopolysaccharide(LPS) from E. coli was added at 10 µg/ml to appropri-ate wells 15 min after addition of the test compounds.For the stimulation of COX-1, the calcium ionophoreA23187 was added to a final concentration of 25 µM toseparate wells 4.5 h after the addition of the test com-pounds. At 30 min after addition of A23187 or 5 h afterLPS addition, all incubations were terminated by cool-ing on ice and adding EGTA to a final concentrationof 2 mM. The blood samples were then transferred to15 ml polypropylene centrifuge tubes and centrifugedat 1200 g for 10 min at 4°C. Then, 100 µl of plasmawas removed from each blood sample and added to1 ml of methanol in a 15 ml polypropylene centrifugetube, mixed vigorously, and stored overnight at –20°C.The next day, the samples were centrifuged at 2000 gfor 10 min at 4°C, and the supernatants were trans-ferred to glass tubes and evaporated to dryness. Afterreconstitution with EIA buffer, and appropriate dilu-tion (2000-fold for COX-1 and 500-fold for COX-2),the samples were assayed for thromboxane B2 usingEIA kits (Cayman, Ann Arbor, Mich., USA) in dupli-cate wells.

Kalajdzic et al. (2002) showed that a preferentialCOX-2 inhibitor suppresses peroxisome proliferator-activated receptor induction of COX-2 gene expressionin human synovial fibroblasts.

Berg et al. (1997) developed a cell assay systemusing the human erythroleukemic cell line HEL asa source for COX-1 and the human monocytic cell lineMono Mac 6 as a source for COX-2.

Kalgutkar et al. (2000) exploited biochemical dif-ferences between the COX isoforms to improve uponthe selectivity of carboxylate-containing NSAIDs asCOX-2 inhibitors.


Faust et al. (2003) recommended human peritonealmacrophages in culture as a model for studying inflam-matory disorders in vitro.

Krause et al. (2003) reported that the NSAIDs in-domethacin and diclofenac and a selective COX-2 in-hibitor uncouple mitochondria in intact cells. To an-alyze the effects on energy metabolism of rat thy-mocytes, top-down elasticity analysis (Brand 1996,1998; Ainscow and Brand 1999) was applied. Energymetabolism was conceptually divided into three blocksof reactions that generated and consumed the centralintermediate mitochondrial membrane potential (ψm).The substrate oxidation subsystem encompassed allcellular catabolic reactions that provide the respira-tory chain with its substrates NADH and succinate(e. g., glucose, fatty acid, and amino acid metabolism)and the electron transport chain itself, which togethergenerate ψm. The ψm -consuming reactions were fur-ther divided into the subsystems proton leak and ATPturnover. The ATP turnover subsystem encompassedATP synthesis by ATP synthase and subsequent ATPconsumption by cellular pathways (e. g., ion pumpsor protein synthesis). Manipulation of the biochemicalproperties of one block of reactions prompted a changeof the intermediate ψm. The whole system evolvedinto a new steady state, which was determined by thekinetic responses of the other blocks to changes ofψm.Successive inhibition of one block of reactions per-mitted determination of these kinetic responses of theother blocks to changes of ψm. A comparison of thekinetic responses in the presence and absence of effec-tors (e. g., drugs) allowed for the identification of sitesof actions.

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Safayhi H, Mack T, Sabieraj J, Anazodo MI, Subramanian LR,Ammon HPT (1992) Boswellic acids: Novel, specific, non-redox inhibitors of 5-lipoxygenase. J Pharmacol Exp Ther261:1143–1146

Salari H, Braquet P, Borgeat P (1984) Comparative effects ofindomethacin, acetylenic acids, 15-HETE, nordihydrogua-jaretic acid and BW 755c on the metabolism of arachidonicacid in human leukocytes and platelets. Prostagl LeukotrMed 13:53–60

Samuelsson B (1986) Leukotrienes and other lipoxygenaseproducts. Prog Lipid Res 25:13–18

Saussy DL Jr, Mais DE, Burch RM, Halushka PV (1986)Identification of a putative thromboxane A2/prostaglandinH2 receptor in human platelet membranes. J Biol Chem261:3025–3029

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Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W,Lee L, Isakson P (1994) Pharmacological and biochemicaldemonstration of the role of cyclooxygenase 2 in inflamma-tion and pain. Proc Natl Acad Sci USA 91:12013–12017

Seibert K, Masferrer J, Zhang Y, Gregory S, Olson G, Hauser S,Leahy K, Perkins W, Isakson P (1995) Mediation of inflam-mation by cyclooxygenase-2. Agents Actions 46:41–50

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Shimizu T, Rådmark O, Samuelsson B (1984) Enzyme withdual lipoxygenase activities catalyzes leukotriene A4 syn-thesis from arachidonic acid. Proc Natl Acad Sci USA81:689–693

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Takeguchi C, Kohno E, Sih CJ (1971) Mechanism of prostaglan-din biosynthesis. I. Characterization and assay of bovineprostaglandin synthetase. Biochemistry 10:2372–2377

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Vane J (1987) The evolution of non-steroidal anti-inflammatorydrugs and their mechanisms of action. Drugs 33(Suppl 1):18–27

Vane J (1998) The mechanism of action of anti-inflammatorydrugs. Naunyn-Schmiedeberg’s Arch Pharmacol 358,Suppl 1, R 8

Vane J, Botting R (1987) Inflammation and the mechanism ofaction of anti-inflammatory drugs. FASEB J 1:89–96

Vane JR, Bakhle YS, Botting RM (1998) Cyclooxygenases 1and 2. Ann Rev Pharmacol Toxicol 38:97–120

Veenstra J, van de Pol H, van der Torre H, Schaafsma G, Ock-huizen T (1988) Rapid and simple methods for the inves-tigation of lipoxygenase pathways in human granulocytes.J Chromatogr 431:413–417

Weithmann KU, Schlotte V, Seiffge D, Jeske S (1993) Con-certed action of pentoxifylline in conjunction with acetyl-salicylic acid on platelet cyclic AMP and aggregation.Thromb Haemorrh Dis 8:1–8

Weithmann KU, Jeske S, Schlotte V (1994) Effect of lefluno-mide on constitutive and inducible pathways of cellulareicosanoid generation. Agents Actions 41:164–170

Winkler JD, Sarau HM, Foley JJ, Mong S, Crooke ST (1988)Leukotriene B4-induced homologous desensitization ofcalcium mobilization and phosphoinositide metabolism inU-937 cells. J Pharmacol Exp Ther 246:204–210

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Xie W, Robertson DL, Simmons DL (1992) Mitogen-inducibleprostaglandin G/H synthase: A new target for nonsteroidalantiinflammatory drugs. Drug Dev Res 25:249–265

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H.3.1.7Influence of Cytokines

H.3.1.7.1Induced Release of Cytokines (Interleukin-1α, IL-1β ,IL-6, IL-8 and TNFα) from Human White Blood CellsIn Vitro

PURPOSE AND RATIONALECytokines represent a class of different, biologicallyhighly potent peptides, that are endogenously syn-


thesized upon stimulation. They are involved in nu-merous cellular processes, such as inflammation, im-munological responses and many others. Their broadpleiotrophic biological activities are best characterizedby their former classification as

• B-cell activating and differentiating factor, endoge-nous pyrogen, osteoclast activating factor, thy-mocyte proliferation factor, monocyte cell factor,leukocyte endogenous factor (IL-1);

• hepatocyte stimulatory factor, hybridoma growthfactor, haematopoietic cell stimulatory factor (IL-6);

• neutrophil chemotactic factor and adhesion in-hibitor (IL-8);

• tumor necrosis factor (TNFalpha).

There are presently 18 cytokines with the name in-terleukin (IL) (Dinarello 2000). The concept of proin-flammatory cytokines and antiinflammatory cytokines(IL-4, IL-10, IL-13) is fundamental to cytokine biol-ogy and novel drug discovery strategies.

Blocking IL-1 or TNF has been highly successful inpatients with rheumatoid arthritis, inflammatory boweldisease, or graft-vs.-host disease.

The following procedure is used to detect com-pounds that interact with the lipopolysaccharide inced cytokine release from human mononuclear bloodcells. The cytokines measured are interleukines 1al-pha, 1beta, 6 and 8, as well as TNFalpha.

PROCEDUREAccording to Böyum (1976)10 ml of freshly preparedhuman citrated blood is diluted 1:1 with PM 16-buffer (Serva, Heidelberg, FRG), and underlayeredwith 15 ml Lymphoprep (Molter GmbH, Heidelberg,FRG), and subsequently centrifuged at 20°C with400 g (Minifuge, Heraeus, Hanau, FRG) for 30 min.The cell fraction appearing as a white ring betweenthe two phases is carefully removed by means of a sy-ringe, diluted 1:1 (v/v) with PM 16 and again cen-trifuged for 15 min. The pellet is washed with 10 mlof RPMI 1640 (Gibco, Berlin, FRG), containing inaddition 300 mg/l L-glutamine. The washed cell frac-tion is taken up in 1 ml RPMI 1640, containing in ad-dition 300 mg/l L-glutamine, 25 mmol/l HEPES, 5%FCS and 100 IU/ml penicillin/streptomycin (Gibco).Using a cell counter (type IT, Coulter Diagnostics,Krefeld, FRG) the cell suspension which consists ofabout 90% lymphocytes and 10% monocytes is ad-justed to approx. 5 × 109 cells/ml.

Synthesis and release of cytokines according toTiku et al. (1986) is performed in 96 wells mi-

crotiter plates. To 0.23 ml of the cell fraction 500 ngLPS (lipopolysaccharide from Salmonella abortusequii, Sigma GmbH, Deisenhofen, FRG), dissolved in0.01 ml dimethylsulfoxide/water (1:10, v/v), and theinvestigational drug, dissolved in 0.01 ml, are added.The cell suspension is now kept at 37°C/5% CO2in a common incubator. Incubation time is usually20 h (for Il-6 and Il-8 only four or one hour, respec-tively). The reaction is stopped by placing the mi-crotiter plate into an ice bath. The plate is then cen-trifuged at 2000 rpm for 2 min. The cytokine levels aredetermined in various aliquots of the supernatant us-ing the appropriate ELISA-kit, which is commerciallyavailable from several distributors.

EVALUATIONThe cytokine-concentration in the sample is deter-mined from appropriate calibration curves. The cy-tokine-concentration is measured as a function of drugconcentration, and related to a control experimentwithout drug. In general, Hostacortin (10 to 0,1 µmol/l)is used as the standard compound.

CRITICAL ASSESSMENTDrugs interfering with LPS-activation, biosynthesisand cellular release of cytokines are to exhibit activ-ity in these assay systems. Usually cell viability is notaltered, as assessed by the lactate-dehydrogenase test.

MODIFICATIONS OF THE METHODVan der Pouw-Kraan et al. (1992) examined the reg-ulation of interleukin (IL)-4 production by human pe-ripheral blood T cells. Production of IL-4 as measuredby ELISA was shown to be regulated differently fromIL-2 and INF-γ (also measured by ELISA).

A proinflammatory role for interleukin-18 (IL-18)in rheumatoid arthritis was attributed by Gracie et al.(1999).

The role of the interleukin-6 family of cytokines ininflammatory arthritis and bone turnover was reviewedby Wong et al. (2003).

Kim et al. (2005) reported an inhibitory effect of lu-teolin, a flavonoid from Lonicera japonica, on tumor-necrosis-factor-α-induced IL-8 production in humancolon epithelia cells.

REFERENCES AND FURTHER READINGBird TA, Saklatvala J (1986) Identification of a com-

mon class of high-affinity receptors for both types ofporcine interleukin-1 on connective tissue cells. Nature324:263–266

Boyum A (1976) Isolation of lymphocytes, granulocytes andmacrophages. Scand J Immunol 5 (Suppl 5):9–15


Chin J, et al (1987) Identification of a high affinity receptor fornative interleukin-1α and interleukin-1β on normal humanlung fibroblasts. J Exp Med 165:70–86

Dinarello CA (1991) Interleukin-1 and interleukin-1 antago-nism. Blood 77:1627–1652

Dinarello CA (2000) Proinflammatory cytokines. Chest118:503–508

Eugui EM, Delustro B, Rouhafza S, Wilhelm R, Allison AC(1993) Coordinate inhibition by some antioxidants ofTNFα, IL-1β and IL-6 production by human peripheralblood mononuclear cells. Ann NY Acad Sci 696:171–184

Gracie JA, Forsey RJ, Chan WL, Gilmour A, Leung BP, GreerMR, Kennedy K, Carter R, Wei XQ, Xu D, Filed M, FoulisA, Liew FY, McInnes IB (1999) A proinflammatory role forIL-18 in rheumatoid arthritis. J Clin Invest 104:1393–1401

Grob PM, David E, Warren TC, DeLeon RP, Farina PR, HomonCA (1990) Characterization of a receptor for human mono-cyte-derived neutrophil chemotactic factor intereukin-8.J Biol Chem 265:8311–8316

Ibelgaufts H (ed) (1992) Lexikon Zytokine, MünchenKillian PL (1986) Interleukin-1α and interleukin-1β bind to the

same receptor on T cells. J Immunol 136:4509–4514Kim JA, Kim DK, Kang OH, Choi YA, Park HJ, Choi SC, Kim

TH, Yun KJ, Nah YH, Lee YM (2005) Inhibitory effectof luteolin on TNF-α-induced IL-8 production in humancolon epithelia cells. Int Immunopharmacol 5:209–217

Lewis GP, Barrett ML (1986) Immunosuppressive actions ofprostaglandins and the possible increase in chronic inflam-mation after cyclooxygenase inhibitors. Agents Actions19:59–65

Maloff BL, Shaw JE, Di Meo TM, Fox D, Bruin EM (1989)Development of a RIA-based primary screen for IL-1 an-tagonists. Clin Chim Acta 180:73–78

Moser B, Schumacher C, von Tscharner V, Clark-Lewis I,Baggiolini M (1990) Neutrophil-activating peptide 2 andgro/melanoma growth-stimulatory activity interact withneutrophil-activating peptide-1/interleukin-8 receptors onhuman neutrophils. J Biol Chem 266:10666–10671

Tiku K, Tiku ML, Skosey JL (1986) Interleukin-1 produc-tion by human polymorphonuclear neutrophils. J Immunol136:3677–3685

Van der Pouw-Kraan T, Van Kooten C, Rensink I, Aarden L(1992) Interleukin (IL)-4 production by human T cells: dif-ferential regulation of IL-4 vs. IL-2 production. Eur J Im-munol 22:1237–1241

Warren JS (1993) Inflammation. DN&P (Drugs, News and Per-spectives) 6:450–459

Whicher JT, Thompson D, Billingham MEJ, Kitchen EA (1989)Acute phase proteins. In: Pharmacological Methods in theControl of Inflammation. Alan R. Liss, Inc., pp 101–128

Wong PKK, Campbell IK, Egan PJ, Ernst M, Wicks IP (2003)The role of the interleukin-6 family of cytokines in in-flammatory arthritis and bone turnover. Arthritis Rheum48:1177–1189

H.3.1.7.2Flow Cytometric Analysis of Intracellular Cytokines

PURPOSE AND RATIONALEFlow cytometry is a powerful analytical technique inwhich individual cells can be simultaneously analyzedfor several parameters, including size an granularity,as well as the expression of surface and intracellu-lar markers defined by fluorescent antibodies. Fluores-

cent anti-cytokine and anti-chemokine monoclonal an-tibodies are very useful for the intracellular stainingand multiparameter flow cytometric analysis of indi-vidual cytokine-producing cells within mixed popula-tions. Multicolor immunofluorescent staining with an-tibodies against intracellular cytokines and cell surfacemarkers provides a high resolution method to identifythe nature and frequency of cells which express partic-ular cytokines (Sander et al. 1991, 1993; Jung 1993).

PROCEDURECells and Cell CulturePeripheral blood is obtained from healthy humanvolunteers. Mononuclear cells are isolated by Ficollgradient centrifugation. For purification of T cellsor further cell sorting, mononuclear cells are incu-bated with neuraminidase treated sheep red bloodcells and centrifuged over Ficoll. Erythrocytes areeliminated by ammonium chloride lysis. For isola-tion of memory cells or naive cells, T cells areincubated with a cocktail of antibodies containinganti-CD16, anti-CD56, anti-CD20, anti-CD14, (anti-CD8), and anti-CD45RA or anti-CD45R0. Cell sort-ing is done with the magnetic cell sorter (MACS)according to Abts et al. (1989), Miltenyi et al.(1990) using rat anti-mouse IgG1 or Ig2a anti-bodies labeled with superparamagnetic beads (Mil-tenyi, Bergisch Gladbach, Germany). Depleted cellsare highly enriched with CD4+(CD3+)CD45R0+ orCD4+(CD3+)CD45R0−CD45RA+ cells (>95%) andare referred to as memory cells and naive cells(CD4+CD45R0−CD45RA+) respectively. Only de-pleted cells are used for experiments.

Cells (2 × 105/100 ml) are cultured in 96 well flatbottom plates for various periods of time at 37°C and8% CO2 in RPMI 1640 supplemented with 2 mM glu-tamine, 1 mM sodium pyruvate, 100 IU/ml penicillin,100 µg/ml streptomycin, 2 × 10−5 M mercaptoethanoland 10% AB serum. Cells are stimulated with phorbol12-myristate 13-acetate 1–10 ng/ml+1 µM ionomycin,phytohemagglutinin 2.4 µg/ml or phytohemagglutinin2.4 µg/ml+phorbol 12-myristate 13-acetate 1 ng/ml inthe presence or absence of 3 µM monensin (Sigma).

StainingCultured cells are washed twice in Hanks’ balancedsolution (HBSS) and then fixed in ice-cold HBSScontaining 4% paraformaldehyde for 10 min. Aftertwo further washes in HBSS the cells are resus-pended to 2 × 105 in 300 µl HBSS containing 0.1%saponin, 10% AB serum, 100 µ g/ml goat IgG and0.01 M HEPES buffer. After 10 min, the cells are spun


down and cytokine specific antibodies diluted in HBSSwith 0.1% saponin and 0.1 M HEPES buffer (saponinbuffer) are added at a concentration of 1 µg/ml for30 min at room temperature. Cells are washed twicein saponin buffer and subsequently incubated with iso-type-specific second step antibodies in a concentrationranging from 0.5 to 5 µg/ml for 20 min in the dark.Cells are washed in saponin buffer and stained withstreptavidin conjugates or in the case of surface stain-ing incubated with 200 µg/ml mouse IgG diluted insaponin buffer for 15 min. After subsequent washingin saponin buffer cells are washed twice in HBSS andstained for 20 min with different antibodies in order todetermine their surface phenotype. As a last step, cellsare washed in HBSS.

Flow CytometryA FACScan flow cytometer (Becton Dickinson, Moun-tain View, USA) equipped with a 15 mW argonion laser and filter settings for fluorescein-isothio-cyanate (530 nm), phycoerythrin (585 nm) and TRI-Color (Medac, Hamburg, Germany) or PerCP (BectonDickinson, USA) emitting in the deep red (>650 nm)is used.

EVALUATION5000–10000 cells are computed in list mode and an-alyzed using the FACScan research software (BectonDickinson)

MODIFICATIONS OF THE METHODSlauson et al. (1999) combined the analytical powerof flow cytometry with mitogen-driven, whole bloodlymphocyte activation and proliferation assays to in-vestigate the in vitro mechanism of action of malonon-itrilamides.

Protocols for immunofluorescent staining of intra-cellular cytokines for flow cytometric analysis are pro-vided by BD Phar Mingen, Life Science Research Eu-rope, Heidelberg, Germany.

Ashcroft and Lopez (2000) highlighted the oppor-tunities in high throughput flow cytometry (HTFC)which are opened by commercial high speed machines.The specifications of these machines are cell analy-sis rates over 100,000 cells/s and cell sorting rates of55,000 cells/s with high purity.

REFERENCES AND FURTHER READINGAbt H, Emmerich M, Miltenyi S, Radbruch A, Tesch H (1989)

CD20 positive human B lymphocytes separated with themagnetic cell sorter (MACS) can be induced to prolif-eration and antibody secretion in vitro. J Immunol Meth125:19–28

Ashcroft RG, Lopez PA (2000) Commercial high speed ma-chines open new opportunities in high throughput flow cy-tometry. J Immunol Meth 243:13–24

Jung T, Schauer U, Heusser C, Neumann C, Rieger C (1993) De-tection of intracellular cytokines by flow cytometry. J Im-munol Meth 159:197–207

Miltenyi S, Möller W, Weichel W, Radbruch A (1990) Highgradient magnetic cell separation with MACS. Cytometry11:231

Sander B, Andersson J, Andersson U (1991) Assessment of cy-tokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immun Rev 119:65–93

Sander B, Hoiden I, Andersson U, Moller E, Abrams JS (1993)Similar frequencies and kinetics of cytokine producingcells in murine peripheral blood and spleen. Cytokine de-tection by immunoassay and intracellular immunostaining.J Immunol Meth 166:201–214

Slauson SD, Silva HT, Sherwood SW, Morris RE (1999) Flowcytometric analysis of the molecular mechanisms of im-munosuppressive action of the active metabolite of lefluno-mide and its malononitrilamide analogues in a novel wholeblood assay. Immunol Lett 67:179–183

H.3.1.7.3Screening for Interleukin-1 Antagonists

PURPOSE AND RATIONALEInterleukin-1α and -1β are potent regulators of inflam-matory processes. The naturally occurring IL-1 recep-tor antagonist (IL-1ra) is effective in vitro and in vivoin modulating biological responses to IL-1 (Carter etal. 1990; Hannum et al. 1990; Schreuder et al. 1995).Using a combination of anion exchange, gel filtra-tion, and reverse-phase HPLC, three species of nativeIL-1ra were identified. An unglycosylated, intracellu-lar isoform is designated as icIL-1ra (Lennard 1995;Arend et al. 1991, 1998).

A cell-free, non-isotopic assay has been developedto discover molecules that compete with the naturalligands for binding to the active sites of the type-I IL-1receptor. The key reagents are the IL-1 receptor an-tagonist, a recombinant soluble form of the receptor(sIL-1R), and a specific anti-sIL-1R non-neutralizingmonoclonal antibody (Sarrubi et al. 1996).

PROCEDUREProteinsThe extracellular portion of the type-I IL-1 receptor(sIL-1R) is expressed on the membrane of Chinesehamster ovary cells using a phosphatidinyl-inositol-glycan linkage (PIG-tail). Its expression, cleavage withphosphoinositol-specific phospholipase C, and purifi-cation are performed according to Whitehorn et al.(1995).

The three IL-1 ligands are expressed in Escherichiacoli using synthetic genes (Dower et al. 1989; Yanof-sky and Zurawski 1990). The purification of IL-1ra


and IL-β is accomplished according to Schreuder et al.(1995) and Yem et al. (1988).

IL-1α is purified as follows: E. coli cell sonicatesare precipitated with 2 M ammonium sulfate, and thepellet is resuspended in TE (25 mM Tris/HCl, pH 8.9,1 mM EDTA), dialyzed against the same buffer andloaded on a DEAE-Sepharose column equilibratedwith TE. The protein is eluted with a linear NaCl gra-dient to 300 mM. Ammonium sulfate to 0.8 M is addedto the IL-1α-containing fractions which are loadedonto a phenyl-Sepharose column equilibrated with TEcontaining 0.8 M ammonium sulfate. The elution isperformed with a linear gradient to TE with no salt.IL-1α-containing fractions are concentrated and chro-matographed on a Sephacryl S-200 column in PBS(phosphate-buffered saline: 20 mM sodium phosphate,pH 7.3, 150 mM sodium chloride).

Fluorescein-labeled IL-1α is obtained by incubat-ing 1 mg/ml IL-1α with 1 mg/ml fluorescein isothio-cyanate in PBS for 2 h at room temperature in thedark. The reaction solution is passed directly over a G-25 column (Pharmacia) equilibrated with PBS to re-move unreacted fluorescein isothiocyanate.

The monoclonal antibody Mab79 is used as directdilutions (1:105–106) of ascitic fluid in PBSA (PBScontaining 0.3% bovine serum albumin). Horseradish-peroxidase-linked anti-mouse IgG polyclonal antibodyis used.

Protein concentrations are determined using theBio-Rad protein assay kit, based on the dye-bindingprocedure according to Bradford (1976). BSA is usedas reference protein.

Immobilized-Ligand IL-1 Receptor Binding AssayEssentially the same procedure can be used for bothmanual and automated versions of the assay, with allsteps and incubations performed at room tempera-ture. In the automated assay a Beckman Biomek 1000Work-Station was used for all steps, from coating tospectrophotometric measurements.

Ligand immobilization is obtained by incubation of3.6 µg/ml IL-1ra in 50 µl PBS in flat-bottomed culture-treated microplate wells, equivalent to 10 pmol/wellof IL-1ra. After overnight incubation microplates areemptied and 250 µl/well of 3% BSA in PBS is addedto block unreacted sites. After 2 h of incubation andthree washes with an excess of PBS, the ligand-coatedmicroplates are ready for the receptor binding reac-tion.

In separate microplates with U-shaped wells, 12 µlof samples (containing up to 50% DMSO or DMF)or controls (the same solution without compound) is

mixed with 48 µl of 150 pM sIL-1R in PBSA. Then50 µl of these mixtures is transferred to the IL-1ra-coated plates (equivalent to 6 fmol/well of sIL-1R) andincubated for 2 h. Microplates are then washed twicewith PBS and 50 µl of 1:500,000 dilution of Mab79ascitic fluid in PBSA is added to each well. After 1 hof incubation, 25 µl of 1:100 dilution of HRP-labeledanti-mouse IgG in PBSA is added and the incuba-tion prolonged for an additional hour. Plates are fi-nally washed four times with PBS and bound perox-idase activity is measured spectrophotometrically, us-ing either o-phenylenediamine (OPD) or tetramethyl-benzidine (TMB) as substrate. In the first case, 150 µlof 1 mg/ml OPD in 0.1 M citric acid, pH 5.0, contain-ing 0.03% of a 35% solution of hydroperoxide is addedand, after color development, the reaction is stoppedwith 50 µl of 4.5 M sulfuric acid. Alternatively, 100 µlof 0.1 mg/ml TMB in 25 mM citric acid and 50 mMsodium phosphate, containing 0.02% hydrogen perox-ide (35% solution), is added and the reaction is stoppedwith 50 ml of 2.5 M sulfuric acid.

EVALUATIONAbsorbance (at 492 nm for OPD and 450 nm forTMB) is measured using either a Titertek microplatereader (for the manual procedure) or directly by theBiomek 1000 WorkStation (in the automated version).IC50 values can be calculated from dose–responsecurves.

CRITICAL ASSESSMENT OF THE METHODSince no cells or cell membranes are used, the assayis very robust, with no interference from membrane-perturbing agents, and has high resistance to the or-ganic solvent normally used to resuspend compoundsof chemical libraries.

MODIFICATIONS OF THE METHODHigh-affinity type I interleukin-1 receptor antagonistswere discovered by screening recombinant peptide li-braries (Yanosfky et al. 1996).

Akeson et al. (1996a) developed an ex vivo methodfor studying inflammation in cynomolgus monkeys us-ing whole blood for analysis of IL-1 antagonists ad-ministered in vivo. Animals were given an i.v. infusionof IL-1ra, and blood samples were taken pre-infusionand during the infusion. The samples were incubatedwith or without IL-1β and the subsequent ex vivo in-duction of IL-6 determined. This allows the analysis ofthe in vivo efficacy of antagonists without exposing theanimals to IL-1.


A novel low-molecular-weight antagonist, selec-tively binding the type I IL-1 receptor and blockingthe in vivo responses to IL-1 was described by Akesonet al. (1996b).

Evaluation of the IL-1 receptor antagonist IL-1rain a rodent abscess model of host resistance was pub-lished by Colagiovanni and Shopp (1996).

Blocking monoclonal antibodies (mAbs) specific tomouse IL-1 receptor antagonist (IL-1ra) were preparedby immunizing Armenian hamsters with recombinantmouse IL-1ra by Fujioka et al. (1995). A sensitive andspecific ELISA against mouse IL-1ra was established.

Miesel et al. (1995) tested the anti-arthritic re-activity of the IL-1 receptor antagonist IL-1ra inmale DBA/1xB10A(4R) mice with arthritis induced byintraplantar injection of potassium peroxochromate.Then 3 µ mol/kg K3CrO8 was administered topicallyinto the left hind paws and 1 h after the induction ofarthritis, 2 mg/kg IL-1ra was administered intraperi-toneally, which was repeated on day 2. An arthritis in-dex was determined daily.

Nakae et al. (2003) found that IL-17 productionfrom activated T cells is required for the spontaneousdevelopment of destructive arthritis in mice deficientin IL-1 receptor antagonist.

Redlich et al. (2003) reviewed rheumatoid arthritistherapy after tumor necrosis factor and interleukin-1blockade.

REFERENCES AND FURTHER READINGAkeson A, Bohnke R, Schroeder K, Kastner P, Seligmann B,

Robinson J (1996a) An ex vivo method for studying inflam-mation in cynomolgus monkeys: analysis of interleukin-1receptor antagonist. J Pharmacol Toxicol Meth 36:155–161

Akeson AL, Woods CW, Hsieh LC, Bohnke RA, Acker-mann BL, Chan KY, Robinson JL, Yanofsky SD, JacobsJW, Barrett RW, Bowlin TL (1996b) AF12198, a novel lowmolecular weight antagonist, selectively binds type I inter-leukin (IL)-1 receptor and blocks in vivo responses to IL-1.J Biol Chem 271:30517–30523

Arend WP (1991) Interleukin 1 receptor antagonist. A newmember of the interleukin 1 family. J Clin Invest88:1445–1451

Arend WP, Malyak M, Guthridge CJ, Gabay C (1998)Interleukin-1 receptor antagonist: role in biology. AnnuRev Immunol 16:27–55

Bradford M (1976) A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the prin-ciple of protein-dye binding. Anal Biochem 72:248–252

Carter DB, Deibel MR Jr, Dunn CJ, Tomich CSC, Laborde AL,Slightom JL, Berger AE, Bienkowski MJ, Sun FF, McE-wan RN (1990) Purification, cloning, expression and bio-logical characterization of an interleukin-1 receptor antag-onist protein. Nature (Lond) 344:633–638

Colagiovanni DB, Shopp GM (1996) Evaluation of theinterleukin-1 receptor antagonist (IL-1ra) and tumor necro-sis factor binding protein (TNF-BP) in a rodent abscess

model of host resistance. Immunopharmacol Immuntoxicol18:397–419

Dower SK, Wignall JM, Schooley K, McMahan CJ, Jackson JL,Prickett KS, Lupton S, Cosman D, Sims JE (1989) J Im-munol 142:4314–4320

Fujioka N, Mukaida N, Harada A, Akiyama M, Kasahara T,Kuno K, Ooi A, Mai M, Matsushima K (1995) Prepara-tion of specific antibodies against murine IL-1ra and theestablishment of IL-1ra as an endogenous regulator of bac-teria-induced fulminant hepatitis in mice. J Leukocyte Biol58:90–98

Hannum CH, Wilcox CJ, Arend WP, Joslin FG, Dripps DJ,Heimdal PL, Armes LG, Sommer A, Eisenberg SP, Thomp-son RC (1990) Interleukin-1 receptor antagonist activ-ity of a human interleukin-1 inhibitor. Nature (Lond)343:336–340

Lennard AC (1995) Interleukin-1 receptor antagonist. CriticalRev Immunol 15:77–105

Miesel R, Ehrlich W, Wohlert H, Kurpisz M, Kröger H (1995)The effects of interleukin-1 receptor antagonist on ox-idant-induced arthritis in mice. Clin Exper Rheumatol13:595–610

Nakae S, Saijo S, Horai R, Sudo K, Mori S, Iwakura Y (2003)IL-17 production from activated T cells is required for thespontaneous development of destructive arthritis in micedeficient in IL-1 receptor antagonist. Proc Natl Acad SciUSA 1000:5986–5990

Redlich K, Schett G, Steiner G, Hayer S, Wagner EF, SmolenJS (2003) Rheumatoid arthritis therapy after tumor necro-sis factor and interleukin-1 blockade. Arthritis Rheum48:3308–3319

Sarrubi E, Yanofsky SD, Barrett RW, Denaro M (1996) A cell-free, nonisotopic, high-throughput assay for inhibitors oftype-I interleukin-1 receptor. Anal Biochem 237:70–75

Schreuder HA, Rondeau JM, Tardif C, Soffientini A, Sarubbi E,Akeson A, Bowlin TL, Yanofsky S, Barrett RW (1995) Re-fined crystal structure of the interleukin-1 receptor antag-onist. Presence of a disulfide link and a cis-proline. Eur JBiochem 227:838–847

Whitehorn E, Tate E, Yanofsky SD, Kochersperger L, Davis A,Mortensen RB, Yonkivich S, Bell K, Dover WJ, Barrett RW(1995) Biotechnology 13:1215–1219

Yanosfky SD, Zurawski G (1990) Identification of key residuesin the amino-terminal third of human interleukin-1α. J BiolChem 265:13000–13006

Yanosfky SD, Baldwin DN, Butler JH, Holden FR, Jacobs JW,Balasubramanian P, Chinn JP, Cwirla SE, Peters-Bhatt E,Whitehorn EA, Tate EH, Akeson A, Bowlin TL, DowerWJ, Barrett RW (1996) High affinity type I interleukin 1receptor antagonists discovered by screening recombinantpeptide libraries. Proc Natl Acad Sci USA 93:7381–7386

Yem AW, Richard KA, Staite ND, Deibel MR (1988) Resolutionand biological properties of three N-terminal analogues ofrecombinant human interleukin-1 beta. Lymphokine Res7:85–92

H.3.1.7.4Inhibition of Interleukin-1β Converting Enzyme (ICE)

PURPOSE AND RATIONALEProgrammed cell death (apoptosis) is effected througha cascade of intracellular proteases known as caspases(Alnemri et al. 1996). The interleukin-1β-convertingenzyme (ICE), alternatively known as capsase-1, was


the first such protein identified on the basis of its se-quence homology to the pro-apoptotic Caenorhabdi-tis elegans gene product, ced-3 (Yuan et al. 1993).The caspase family includes 10 reported human ho-mologs of ICE. By sequence homology comparisonsbetween three caspase subfamilies have been identi-fied. The ICE subfamily includes three caspases: ICE,TX (caspase-4), and TY (caspase-5). The CPP32 sub-family includes CPP32 (caspase-3), CMH-1 (caspase-7), and MCH-2 (caspase-6). A third caspase subfam-ily includes ICH-1 (caspase-2), FLICE (caspase-8) andcaspases-9 and -10.

ICE processes pro-IL-1β to yield active IL-1β,which plays a pivotal role in inflammatory cell acti-vation (Dinarello 1996) and is known to inhibit the ex-pression of apoptosis (Tatsuda et al. 1996). Inhibitionof IL-1β formation is an approach for the treatmentof inflammatory disorders such as rheumatoid arthri-tis. Livingstone (1997) presented a review on in vitroand in vivo studies of peptidyl ICE inhibitors.

PROCEDURENeutrophil IsolationNeutrophils are isolated from healthy volunteersby dextran sedimentation and centrifugation througha discontinuous Ficoll gradient (Lee et al. 1993).Isolated neutrophils are resupended in polypropylenetubes at a concentration of 1 × 106 cells/ml in DMEMsupplemented with 10% FCS, 1% glutamine, and1% penicillin/streptomycin solution. Neutrophil purityis assessed by size and granularity on flow cytome-try.

Quantification of ApoptosisNeutrophil apoptosis is quantified by flow cytome-try as the percentage of cells with hypodiploid DNA(Nicoletti et al. 1991). Cells are centrifuged at 200 gfor 10 min, gently resuspended in 500 µl of hypotonicfluorochrome solution (50 µg/ml propidium iodide,3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA,and 0.1% Triton X-100) and stored in the dark at 4°Cfor 3–4 h before analysis using a Coulter Epics XL-MCL cytofluorometer. A minimum of 5000 events arecollected and analyzed. Apoptotic nuclei are distin-guished from normal neutrophil nuclei by their hy-podiploid DNA; neutrophil debris is excluded fromanalysis by raising the forward threshold. Apoptoticnuclei appear as a broad hypodiploid DNA peak whichis easily discernible from the narrow peak of cells withnormal diploid DNA content. Apoptosis is assessed at24 h after treatment.

Assay of Caspase-1 ActivityCell lysates are prepared from the membrane fractionof 20 × 106 neutrophils following experimental manip-ulation. Aliquots of the lysates (10 µl) are diluted in as-say buffer 100 mM HEPES (pH 7.4), 10% sucrose, and0.1% 3-([[3-cholamidopropyl]dimethylammonio]-1-propanesulfonate) containing 20 µM Ac-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Calbiochem) andthen incubated for 45 min at room temperature. Therelease of 7-amino-4-methylcoumarin is detected bycontinuous measurement using a Perkin-Elmer LS50luminescence spectrometer with an excitation of380 nm and an emission slit at 460 nm. Specific ICE(caspase-1) activity is measured as pmol/s per mil-ligram of protein.

EVALUATIONIndividual experiments are repeated a minimum offour times; results are expressed as the mean ±SD.Analysis is performed using the Student’s t-test orANOVA with Scheffè’s correction.

MODIFICATIONS OF THE METHODNorman et al. (1997) found that the severity and mor-tality of experimental pancreatitis are dependent oninterleukin-1 converting enzyme.

REFERENCES AND FURTHER READINGAlnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Thorn-

berry NA, Wong WW, Yuan JY (1996) Human ICE/CED3nomenclature. Cell 87:171

Dinarello CA (1996) Biological basis for interleukin-1 in dis-ease. Blood 87:2095–2147

Lee A, Whyte MKB, Haslett C (1993) Inhibition of apoptosisand prolongation of neutrophil functional longevity by in-flammatory mediators. J Leukocyte Biol 54:283–288

Livingstone DJ (1997) In vitro and in vivo studies of ICE in-hibitors. J Cell Biochem:19–26

Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C(1991) A rapid and simple method for measuring thymo-cyte apoptosis by propidium iodide staining and flow cy-tometry. J Immunol Methods 139:271–279

Norman J, Yang J, Fink G, Carter G, Ku G, Denham W, Liv-ingston D (1997) Severity and mortality of experimentalpancreatitis are dependent on interleukin-1 converting en-zyme (ICE). J Interferon Cytokine Res 17:113–118

Tatsuda T, Cheng J, Mountz JD (1996) Intracellular IL-1βis an inhibitor of Fas-mediated apoptosis. J Immunol157:3949–3957

William R, Watson G, Rotstein OD, Parodo J, Bitar R, Mar-shall JC (1998) The IL-1β-converting enzyme (caspase-1)inhibits apoptosis on inflammatory neurophils through ac-tivation of IL-1β. J Immunol 161:957–962

Yuan JS, Shaham S, Ledoux S, Ellis HM, Horvitz HR (1993)The C. elegans cell death gene ced-3 encodes a protein sim-ilar to mammalian interleukin 1β-converting enzyme. Cell75:641


H.3.1.7.5Nuclear Factor-κB

H.3.1.7.5.1General ConsiderationsNuclear factor kappa B (NF-κB) is an inducible tran-scription factor of the Rel family, sequestered in thecytoplasm by the IκB family of proteins. NF-κB ex-ists in several dimeric forms, but the p50/p65 het-erodimer is the predominant one. Activation of NF-κB by a range of physical, chemical, and biologicalstimuli leads to phosphorylation and proteasome de-pendent degradation of IκB, leading to the release offree NF-κB. This free NF-κB then binds to its targetsite (κB sites in the DNA), to initiate transcription.This transcription is involved in a number of diseasesincluding cancer, AIDS, autoimmune diseases, and in-flammatory disorders. The nuclear factor κB is essen-tial for the transcriptional regulation of the proinflam-matory cytokines IL-1, IL-6, IL-8 and tumor necro-sis factor α. NF-κ B is also the target for glucocor-ticoid–mediated IL-8 repression. Reduction-oxidation(redox) regulation is implicated in the activation ofNF-κB. (Mukaida et al. 1994, Aupperle et al. 1999,Christman et al. 2000a, 2000b, Nichols et al. 2001,D’Acquisto et al. 2002, Nishi et al. 2002, Palanki 2002,Tian et al. 2002, Heynink et al. 2003, Aggarwal et al.2004, Pande and Ramos 2003, 2005, Kaltschmidt et al.2005). The IKK complex, as a critical activator of NF-κB function, consists of a core of three subunits, two ofwhich, namely IKKα and IKKβ contain functional ki-nase domains and are capable of phosphorylating IκBat specific N-terminal residues to initiate its ubiquitina-tion. In contrast, the third core subunit of the IKK com-plex, called NEMO (also known as IKKγ or IKKAP)is a non-catalytic component that functions as a keyregulator of IKK activity (Gosh and Karin 2002, Karinet al. 2004).

Studying joint erosion in rheumatoid arthritisO’Gradaigh et al. (2003) found that interactions be-tween tumor necrosis factor α, interleukin, and recep-tor activator of nuclear factor κB ligand (RANKL) reg-ulate osteoclasts

REFERENCES AND FURTHER READINGAggarwal BB, Takada Y, Shishodia S, Gutierrrez AM, Oom-

men OV, Ichikawa H, Baba Y, Kumar A (2004) Nu-clear transcription factor NF-kappa B. Role in biology andmedicine. Indian J Exp Biol 42:341–353

Aupperle KR, Bennett BL, Boyle DL, Tak PP, Manning AM,Firestein GS (1999) NF-κB regulation by IκB kinase inprimary fibroblast-like synoviocytes. J Immunol 163:427–433

Christman JW, Blackwell TS, Juurlink BH (2000a) Redoxregulation of nuclear factor kappa B: therapeutic poten-tial for attenuating inflammatory responses. Brain Pathol10:153–162

Christman JW, Sadikot RT, Blackwell TS (2000b) The roleof nuclear factor κB in pulmonary diseases. Chest117:1482–1487

D’Acquisto F, May MJ, Gosh S (2002) Inhibition of nuclear fac-tor kappa B (NF-κB): An emerging ther in antiinflamma-tory therapies. Mol Interventions 2:22–35

Gosh S, KarinM (2002) Missing pieces in the NF-κB puzzle.Cell 109:S81-S96

Heynink K, Wullaert A, Beyaert R (2003) Nuclear factor-kappaB plays a central role in tumor necrosis factor-mediatedliver disease. Biochem Pharmacol 66:1409–1415

Kaltschmidt B, Widera D, Kaltschmidt C (2005) Signaling viaNF-kappa B in the nervous system. Biochim Biophys Acta1745:287–299

Katin M, Yamamoto Y, Wang QM (2004) The IKK NF-κB sys-tem: a treasure trove for drug development. Nature Rev3:17–26

Mukaida N, Morita M, Ishikawa Y, Rice N, Okamoto SI,Kasahara T, Matsushima K (1994) Novel mechanism ofglucocorticoid-mediated gene repression. J Biol Chem269:13289–13295

Nichols TC, Fischer TH, Deliagyris EN, Baldwin AS Jr (2001)Role of nuclear factor kappa B (NF-κB) in inflammation,periodontitis, and atherogenesis. Ann Periodontol 6:20–29

Nishi T, Shimizu N, Hiramoto M, Sato I, Yamaguchi Y,Hasegawa M, Aizawa S, Tanak H, Kataoka K, Watan-abe H, Handa H (2002) Spatial redox regulation of a crit-ical cysteine residue of NF-κB in vivo. J Biol Chem277:44548–44556

O’Gradaigh D, Ireland D, Bord S, Compston JE (2003) Jointerosion in rheumatoid arthritis: interactions between tumornecrosis factor α, interleukin, and receptor activator of nu-clear factor κB ligand (RANKL) regulate osteoclasts. AnnRheum Dis 63:354–359

Palanki MSS (2002) Inhibitors of AP-1 and NF-κB mediatedtranscriptional activation: therapeutic potential in autoim-mune diseases and structural diversity. Curr MedicinalChem 9:219–227

Pande Y, Ramos MJ (2003) Nuclear factor kappa B: a poten-tial target for anti-HIV chemotherapy. Curr Med Chem10:1603–1615

Pande Y, Ramos MJ (2005) NF-κB in human disease: Currentinhibitors and prospects for de novo structure based designof inhibitors. Curr Med Chem 12:357–373

Tian Y, Rabson AB, Gallo MA (2002) Ah receptor and NF-κBinteractions: mechanisms and physiological implications.Chem Biol Interact 141:97–115

H.3.1.7.5.2Inhibition of Nuclear Factor-κB

PURPOSE AND RATIONALESeveral authors studied the inhibition of NF-κB bycompounds.

Staal et al. (1990) found that intracelluar thiols reg-ulate activation of nuclear factor κB and transcrip-tion of human immunodeficiency virus. Schrenk et al.(1992) reported dithiocarbamates as potent inhibitorsof nuclear factor κB activation in intact cells. Natara-


jan et al. (1996) described caffeic acid phenyl esteras a potent and specific inhibitor of activation of nu-clear transcription factor NF-κB. Geng et al. (1997)reported that S-allyl cysteine inhibits activation of nu-clear factor κB in human T cells. Hiramoto et al.(1998) described nuclear-targeted suppression of NF-κB by a quinone derivative. Ichiyama et al. (1999)found inhibition of peripheral NF-κB activation bya central action of α-melanocyte-stimulating hormone.Castrillo et al. (2001) described inhibition of the nu-clear factor κ B pathway by tetracyclic kaurene diter-penes in macrophages with specific effects on NF-κB-inducing kinase activity and on the coordinated acti-vation of ERK and p38 MAPK. Kang et al. (2001)reported that genistein prevents nuclear factor kappaB activation and acute lung injury induced by in-tratracheal treatment of rats with lipopolysaccaride.Lee et al. (2002) found that kamebakaurin, a kau-rane diterpene, inhibits NF-κB by directly targeting theDNA-binding activity of p50 and blocks the expres-sion of antiapoptotic NF-κB target genes. Palanki et al.(2002) reported structure–activity relationship studiesof ethyl 2-[3-methyl-2,5-dioxo(3-pyrrolinyl) amino]-4-(trifluoromethyl)pyrimidine-5-carboxylate, an in-hibitor or AP-1 and NF-κB mediated gene expres-sion. Kim et al. (2004) found that tripolide, a naturalcompound extracted from the Chinese herb Triptery-gium wilfordii, inhibits murine-inducible nitric syn-thase expression by down-regulating lipopolysaccha-ride-induced activity of nuclear factor κB and c-JunNH2-terminal kinase. Jancso et al. (2005) studied theeffect of acetylsalicylic acid on nuclear factor-κB ac-tivation and on late preconditioning against infarc-tion of the myocardium. Kunsch et al. (2005) de-scribed redox-sensitive inflammatory gene expressionof AGIX-4207, an antioxidant and anti-inflammatorycompound.

Tse et al. (2005) found that honokiol, a small molecular weight lignan isolated from Magnolia offici-nalis, inhibits tumor-necrosis-factor-α-stimulated NF-κB activation and NF-κB-regulated gene expressionthrough suppression of inhibitor κB kinase (IKK) ac-tivation.

PROCEDUREHonokiol was dissolved in DMSO as a 100 mM stocksolution and stored at -20oC.

Cell CultureThe cell lines used in this experiment were ob-tained from American Type Culture Collection (Man-assas, Va., USA). U937 and HL-60 cells were grown

in RPMI-1640 medium containing 10% fetal bovineserum, 100 U/ml penicillin, and 100 µg/ml strepto-mycin (Gibco, NY, USA) at 37°C in humidified 5%CO2 atmosphere. MCF-7 and HeLa cells were culturedin Eagles’ minimum essential medium containing 10%fetal bovine serum under the same condition.

Electrophoretic Mobility Shift Assay (EMSA)For the electrophoretic mobility shift assay accord-ing to Chaturvedi et al. (2000), equal quantities ofnuclear protein (5 µg) from each sample was incu-bated with radiolabeled gel shift oligonucleotides for15 min at 37°C and then resolved on a non-denaturing5% (w/v) polyacrylamide gel. The gel was dried onto3 MM blotting paper and used to expose X-ray filmfor overnight at −70°C. For supershift assays, 1 µlof antiserum recognizing each of the NF-κB subunitswas added to the EMSA reaction 30 min before elec-trophoresis.

Western Blot AnalysisTo obtain the whole-cell lysates, samples containing1 × 107 cells were pelleted, washed twice with ice-cold PBS, then lysed in 150 µ l of modified RIPAbuffer [50 mM Tris–Cl, 1% (v/v) NP-40, 0.35% (w/v)sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1mM EGTA, pH 7.4] supplemented with 1 mM phenyl-methylsulfonyl fluoride (PMSF), 1 mM NaF, 1 mMNa3VO4, 10 µg/ml each of aprotinin, leupeptin andpepstatin A for 20 min at 4°C. Supernatants after cen-trifugation at 14,000 g for 15 min at 4°C were col-lected. Alternatively, cytoplasmic extracts were pre-pared. Samples containing 30–50 µg of protein wereseparated on SDS-polyacrylamide gel and then trans-ferred onto nitrocellulose membrane (0.45 µm, Bio-Rad). Membranes were immunoblotted with primaryantibodies and followed by horseradish-peroxidase-conjugated secondary antibodies (1:5000) and visual-ized by ECL (Amersham Biosciences) according tomanufacturer’s instructions.

IKK AssayWhole-cell lysates (500 mg) were collected in mod-ified RIPA buffer without sodium deoxycholate, andcellular debris was removed by high-speed centrifuga-tion. Lysates were pre-cleared by incubation with 0.25µg of the appropriate control IgG together with 20 µl ofprotein A/G plus (25%, v/v) agarose conjugate for 30min at 4°C, followed by centrifugation. Supernatantswere then incubated with 1 µg of anti-IKKα/β for 2h at 4°C, and then 20 µl of protein A/G plus agarosewas added and incubated at 4°C on a rocker platform


overnight. After several washes with IP buffer andPBS, beads containing IKKα/β were incubated with0.5 µg GST- Iκ Bα substrate, 200 µM ATP in 20 µl ki-nase buffer (50 mM Tris–Cl pH 7.4, 20 mM MgCl2, 20mM β-glycerophosphate, 1 mM NaF, 1 mM Na3VO4,1 mM PMSF, 0.5 mM DTT, 1 mM benzamidine, 10µg/ml aprotinin and 1 µg/ml leupeptin) at 30°C for 30min. Kinase reactions were stopped by the addition of5 µl 5 × Laemmli’s loading buffer and heated at 100°Cfor 5 min. The samples were resolved by 8% SDS-PAGE, electro-transferred to nitrocellulose membraneand probed with anti-phosphor-Iκ Bα (Ser32) antibody(1:1000). Membranes were re-probed with anti-IKKto ensure equal loading and the presence of total IKKprotein.

Plasmids, Transfection and NF-κB-Dependent-LuciferaseReporter AssayTo measure the effect of honokiol on TNF-α-inducedNF-κ B-dependent gene reporter transcription, HeLacells were seeded into 24-well plates at a den-sity 1.6 × 105 cells/well for 24 h. Subsequently, cellswere transiently transfected with p3EnhConA-Lucor pControl-Luc (0.75 µg) using LipofectAMINE2000 (Invitrogen). To normalize the transfection effi-ciency, cells were co-transfected with 0.25 µg of β-galactosidase control vector. After overnight incuba-tion, cells were pre-treated with honokiol for 12 hfollowing by 5 ng/ml TNF-α for 15 h and then har-vested with 1 × reporter lysis buffer (Promega, Madi-son, Wis., USA). Relative luciferase activity was mea-sured with a Bright-GLO luciferase assay system usingPOLARStar OPTIMA luminometer (BMG Labtech-nologies). Luciferase activity was normalized with β-galactosidase activity, as measured by the Beta-GLOluciferase assay system according to the manufac-turer’s instructions.

To measure the effect of honokiol on NF-κB-dependent gene reporter transcription induced byvarious kinases, HeLa cells were transfected withp3EnhConA-Luc and β-galactosidase control vectortogether with 0.2 µg of expression vectors. After 5 hof incubation, cells were treated with honokiol for 24h and then harvested and assayed as described above.

EVALUATIONStatistical analyses were performed using an unpairedtwo-tailed Student’s t-test. Two compounds (A and B)were considered enhancing each other’s actions if theeffect of combined treatment (AB) was larger then thesum of their individual effects (AB > A + B) after sub-traction of the respective background control values.

MODIFICATIONS OF THE METHODMortellaro et al. (1999) reported that the immunosup-pressive drug PNU156804 blocks IL-2-dependent pro-liferation and NF-κB and AP-1 activation in humanprimary T lymphocytes.

Spencer et al. (1999) found in murine NIH3T3 fi-broblasts and primate COS-7 cells that taxol selec-tively blocks microtubule-dependent NF-κB activationby phorbol ester via inhibition of Iκ Bα phosphoryla-tion and degradation.

Yan and Polk (1999) reported that aminosalicylicacid inhibits IκB kinase α phosphorylation of Iκ Bα inmouse intestinal epithelial cells.

Acarin et al. (2000) found that oral administrationof the anti-inflammatory substance triflusal results inthe downregulation of constitutive transcription factorNF-κB in the postnatal rat brain.

Eberhardt et al. (2002) studied involvement of nu-clear factor κB and Ets transcription factors in glu-cocorticoid-mediated suppression of cytokine-inducedmatrix metalloprotease-9 expression in rat mesangialcells.

Kang et al. (2002) showed that inhaled nitric ox-ide attenuated acute lipopolysaccharide-induced lunginjury in rabbits via inhibition of nuclear factor-κB.

Macotela et al. (2002) found on rat pulmonary fi-broblasts that 16K prolactin induces NF-κB activation.

Roshak et al. (2002) reported small-molecule in-hibitors of NF-κB for the treatment of inflammatoryjoint disease.

Burke et al. (2003) found that BMS-345541 isa highly selective inhibitor of IκB kinase that bindsat an allosteric site of the enzyme and blocks NF-κB–dependent transcription in mice.

Castro et al. (2003) described β-carbolines as in-hibitors of the NF-κB kinase.

Clarke et al. (2003) reported that two distinct phasesof virus-induced nuclear factor κB regulation enhancetumor necrosis factor-related apoptosis-inducing lig-and-mediated apoptosis in virus-infected cells.

Murata et al. (2003) described discovery of selec-tive IKK-β serine-threonin protein kinase inhibitors.

Yadav et al. (2003) reported that a diarylheptanoidfrom lesser galangal (Alpinia officinarum) inhibitsproinflammatory mediators via inhibition of mitogen-activated protein kinase, P44/42, and transcription fac-tor nuclear factor-κB.

HMG-CoA reductase inhibitors (statins) inhibitedthe binding of nuclear proteins to both NF-κB andAP-1 DNA consensus oligonucleotides in human en-dothelial and vascular smooth muscle cells as assed byEMSA (Dichtl et al. 2003).


Gupta et al. (2004) discussed the essential role ofcaspases in epigallocatechin-3-gallate-mediated inhi-bition of nuclear factor kappaB and induction of apop-tosis.

Mühlbauer et al. (2004) studied differential effectsof deoxycholic acid and taurodeoxycholic acid on NF-κB signal transduction and IL-8 gene expression in hu-man colonic epithelial cells.

Syrovets et al. (2005) found that acetyl-boswellicacids inhibit lipopolysaccaride-mediated TNF-α in-duction by direct interaction with IκB kinases.

Eberhardt et al. (2005) found that dissociated glu-cocorticoids equipotently inhibit cytokine- and cAMP-induced matrix degrading proteases in rat mesangialcells.

Matsubara et al. (2005) found that a histamine H1receptor antagonist blocks histamine-induced proin-flammatory cytokine production through inhibition ofCa2+-dependent protein kinase C, Raf/MEK/ERK andIKK/ Iκ B/NF-κB signal cascades.

Mendoza-Milla et al. (2005) reported that NF-κBactivation but not PIK/Akt is required for dexametha-sone-dependent protection against TNF-α cytotoxicityin L929 cells.

APPENDIXElectrophoretic Mobility Shift Assay (EMSA)Most papers mentioned above used the electrophoreticmobility shift assay (EMSA) for determination of nu-clear factor κB. This method is one of the most sensi-tive ones for studying the DNA-binding properties ofa protein. It can be used to deduce the binding param-eters and relative affinities of a protein for one or moreDNA sites or for comparing the affinities of differentproteins to the same sites (Fried 1989). It is also usefulfor studying higher-order complexes containing sev-eral proteins, observed as a “supershift assay.” EMSAcan also be used to study protein- or sequence-depen-dent DNA bending (Crothers et al. 1991).

In an EMSA, or simple “gel shift,” a 32P-labeledDNA fragment containing a specific DNA site isincubated with a candidate DNA-binding protein.The protein–DNA complexes are separated from free(unbound) DNA by electrophoresis through a non-denaturing polyacrylamide gel. The protein retards themobility of the DNA fragments to which it binds; thus,the free DNA migrates faster through the gel than doesthe DNA–protein complex. An image of the gel revealsthe positions of the free and bound 32P-labeled DNA.

A protocol of the detailed procedure was describedby Carey and Smale (2000).

PROCEDURE1. Prepare a 40-ml 4.5% native acrylamide gel (using

1- to 1.5-mm spacers)

Acrylamide mix (30%:29:1acrylamide:bisacrylamide)

6 ml

5 × Tris-borate/EDTA (TBE) buffer 4 ml

Glycerol (20% vol/vol) 2 ml

Water 28 ml

Ammonium persulfate (10% solution in water) 300 µl

N,N,N,N-tetramethylenediamine(add just before pouring the gel)

30 µl

Pre-run the gel for 2 h at 10 mA.2. Set up binding reactions in 0.5-ml siliconized

microcentrifuge tubse

Recombinant protein (0.5–100 ng) 1.00 µl32P-labeled DNA template (ideally 1 fmol) 1.00 µl

Poly(dl:dC) (1 µg/µl) 0.20 µl

Dimethylsulfoxide (0.1 M) 0.10 µl

MgCl2 (0.1 M) 0.75 µl

Buffer D 6.70 µl

Water to 10 µl

Buffer D is 20 mM HEPES-KOH (pH 7.9), 20%glycerol (vol/vol), 0.2 mM EDTA, 0.1 M KCl, 0.5mM phenylmethylsulfonyl fluoride (PMSF), 1 mMDTT. Add PMSF and DTT just before use.

3. Incubate the reaction at 30°C (incubate at15oC–25°C or on ice for crude extracts) for1 h.

4. Load the samples directly (with no dye) onto gel.Carefully layer the mix onto the bottom of the welland observe the schlieren line form at the glyc-erol–buffer interface.

5. Rune the gel for desired time at 10 mA; for a 30-bpfragment, allow the bromophenol blue dye to mi-grate about two-thirds of the way down the gel.

6. When the electrophoresis run is complete, carefullypour out the buffer into the sink and remove the gelfrom the apparatus. Remove the comb and split theplates, leaving the gel attached to one plate.

7. (Optional) Fix the gel in gel fixing solution (200ml methanol, 100 ml acetic acid, 700 ml water) at15oC–25°C for 15 min.

8. Place the gel on two sheets of Whatman 3MM pa-per. Cover the other side of the gel with Saran Wrapand dry on a gel dryer at 70°C for 1 h.

9. Expose the gel to autoradiography film or to phos-phorimager screen overnight.


REFERENCES AND FURTHER READINGAcarin L, González B, Castellano B (2000) Oral administration

of the anti-inflammatory substance triflusal results in thedownregulation of constitutive transcription factor NF-κBin the postnatal rat brain. Neurosci Lett 288:41–44

Burke JR, Pattoli MA, Gregor KR, Brassil PJ, MacMaster JF,McIntyre KW, Yang X, Iotzova VS, Clarke W, Strnad J,Qiu Y, Zusi FC (2003) BMS-345541 is a highly selectiveinhibitor of IκB kinase that binds at an allosteric site ofthe enzyme and blocks NF-κB–dependent transcription inmice. J Biol Chem 278:1450–1456

Carey M, Smale ST (2000) Electrophoretic mobility shift as-says. In: Transcriptional regulation in eukaryotes: concepts,strategies, and techniques. Chap 13, Protocol 13.5:493–496(Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, New York, USA, 2000). Nature Methods 2:557–558

Castrillo A, de las Heras B, Hortelano S, Rodríguez B, Vil-lar A, Boscá L (2001) Inhibition of the nuclear factor κB(NF-κ B) pathway by tetracyclic kaurene diterpenes inmacrophages. J Biol Chem 276:15854–15860

Castro AC, Dang LC, Soucy F, Grenier L, Mazdiyasni H, Hot-telet M, Parent L, Pien C, Palombella V, Adams J (2003)Novel IKK inhibitors: β-carbolines. Bioorg Med ChemLett 13:2419–2422

Chaturvedi MM, Mukhopadhyay A, Aggarwal BB (2000) Assayfor redox-sensitive transcription factor. Methods Enzymol319:585–602

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H.3.1.8TNF-α Antagonism


TNF-α has been cloned and identified by Beutler et al.(1985). It is primarily produced in macrophages, lym-phocytes, neutrophils, endothelial cells, keratinocytesand fibroblasts during acute inflammatory reactions.TNF-α is a member of a large family of proteins andreceptors that are involved in immune regulation suchas kinases, including nuclear factor kappa B (NF-κ B),p38 MAP kinase, JUN kinases and others. Therefore, itis a therapeutic target for immune-mediated inflamma-tory diseases (Pfizenmaier et al. 1996; Van Deventer1997; Rath and Aggarwal 1999; Feldmann et al. 2001;Furst et al. 2001; Taylor 2001; Doggrell 2002; Braunet al. 2003; Sharma and Anker 2003; Louie et al. 2003;Nanes 2003; Peng et al. 2003; Chen et al. 2003; Tay-

lor et al. 2004; Gupta et al. 2005; Pfeifer et al. 2006;Reber et al. 2006; Wagner and Laufer 2006). This maybe achieved by small molecular anti-cytokine agentsinhibiting cytokine production, which target p38 mi-togen activated protein (MAP) kinase, TNF-α con-verting enzyme (TACE), or IL-1β converting enzyme(ICE).

Several so-called “biologicals” are in clinical use:Etanercept (Enbrel), a fully human soluble TNF

receptor fusion protein consisting of the extracellularligand-binding domain of the 75-kDa receptor for hu-man tumor necrosis factor-α and and the constant por-tion of human IgG1 (Jarvis and Faulds 1999; Pugsley2001; Scallon et al. 2002; Agnholt et al. 2003; Coleand Rabasseda 2004; Goffe 2004; Moe et al. 2004;Vallejo et al. 2005). The compound has been approvedfor treatment of psoriasis, psoriatic arthritis, ankylos-ing spondylitis and rheumatoid arthritis.

Infliximab (Remicade), a chimeric anti humanTNF-α monoclonal antibody (Scallon et al. 2002;Agnholt et al. 2003; Di Sabatino et al. 2004; Wagneret al. 2004; Panaccione et al. 2005; Shen et al. 2005;Pfeifer et al. 2006). The compound is used for treat-ment of rheumatoid arthritis and Crohn’s disease.

Adalimumab (HUMIRA), a recombinant humananti-human TNF-α monoclonal antibody (Gordonet al. 2005; Aggarwal et al. 2006; Scheinfeld 2006;Shen et al. 2006). The compound is used for treatmentof rheumatoid arthritis and psoriatic arthritis.

Imatinib mesylate (STI571, Gleevec), a kinase in-hibitor of TNF-α production (Kilic et al. 2000; Traxleret al. 2001; Dietz et al. 2004; Kaelin 2004; Lassila et al.2005; Wolf et al. 2005; Adcock et al. 2006). The com-pound has been found to be active in the treatment ofchronic myelogenous leukemia, gastrointestinal stro-mal tumors, eosinophilic disorders, and systemic mastcell disease.

Omalizumab (Xolair), a recombinant humanizedmonoclonal antibody which specifically binds the Cε3domain of IgE, the site of high-affinity IgE receptorbinding (Easthope and Jarvis 2001; Anonymous 2002;Johansson et al. 2002; Davis 2004; Richards et al.2004; Belliveau 2005; D’Amato 2006). The compoundis used for treatment of bronchial asthma and allergicrhinitis.

Anakinra (Kineret) is a specific recombinant hu-man interleukim-1 receptor antagonist that differsfrom naturally occurring IL-1 receptor antagonistby the presence of a methionine group (Cvetkovicand Keating 2002; Fleischmann et al. 2004; Le andAbbenante 2005; Waugh and Perry 2005). The com-pound is effective in patients with active rheumatoid


arthritis, either when given alone or in combinationwith methotrexate.

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Furst DE, Keystone EC, Breedveld FC, Kalden JR, Smolen JS,Antoni CE, Burmester GR, Crofford LJ, Kavanaugh A(2001) Updated consensus statement on tumor necrosis fac-tor blocking agents for the treatment of rheumatoid arthritisand other rheumatic disorders.. Ann Rheum Dis 60:2–5

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H.3.1.8.2Inhibition of TNF-α Release

PURPOSE AND RATIONALEThere are two distinct types of tumor necrosis factors,TNF-alpha (cachectin) and TNF-beta (lymphotoxin),with biological activities going beyond the necrosis oftumor cells. Some of the known activities include theinduction of interleukin 1, activation of PMNs, modu-lation of endothelial cell functions, and augmentationof specific immune functions. The complex sequenceof hemodynamic and metabolic collapse, which leadsto shock and death during lethal endotoxinemia, ap-pear to represent the response of the infected hostto the acute, systemic release of TNF-alpha. Thus,drugs that antagonize the activity of this mediatorcould be of clinical value in combating its fatal ef-fects.

PROCEDURETwenty hours before the initiation of the experiments,L 929 cells are harvested from stock cultures and areplated in 96 well culture plates (2 × 104 cells/well)and incubated at 37°C and 5% CO2 in air. For eachgroup 6 wells are set up. The cells are then preincu-bated for 30 min with test substances or solvent beforeTNF-alpha is added (between 1 and 10 IU/well). Af-ter an additional incubation time of 20 h, the cultureplates are flicked out and the remaining living cells arelysed by the addition of bidistilled water (100 µl). After30 min incubation at room temperature, 100 µl of LDHreagent are given to each culture well. After 15 min,the enzyme activity is determined photometrically at490 nm.

EVALUATIONThe percent inhibition is calculated according to theformula:

% inhibition = 100%

− ext. test group − ext. spontaneous lysis

ext.positive control − ext. spontaneous lysis.

The positive control is the group which receives vehi-cle and TNF-alpha. The spontaneous lysis is based oncultures which receive vehicle without TNF-alpha.

MODIFICATIONS OF THE METHODMaloff and Delmendo (1991) measured the binding oftumor necrosis factor (TNF-α) to the human TNF re-ceptor. Membranes were prepared from HeLa S3 hu-man cervical epithelioid carcinoma cells. An aliquotof 0.2 mg of membrane preparation was incubated with62 pM [125I]TNF-α for 3 h at 4°C. Nonspecific bindingwas measured in the presence of 50 nM TNF-α. Mem-branes were filtered and washed 3 times and the filterswere counted to determine the bound [125I]TNF-α.

Golebiowski et al. (2005) tested pyrazolone-basedcytokine synthesis inhibitors for the inhibition ofTNF-α production using lipopolysaccharide-stimu-lated monocytic cells. Duplicate cultures of humanmonocytic cells (2.0 × 106/well) were incubated for 15min in the presence or absence of various concentra-tions of inhibitor before the stimulation of cytokine re-lease by the addition of lipopolysaccharide (1 µg/ml).The amount of TNF-α released was measured 4 h laterusing an ELISA system.

Kumar et al. (1997) described homologs ofCSBP/p38 MAP kinase, their activation, as well assubstrate specificity and sensitivity to inhibition bypyridyl imidazoles.


McLay et al. (2001) reported the discovery of a p38MAP kinase inhibitor displaying a good oral anti-arthritic efficacy.

Ignar et al. (2003) described the regulation of TNF-α secretion by a specific melanocortin-1 receptor pep-tide agonist.

Kinases AssayThe p38 enzyme assay is carried out at room tem-perature for 1 h, using 40 ng/well of the mouse en-zyme. The substrate, 50 µg/ml ATF-2 transformationfactor, is coated onto 96-well plates, and the assayis carried out in 25 mM Hepes buffer, pH 7.7 con-taining 25 mM magnesium chloride, 2 mM dithiothre-itol, 1 mM sodium orthovanadate and 100 µM ATP.Phosphorylated ATF-2 is quantitated using a phospho-specific ATF-2 primary antibody (rabbit anti-human)followed by a europium-labeled secondary antibody(sheep anti-rabbit IgG) with addition of the DELFIAenhancement solution resulting in fluorescence. ERKwas measured using a [33P]ATP filtration assay formatusing for substrates myelin basic protein. ZAP-70, Sykand Lck kinase activities were measured using the ho-mogeneous time-resolved fluorescence methodology(HTRF) with the catalytic domains of each of the tyro-sine kinases, biotinylated, specific peptide substrates,streptavidin-linked APC and europium cryptate-conju-gated anti-phosphotyrosine antibody.

Monocyte TNF-α Release AssayAdherent human monocytes (100,000 cells/well) wereincubated with LPS (10 ng/ml) in the absence and pres-ence of compound for 18 h. Individual experimentswere carried out in quadruplicate samples. TNF-αwas measured by sandwich ELISA and IC50 valuescalculated for the activity of individual compounds.IC50 values shown from repeat experiments are means±SEM.

Mouse TNF-α Release AssayCompound was administered orally to BALB/c mice30 min prior to LPS (0.1 mg/kg i.p.) challenge. SerumTNF-α levels were determined 90 min after LPS insult.Results represent means ±SEM.

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Golebiowski A, Towner J, Laufersweiler MJ, Brugel TA,Clark MP, Clark CM, Djung JF, Laughlin SK, Sabat MP,Bookland RG, VanRens JC, De B, Hsieh LC, Janusz MJ,

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Ignar D, Andrews JL, Jansen M, Eilert MM, Pink HM, Lin P,Sherrill RG, Szewczyk JR, Conway JG (2003) Regulationof TNF-α secretion by a specific melanocortin-1 receptorpeptide agonist. Peptides 24:709–716

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McLay IM, Halley F, Souness JE, McKenna J, Benning V, Bir-rell M, Burton B, Belvisi M, Collis A, Constan A, Fos-ter M, Hele D, Jayyosi Z, Kelley M, Maslen C, Miller G,Ouldelhkim MC, Page K, Phipps S, Pollock K, Porter B,Ratcliffe AJ, Redford EJ, Webber S, Slater B, Thybaud V,Wilsher N (2001) The discovery of RPR 200765A, a p38MAP kinase inhibitor displaying a good oral anti-arthriticefficacy. Bioorg Med Chem 9:537–554

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H.3.1.8.3Effect of TNF-α Binding

PURPOSE AND RATIONALESeveral authors compared the effects of the prototypesof the tumor necrosis factor (TNF) antagonists in-fliximab or adalimumab and etanercept (Vuolteenahoet al. 2002; Kirchner et al. 2004; Shen et al. 2005).

Scallon et al. (2002) compared the binding andfunctional properties of the two prototypes of the TNFantagonists infliximab and etanercept-

Although both infliximab and etanercept are potentneutralizers of TNF bioactivity, there are fundamen-tal differences in their molecular structures, their bind-ing specificities, and the manner in which they neu-tralize TNF. Infliximab is a chimeric monoclonal anti-body (mAb) with murine variable regions and humanIgG1 and κ constant regions (Knight et al. 1993). Thesize (149 kDa) and structure of infliximab are there-fore similar to those of naturally occurring antibodies.Etanercept is a fusion protein made up of the extra-cellular domain of the p75 TNF receptor (CD120b)and the hinge and Fc domains of human IgG1 (Mohleret al. 1993), a structure distinct from any known natu-rally occurring molecule. Importantly, infliximab is notknown to bind to any antigen other than TNF, whereasetanercept binds equally well to both TNF and lym-photoxin α (LT α), consistent with observations re-ported for the cellular p75 TNF receptor (Schall et al.1990; Smith et al. 1990). Each infliximab molecule iscapable of binding to two TNF molecules, and up tothree infliximab molecules can bind to each TNF ho-


motrimer, thereby blocking all receptor binding siteson TNF. In contrast, it is believed that the bivalent etan-ercept molecule forms a 1:1 complex with the TNFtrimer in which two of the three receptor binding siteson TNF are occupied by etanercept, and the third re-ceptor binding site is open. In addition, the p75 TNFreceptor is known to have fast rates of association anddissociation with TNF (Evans et al. 1994), which sug-gests that etanercept may only transiently neutralizethe activity of an individual TNF molecule.

PROCEDURECell CultureKYM-1D4 cells that endogenously express TNF re-ceptors (Butler et al. 1994) were maintained in RPMI-1640 medium supplemented with 2 mM L-glutamineand 10% FBS. Human umbilical vein endothelial(HUVE) cells from Cell Systems (Seattle, Wash.,USA) were maintained in HUVE cell medium sup-plied by Cell Systems. K2 cells were maintainedin Iscove’s modified Dulbecco’s medium (IMDM)supplemented with 5% FBS, 2 mM L-glutamine,0.5 µg/ml mycophenolic acid, 2.5 µg/ml hypoxanthine,and 50 µg/ml xanthine. All cells were cultured in a hu-midified incubator maintained at 37°C and 5% CO2.

Binding to Monomer Subunits of TNFDimethylsulfoxide (DMSO) was added to 125I-TNF(40–60 µCi/µ g; 1.48–2.2 MBq/µg) to a final con-centration of 10% DMSO and incubated at 20°C for30 min to allow dissociation of TNF trimers. The mix-ture was passed over a 10 × 300-mm Superose 12 col-umn equilibrated with PBS, and 125I-TNF trimer andmonomer were collected separately. Polystyrene 96-well microtiter plates were coated by incubating 50 µlof 1 µg/ml of infliximab, etanercept, or an isotype-matched, negative control antibody (cM-T412) in thewells overnight at 4°C. After washing with PBS-0.05%Tween 20 (PBS-T), all wells were blocked for 1 h at37°C with PBS-1% bovine serum albumin and washedthree times with PBS-T. Triplicate wells were thenincubated with 125I-TNF trimer [0.4 µCi (14.8 kBq),10 ng/ml] or 125I-TNF monomer [0.1 µCi (3.7 kBq),2.5 ng/ml] alone or with 5 µg/ml unlabeled TNF. Af-ter 1 h at 37°C, the wells were washed with PBS-T andcounted for 125I.

Binding Assay to Measure Stabilityof Complexes with Soluble TNFEach well of a 96-well enzyme immunoassay plate wasincubated overnight at 4°C with 100 µl of 0.1 M car-bonate, pH 9.6, containing 10 µ g/ml goat anti-human

γ Fc antibody. Plates were washed three times withPBS-T and then incubated for 1 h at 37°C in block-ing buffer (10 mM HEPES, pH 7.5, containing 0.1%porcine gelatin, 150 µl/well). Wells were incubated for1 h at 37°C with 100 µl/well of blocking buffer con-taining 1 µg/ml infliximab or etanercept. Plates werewashed three times with PBS-T, and then all TNFbinding sites were saturated by incubating the wellsfor 1 h at 37°C in 100 µl/well of blocking buffer con-taining 10 ng/ml 125I-TNF (40–60 µCi/µg). Wells werewashed three times with PBS-T and then filled with100 µl/well of blocking buffer alone or containing anexcess of soluble, unlabeled competitor such as inflix-imab, etanercept, or human TNF, and subsequently in-cubated at 37°C. At the indicated time points, tripli-cate wells were washed three times with PBS-T to re-move free 125I-labeled TNF. The last wash was aspi-rated and replaced with 50 µl of scintillation fluid andthe entire plate counted in a Packard TopCount gammacounter.

Assay for Bioactivity of Dissociated Soluble TNFMicrotiter plates were coated with goat anti-humanγ Fc antibody and used to capture etanercept as de-scribed above. Wells were washed three times withPBS-T and incubated with 100 µl of 10 ng/ml unla-beled human TNF in 100 µl/well of blocking bufferfor 1 h at 37°C. Wells were washed three times withKYM media, filled with 100 µl of KYM media, and500 ng/ml mouse TNF was added to each well asa competitor. After a 1-h incubation at 37°C, the sol-uble fraction was removed and pre-incubated for 1 hin fresh wells with either no mAb, 10 µg/ml anti-human TNF mAb (infliximab), 85 µg/ml anti-mouseTNF mAb (cV1q huG3), or a combination of 10 µg/mlanti-human TNF and 85 µg/ml anti-mouse TNF mAb.After the pre-incubation, the soluble fractions wereadded to cultures of KYM-1D4 cells (50,000 cells/wellin a 96-well plate) and the cells incubated for 16 h at37°C in the presence of 0.5 µg/ml actinomycin D. Toquantitate cell viability, MTT dye was added to a fi-nal concentration 0.5 mg/ml and the cells incubated at37°C for 4 h. The medium was aspirated and 100 µl of100% DMSO was added to the cells. The differencebetween the absorbance at 550 and 650 nm was thendetermined.

HUVE Cell Assay to Measure Stabilityof Complexes with Soluble TNFInfliximab or etanercept was mixed with 1 µg/ml hu-man TNF at 10:1 or 30:1 M ratios in HUVE cellmedium and incubated for 30 min at 37°C. Serial di-


lutions of the preformed complexes were then addedto confluent HUVE cells cultured in 96-well plates.Cells were incubated with the preformed complexesin 100 µl of HUVE cell medium for 4 h at 37°C andthen washed three times with HBSS. The cells werethen incubated for 1 h at 37°C in HBSS containing1 µg/ml 125I-labeled anti-E-selectin (20 µCi/µg). Cellswere washed three times with HBSS, the last washwas aspirated and replaced with 30 µl of scintillationfluid, and the entire plate was counted in a PackardTopCount gamma counter.

Binding Assay to Measure Stabilityof Complexes with Transmembrane TNFK2 cells, which stably express an uncleavable and thuspermanently transmembrane (tm) form of TNF, wereseeded at a density of 5 × 104 cells/well in a 96-wellround-bottom plate in 100 µl of IMDM, 5% FBS. Sub-sequently, 125I-labeled infliximab or 125I-labeled etan-ercept (both at 8.5 µCi/µg) was added to a final con-centration of 0.5 µg/ml (enough to saturate all TNFbinding sites on the cells). After a 1-h incubationat 25°C, unbound infliximab and etanercept were re-moved by washing three times with IMDM medium.Fresh IMDM, 5% FBS medium (100 µl) alone, or con-taining 50 µg/ml of an unlabeled soluble competitor,was added to the cells. Soluble competitors were ei-ther infliximab or etanercept for samples treated withradiolabeled infliximab and one of infliximab, etaner-cept, or human LTα for samples treated with radio-labeled etanercept. The cells were then incubated at37°C in 5% CO2. At different time points, cells in se-lected wells were washed with PBS, and the numberof counts bound to the cells determined using a gammacounter (PerkinElmer Wallac, Wellesley, Mass., USA).

Characterization of Infliximaband Etanercept Binding to tmTNFK2 cells or TNF-negative Sp2/0 control cells wereseeded in 96-well round-bottom plates at a densityof 5 × 104 cells/well in IMDM, 5% FBS. Varyingamounts of 125I-labeled infliximab (23.4 µ Ci/µg) or125I-labeled etanercept (22.4 µCi/µg) were added tothe cells. After a 16-h incubation at 4°C, cells werewashed four times with culture medium (IMDM, 5%FBS), the last wash was aspirated, and 50 µl of culturemedium was added to each well. The cells were thenremoved with cotton swabs and the number of countsper well was determined using a gamma counter(PerkinElmer Wallac). The resulting binding data wereanalyzed by non-linear regression using Prism soft-ware (GraphPad Software, San Diego, Calif., USA).

HUVE Cell Assay to Compare Abilityto Inhibit tmTNF BioactivityK2 cells or Sp2/0 control cells were seeded in 96-wellround-bottom plates at a density of 1 × 105 cells/wellin IMDM, 5% FBS. Varying amounts of infliximabor etanercept in IMDM, 5% FBS were added and themixture incubated for 1 h at 37°C. This mixture wasthen added to confluent cultures of HUVE cells in96-well plates. The resulting cell-cell mixture was in-cubated for an additional 4 h at 37°C in a 5% CO2incubator. Cells were then washed three times withHBSS and incubated for 1 h with 1 µg/ml 125I-anti-E-selectin (20 µCi/µg). Cells were washed three timeswith HBSS, the last wash was aspirated and replacedwith 30 µl of scintillation fluid, and the plate countedin a Packard TopCount gamma counter.

EVALUATIONData were analyzed using a paired Student’s t-test todetermine whether there was a statistically significantdifference between the capacities of infliximab andetanercept to block the bioactivity of tmTNF.

MODIFICATIONS OF THE METHODMaloff and Delmendo (1991) developed high-throughput radioligand binding assays for interleukin1-α (IL-1-α) and tumor necrosis factor (TNF-α) in iso-lated membrane preparations.

Zhang et al. (2002) described identification andcharacterization of a dual tumor necrosis factor con-verting enzyme/matrix metalloprotease inhibitor forthe treatment of rheumatoid arthritis.

Transgenic mice expressing human tumor necro-sis factor develop severe polyarthritis (Keffer et al.1991; Kollias et al. 1999; Kontoyiannis et al. 1999;Mijatovic et al. 2000; Akassoglou et al. 2003; Li andSchwarz 2003). A targeting vector containing a ge-nomic fragment encoding the entire TNF-α with theARE-containing 3′ UTR was replaced with the 3′ UTRfrom the β-globin gene. This mutation increases thestability and translational efficiency of TNF-α mRNAand thus results in chronic TNF-α over-expression thatleads to severe erosive polyarthritis. Administration ofanti- TNF-α antibodies completely prevents the dis-ease.

REFERENCES AND FURTHER READINGAkassoglou K, Douni E, Bauer J, Lassmann H, Kollias G,

Probert L (2003) Exclusive tumor necrosis factor (TNF)signaling by the p75TNF receptor triggers inflammatory is-chemia in the CNS of transgenic mice. Proc Natl Acad SciUSA 100:709–714


Butler DM, Scallon B, Meager A, Kissonerghis M, Corcoran A,Chernajovsky Y, Feldmann M, Ghrayeb J, Brennan FM(1994) TNF receptor fusion proteins are effective inhibitorsof TNF-mediated cytotoxicity on human KYM-1D4 rhab-domyosarcoma cells. Cytokine 6:616–623

Evans TJ, Moyes D, Carpenter A, Martin R, Loetscher H, Less-lauer W, Cohen J (1994) Protective effect of 55- but not75-kD soluble tumor necrosis factor receptor-immunoglob-ulin G fusion proteins in an animal model of gram-negativesepsis. J Exp Med 180:2173–2179

Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E,Kioussis D, Kollias G (1991) Transgenic mice expressinghuman tumour necrosis factor: a predictive model of arthri-tis. EMBO J 10:4025–4031

Kirchner S, Holler E, Haffner S, Andreesen R, Eissner G (2004)Effect of different tumor necrosis factor (TNF) reactiveagents on reverse signaling of membrane integrated TNFin monocytes. Cytokine 28:67–74

Knight DM, Trinh H, Le J, Siegel S, Shealy D, McDonough M,Scallon B, Moore MA, Vilcek J, Daddona P (1993) Con-struction and initial characterization of a mouse-humanchimeric anti-TNF antibody. Mol Immunol 30:1443–1453

Kollias D, Douni E, Kassiotis G, Kontoyannis D (1999) Thefunction of tumour necrosis factor and receptors in modelsof multi-organ inflammation, rheumatoid arthritis, multiplesclerosis and inflammatory bowel disease. Ann Rheum Dis58 [Suppl 1]:I32–I39

Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelle F, Kol-lias G (1999) Impaired on/off regulation of TNF biosyn-thesis in mice lacking TNF AU-rich elements: implicationsfor joint and gut-associated immunopathologies. Immunity10:387–398

Li P, Schwarz EM (2003) The TNF-α transgenic mouse modelin inflammatory arthritis. Springer Semin Immonopathol25:19–33

Maloff BL, Delmendo RE (1991) Development of high-throughput radioligand binding assays for interleukin 1-α (IL-1-α) and tumor necrosis factor (TNF-α) in isolatedmembrane preparations. Agents Actions 34:132–134

Mijatovic T, Houzet L, Defence P, Droogmans L, Huez G,Kruys V (2000) Tumor necrosis factor-α mRNA re-mains unstable and hypoadenylated upon stimulationof macrophages by lipopolysaccharides. Eur J Biochem267:6004–6011

Mohler KM, Torrance DS, Smith CA, Goodwin RG, StremlerKE, Fung VP, Madani H, Widmer MB (1993) Soluble tu-mor necrosis factor (TNF) receptors are effective therapeu-tic agents in lethal endotoxemia and function simultane-ously as both TNF carriers and TNF antagonists. J Immunol151:1548–1561

Scallon B, Cai A, Solowski N, Rosenberg A, Song XY,Shealy D, Wagner C (2002) Binding and functional com-parisons of two types of tumor necrosis factor antagonists.J Pharmacol Exp Ther 301:418–426

Schall TJ, Lewis M, Koller KJ, Lee A, Rice GC, Wong GH,Gatanaga T, Granger GA, Lentz R, Raab H (1990) Molec-ular cloning and expression of a receptor for human tumornecrosis factor. Cell 61:361–370

Shen C, Assche GV, Colpaert S, Maerten P, Geboes K, Rut-geerts P, Ceuppens JL (2005) Adalimumab induces apop-tosis of human monocytes: a comparative study with inflix-imab and etanercept. Aliment Pharmacol Ther 21:251–258

Smith CA, Davis T, Anderson D, Solam L, Beckmann MP, JerzyR, Dower SK, Cosman D, Goodwin RG (1990) A receptorfor tumor necrosis factor defines an unusual family of cel-lular and viral proteins. Science 248:1019–1023

Vuoltteenaho K, Moilanen T, Hämäläinen M, Moilanen T (2002)Effects of TNF-α antagonists on nitric oxide production inhuman cartilage. Osteoarthr Cartil 10:327–332

Zhang Y, Xu J, Levin J, Hegen M, Li G, Robertshaw H,Brennan F, Cummons T, Clarke D, Vansell N, Nickerson-Nutter C, Barone D, Mohler K, Black R, Skotnicki J,Gibbons J, Feldmann M, Frost P, Larsen G, Lin LL(2002) Identification and characterization of 4-[[4-(2-butynyloxy]phenyl]sulfonyl)-N-hydroxy-2,2-dimethyl-(3S)-thiomorpholinecarboxamide (TMI-1), a novel dualtumor necrosis factor converting enzyme/matrix metallo-protease inhibitor for the treatment of rheumatoid arthritis.J Pharmacol Exp Ther 309:348–355

H.3.1.9Binding to Interferon Receptors

PURPOSE AND RATIONALEThe interferons (IFNs) were discovered in 1957 as bi-ological agents interfering with virus replication. Theyare a family of secreted proteins occurring in verte-brates and can be classified as cytokines. The IFNsare multifunctional and are components of the host de-fense against viral and parasitic infections and certaintumors. They affect the functioning of the immune sys-tem in various ways and also affect cell proliferationand differentiation.

IFNs were initially classified by their sources asleukocyte, fibroblast, and immune IFNs. Leukocyteand fibroblast IFNs, together, were also designated asType 1 IFNs and immune IFN as Type 2 IFN. The re-cent nomenclature designates leukocyte IFNs as IFN-αand IFN-ω (earlier α-1 and α-2, respectively) fibrob-last IFN as IFN-β, and immune IFN as IFN-γ .

Interferons bind to receptors on the cell surface andinduce the synthesis of specific proteins. Littman et al.(1985) found that recombinant IFN-γ produced in bac-teria, which is not glycosylated, binds to cellular recep-tors with an affinity similar to that of natural IFN-γ .

PROCEDUREHuman lymphoblastoid cells (Daudi, MOLT-4 andRaji) are grown in stationary cultures in Dulbecco’smedium with 10% heat-inactivated horse serum. HeLacells are grown in Eagle’s medium with 7% horseserum.

The following interferons are used: Purified recom-binant interferon-γ (rIFN-γ ) (Genentech, antiviral ac-tivity 1.2 × 107 units/mg); natural human INF-β (In-terferon Working Group of the NCI, antiviral activity2 × 105 roentgen units/mg); rIFN-2α (Schering, antivi-ral activity 2 × 108 reference units/mg); rIFN-β (CetusCorp., antiviral activity 2.6 × 108 reference units/mg).

Fifty micrograms of rIFN-γ are reacted for 2 hat 0°C with 1 mCi of 125I-Bolton-Hunter reagent


(2000 Ci/mmol) in 0.25 ml of sodium borate buffer,pH 8.0. The reaction is stopped by the addition ofglycine to a final concentration of 0.2 M and appliedto a 26 × 0.7 cm column of Sephadex G-75 equili-brated with phosphate-buffered saline, pH 7.4, con-taining 0.25% gelatin. The reaction vial is washedwith 20-µl aliquots of this buffer containing 40% ethy-lene glycol and then with buffer alone. The washesare added to the column and 0.32 ml fractions arecollected. The fractions containing 125I-rIFN-γ arepooled and diluted with 1/10 volume of 10-fold con-centrated Eagle’s medium containing 10 mg/ml bovineserum albumin and 0.1 mM dithiothreitol.

Cells harvested from exponentially growing cul-tures are centrifuged and resuspended at 8 × 106/ml intheir own medium supplemented with 10 mM HEPESbuffer, pH 7.4. Standard binding assays contain 3 to 5 ×106 cells and 0.46 nM 125I-rIFN-γ . At the end of thereaction, the cells are centrifuged through 10% sucroseat 10,000 rpm in Microfuge tubes and the cell pelletis counted. A blank value is determined by incubatingand processing in the same way an equal amount of125I-rIFN-γ in the absence of cells; this blank is sub-tracted from the cpm bound.

EVALUATIONThe binding data are analyzed using the LIGAND pro-gram developed by Munson and Rodbard (1980).

MODIFICATIONS OF THE METHODBlatt et al. (1996) described the biological activity ofconsensus interferon, a wholly synthetic type I inter-feron, developed by scanning several interferon-alphanonallelic subtypes and assigning the most frequentlyobserved amino acid in each position.

IFN-τ , a new class of type I interferon was de-scribed by Pontzer et al. (1994), Alexenko et al. (1997,1999), Martal et al. (1998), Swann et al. (1999).

Thiam et al. (1998) reported the agonist activities ofa lipopeptide derived from INF-γ on murine and hu-man cells by analysis and quantification of cell surfacemarkers using flow cytometry and cell-ELISA.

Bosio et al. (1999) reported efficacy of type I in-terferon in cytomegalovirus infections in vivo. Oraladministration of type I interferons (murine INF-αand INF-β) reduced early replication of murine cy-tomegalovirus in both the spleen and liver of infectedBLB/c mice.

Tovey and Maury (1999) found a marked antivi-ral activity of murine interferon-α/β or individual re-combinant species of murine INF-α, INF-β, or INF-γ

or recombinant human INF-α1–8 in mice challengedsystemically with a lethal dose of encephalomyocardi-tis virus, vesicular stomatitis virus, or varicella zostervirus. Oromucosal administration of INF-α also ex-erted a marked antitumor activity in mice injectedi.v. with highly malignant Friend erythroleukemiacells or other transplantable tumors, such as L1210leukemia, the EL4 tumor, or the highly metastaticB16 melanoma.

To gain more insight into similarities of differentINF-α species, Viscomi et al. (1999) evaluated neutral-ization and immunoactivity of a variety of INF prepa-rations with various monoclonal antibodies obtainedthrough immunization with recombinant, lymphoblas-toid, and leukocyte INF-α.

Reporter transgenic mice expressing the luciferasegene under the control of separate TCR-response el-ements from the INF-γ promoter or expressing thegreen fluorescent protein gene under the control of anINF-γ minigene were employed by Zhang et al. (1999)to explore the basis for IL-12 regulation of INF-γ genetranscription.

Poynter and Daynes (1999) studied the influence ofconstitutively expressed INF-γ on age-associated al-terations in inducible nitric oxide synthase regulationusing cell cultures from mouse spleen for nitrite andcytokine analysis.

REFERENCES AND FURTHER READINGAlexenko AP, Leaman DW, Li J, Roberts RM (1997) The anti-

proliferative and antiviral activities of IFN-τ variants in hu-man cells. J Interferone Cytokine Res 17:769–779

Alexenko AP, Ealy AD, Roberts RM (1999) The cross-speciesantiviral activities of different IFN-τ subtypes on bovine,murine, and human cells: contradictory evidence for ther-apeutic potential. J Interferon Cytokine Res 19:1335–1341

Blatt LM, Davis JM, Klein SB, Taylor MW (1996) The biolog-ical activity and molecular characterization of a novel syn-thetic interferon-alpha species, consensus interferon. J In-terferon Cytokine Res 16:488–499

Bosio E, Beilharz MW, Watson MW, Lawson CM (1999) Ef-ficacy of low-dose oral use of type I interferon in cy-tomegalovirus infections in vivo. J Interferon Cytokine Res19:869–876

Littman SJ, Faltynek CR, Baglioni C (1985) Binding of humanrecombinant 125I-interferon to receptors on human cells.J Biol Chem 260:1191–1195

Martal JL, Chene NM, Huynh LP, L’Haridon RM, Rein-aud PB, Guillomot MW, Charlier MA, Charpigny SY(1998) IFN-τ : A novel subtype I IFN1. Structural charac-teristics, non-ubiquitous expression, structure-function re-lationships, a pregnancy hormonal embryonic signal andcross-species therapeutic potentialities. Biochemie 80:755–777

Munson PJ, Rodbard D (1980) LIGAND, a versatile comput-erized approach for characterization of ligand binding sys-tems. Anal Biochem 107:220–239


Pontzer CH, Ott TL, Bazer FW, Johnson HM (1994) Struc-ture/function studies with interferon τ : evidence for multi-ple active sites. J Interferon Res 14:133–141

Poynter ME, Daynes RA (1999) Age-associated alterationsin splenic iNOS regulation: influence of constitutivelyexpressed INF-γ and correction following supplementa-tion with PPARα activators of vitamin E. Cell Immunol195:127–136

Sen GC, Lengyel P (1992) The interferon system. A bird’s eyeview of its biochemistry. J Biol Chem 267:5017–5020

Swann SL, Bazer FW, Villarete LH, Chung A, Pontzer CH(1999) Functional characterization of monoclonal antibod-ies to interferon-τ . Hybridoma 18:399–405

Thiam K, Loing E, Delanoye A, Diesis E, Gras-Masse H, Au-riault C, Verwaerde C (1998) Unrestricted agonist activityon murine and human cells of a lipopeptide derived fromINF-γ . Biochem Biophys Res Commun 253:639–647

Tovey MG, Maury C (1999) Oromucosal interferon therapy:marked antiviral and antitumor activity. J Interferon Cy-tokine Res 19:145–155

Viscomi GC, Antonelli G, Bruno C, Scapol L, Malavasi F, Fu-naro A, Simeoni E, Pestka S, de Pisa F, Dianzani F (1999)Antigenic characterization of recombinant, lymphoblas-toid, and leukocyte INF-α by monoclonal antibodies. J In-terferon Cytokine Res 19:319–326

Zhang F, Nakamura T, Aune TM (1999) TCR and IL-12 re-ceptor signals cooperate to activate an individual responseelement in the INF-γ promoter on Th cells. J Immunol163:728–735

H.3.1.10Chemokine Antagonism

PURPOSE AND RATIONALEThe human chemokine system comprises about50 distinct chemokines and 20 G-protein-coupledchemokine receptors (Rossi and Zlotnik 2000; Sal-lusto et al. 2000; Zlotnik and Yoshie 2000; Fernan-dez and Lolis 2002; D’Ambrosio et al. 2003; Housh-mand and Zlotnik 2003; Ono et al. 2003; Proudfootet al. 2003; Chen et al. 2004; Haringman and Tak2004; Cunha et al. 2005). The biological activitiesof chemokines range from the control of leukocytetrafficking in basal and inflammatory conditions toregulation of hematopoiesis, angiogenesis, tissue ar-chitecture and organogenesis. Grainger and Reckless(2005) studied the anti-inflammatory effects of broad-spectrum chemokine inhibitors. Several groups de-scribed CC chemokine receptor-1 antagonists (Lianget al. 2000a, 2000b; Naya et al. 2001; Eltayeb et al.2003; Gladue et al. 2006). CC chemokine receptor-3 (CCR3) antagonists were reported by De Luccaet al. (2005) and Fryer et al. (2006). CC chemokinereceptor-5 (CCR5) antagonists were described by Rosiet al. (2005) and Saita et al. (2005).

De Lucca et al. (2005) described the discovery ofCC chemokine receptor-3 (CCR3) antagonists with pi-comolar potency.

PROCEDUREBiological Assays

CCR3-Receptor BindingMillipore filter plates (no. MABVN1250) are treatedwith 5 µg/ml protamine in phosphate-buffered saline,pH 7.2, for 10 min at room temperature. Plates arewashed three times with phosphate-buffered saline andincubated with phosphate-buffered saline for 30 min atroom temperature. For binding, 50 µl of binding buffer(0.5% bovine serum albumen, 20 mM HEPES buffer,and 5 mM magnesium chloride in RPMI 1640 media)with or without a test concentration of a compoundpresent at a known concentration is combined with 50µl of 125I-labeled human eotaxin (to give a final con-centration of 150 pM radioligand) and 50 µl of cellsuspension in binding buffer containing 5 × 105 totalcells. Cells used for the binding assay are CHO celllines transfected with a gene expressing human CCR3(Daugherty et al. 1996). The mixture of compound,cells, and radioligand is incubated at room temperaturefor 30 min. Plates are placed onto a vacuum manifold,vacuum is applied, and the plates are washed threetimes with binding buffer with 0.5 M NaCl added. Theplastic skirt is removed from the plate, and the plate isallowed to air-dry; the wells are punched out, and theradioactivity counted (cpm).

EVALUATIONThe percent inhibition of binding is calculated usingthe total count obtained in the absence of any com-peting compound or chemokine ligand and the back-ground binding determined by addition of 100 nM eo-taxin in place of the test compound.

Human Eosinophil Chemotaxis AssayNeuroprobe MBA96 96-well chemotaxis chamberswith Neuroprobe poly(vinylpyrrolidone)-free polycar-bonate PFD5 5-µm filters in place are warmed ina 37°C incubator prior to the assay. Freshly isolatedhuman eosinophils are suspended in RPMI 1640 with0.1% bovine serum albumin at 1 × 106 cells/ml andwarmed in a 37°C incubator prior to the assay. A 20nM solution of human eotaxin in RPMI 1640 with0.1% bovine serum albumin is warmed in a 37°C incu-bator prior to the assay. The eosinophil suspension andthe 20 nM eotaxin solution are each mixed 1:1 withprewarmed RPMI 1640 with 0.1% bovine serum albu-min with or without a dilution of a test compound thatis at twofold the desired final concentration. The fil-ter is separated, and the eotaxin/compound mixture is


placed into the bottom part of the chemotaxis cham-ber. The filter and upper chamber are assembled, and200 µL of the cell suspension/compound mixture isadded to the appropriate wells of the upper chamber.The upper chamber is covered with a plate sealer, andthe assembled unit is placed in a 37oC incubator for45 min. After incubation, the plate sealer is removedand all remaining cell suspension is aspirated off. Thechamber is disassembled, and unmigrated cells arewashed away with phosphate-buffered saline and thenthe filter is wiped with a rubber-tipped squeegee. Thefilter is allowed to completely dry and stained withWright Giemsa. Migrated cells are enumerated by mi-croscopy.

Calcium Mobilization AssayIntracellular calcium flux was measured as the increasein fluorescence emitted by the calcium-binding flu-orophore, fluo-3, when preloaded cells were stimu-lated with CCR3 ligand. Freshly isolated eosinophilswere loaded with fluorophore by resuspending themin a HEPES-buffered PBS solution containing 5 µMfluo-3 and incubating for 60 min at 37oC. After beingwashed twice to remove excess fluorophore, cells wereresuspended in binding buffer (without phenol red) andplated into 96-well plates at 2 × 105/well. Plates wereplaced individually in a FLIPR-1 (Molecular Devices)that uses an argon-ion laser to excite the cells androbotically adds reagents while monitoring changesin fluorescence in all wells simultaneously. To deter-mine the IC50, compound or buffer alone was addedand cells were incubated for 5 min; eotaxin was thenadded to a final concentration of 10 nM. The fluores-cence shift was monitored, and the base-to-peak excur-sion was computed automatically. All conditions weretested in duplicate, and the mean shift per conditionwas determined. The inhibition achieved by gradedconcentrations of compound was calculated as a per-centage of the compound-free eotaxin control.

MODIFICATIONS OF THE METHODChen et al. (1998) reported in vivo inhibition of CCand CX3C chemokine-induced leukocyte infiltrationand attenuation of glomerulonephritis in Wistar–Kyoto(WKY) rats by the viral protein vMIP-II.

Ruth et al. (2001) investigated fractalkine,a chemokine, in rheumatoid arthritis and in rat adju-vant-induced arthritis. Fractaline in vascular biologywas discussed by Umehara et al. (2004).

Laudanna and Constanin (2003) described newmodels of intravital microscopy for analysis of che-

mokine receptor-mediated leukocyte vascular recogni-tion.

REFERENCES AND FURTHER READINGChen L, Pei G, Zhang W (2004) An overall picture of chemokine

receptors: basic research and drug development. CurrPharm Design 10:1045–1055

Chen S, Bacon KB, Li L, Garcia GE, Xia Y, Lo D, Thomp-son DA, Siani MA, Yamamoto T, Harrison Jk, Feng L(1998) In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomeru-lonephritis in Wistar–Kyoto (WKY) rats by vMIP-II. J ExpMed 188:193–198

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H.3.1.11Influence of Peroxisome Proliferator-ActivatedReceptors (PPARs) on Inflammation

PURPOSE AND RATIONALEPeroxisome proliferator-activated receptors (PPARs)play an important role not only in lipid metabolismand diabetes but also in the inflammation process (De-vchand et al. 1996; Delerive et al. 2001; Cabrero et al.2002; Clark 2002; Blanquart et al. 2003; Moller andBerger 2003; Nencioni et al. 2003; Tai et al. 2003; Wo-erly et al. 2003; Diep et al. 2004).

Jiang et al. (1998) reported that PPAR-γ antago-nists inhibit production of monocyte inflammatory cy-tokines.

PROCEDUREMonocyte PreparationHuman monocytes are isolated from freshly collectedbuffy-coat preparations of whole human blood. Themononuclear cell fraction is prepared by dilution withan equal volume of phosphate-buffered saline (PBS)at room temperature and layered over a solution con-taining 3 ml Ficoll-Hypaque per 10 ml blood and PBSand centrifuged for 10 min at 900 g. The mononuclearcell layer is transferred to a fresh tube, mixed with 3vols of PBS, and centrifuged for 10 min at 400 g. Thesupernatant is removed and the dilution and centrifu-gation are repeated three times. Mononuclear cells areresuspended in RPMI medium 1640, counted and di-luted to 5 × 106 cell per ml, after which 1 ml is trans-ferred to each well of a 24-well tissue culture plateand incubated for 37°C in a 5% CO2 humidified in-cubator. The non-adherent cells are removed and themonocytes washed once with PBS before adding 1 mlof fresh RPMI medium 1640 with 10% fetal bovineserum. Experiments are initiated on the day blood iscollected, and all manipulations are carried out underendotoxin-free conditions.

Cytokine AssayMonocytes in fresh medium are treated with induc-ers and candidate induction inhibitors at the time ofculture initiation. Medium is collected from triplicatewells 18–20 h after the test compounds are added. Su-pernatant concentrations of TNF-α, IL-6 and IL-1β aremeasured by ELISA.

RNA BlotTotal RNA of human monocyte cultures is isolated 15h after stimulation with 25 nM PMA in the absenceor presence of PPARγ agonists. RNA blot analysesare performed with standard procedures and labeledprobes prepared from human TNF-α and GAPDHcDNA with the Megaprime DNA labeling Kit (Amer-sham Life Science).

Luciferase AssayU937 cells (2 × 107) are transfected with 10–20 µgluciferase reporter DNA by electrophoresis at 875 Vcm−1, 960 µF (Bio-Rad Laboratories). Transfectedcells are allowed to recover for 1 h and triplicate sam-ples are either untreated or treated with 25 nM PMAin the absence or presence of indicated drugs. Lu-ciferase activity is measured 18–36 h later using theDual-luciferase reporter assay system (Promega) withthe pRL-TK vector as an internal reporter control.


EVALUATIONTo obtain IC50 values, data are fitted to a four-parameter exponential and the derived parameters usedto calculate the concentration at which 50% of themaximal activity is observed.

MODIFICATIONS OF THE METHODKojo et al. (2003) evaluated human PPAR subtype se-lectivity of a variety of anti-inflammatory drugs basedon a novel assay for PPARδ (β).

PROCEDUREPlasmidsThe cDNAs for human PPAR α, δ, and γ were syn-thesized using the polymerase chain reaction (PCR)with human liver cDNA for human PPARα, humanheart cDNA for human PPARδ (β), and human fatcell cDNA for human PPARγ 1 as templates. RetinoidX receptor (RXR)α expression plasmids were con-structed by inserting a coding sequence of RXRα intothe expression vector pcDNA3.1(+) (Invitrogen, Carls-bad, Calif., USA). The cDNA for human RXRα wassynthesized with human kidney cDNA as a template.The template cDNAs used were purchased from Clon-tech Laboratories (Palo Alto, Calif., USA). The ampli-fied cDNA fragments were cloned into pCRII (Invitro-gen) and the nucleotide sequences of the cDNAs weredetermined by the dideoxy chain termination methodusing an automated laser fluorescent DNA sequencer.Multiple clones of each cDNA were sequenced and ar-tifacts generated by the Taq polymerase used in thePCR were revised by replacement of the specified re-gion among the clones. The PPAR and RXRα ex-pression plasmids were constructed by inserting eachfull-length cDNA at a multiple cloning site of themammalian expression vector pCDM8, pcDNAI, orpcDNA3.1 (+) (Invitrogen). Coactivator expressionplasmids pcDNA3.1-CBP and pcDNA3.1-SRC-1 wereprovided by Fujimura and Aramori of the Pharmaco-logical Research Laboratories of Fujisawa Pharm. Thehuman RXRγ expression plasmid was purchased fromInvitrogen.

The reporter gene plasmid pGVPPREluc-1 wasconstructed as follows. Three copies of a 33-bpPPRE identical to that of acyl CoA oxidase werefirst cloned at the SalI site of pBLCAT-2 to gener-ate pBLPPRECAT-1. The luciferase reporter plasmidpGVPPRELuc was constructed by inserting the PPRE-containing fragment of pBLPPRECAT-1 at a multiplecloning site of PGV-P2 (Wako Pure Chemical, Osaka).

Gal4-PPARδ (β) fusion expression plasmid forone-hybrid assay was constructed as follows: full-

length PPARδ (β) cDNA and PPARδ (β)-LBD cDNAwere prepared by PCR amplification using pCDM8-hPPARδ as a template, and the amplified cDNA wascloned into a multiple cloning site of the pBIND vector(Promega, Madison, Wis., USA). The nucleotide se-quence of each fusion expression plasmid was checkedwith an automated DNA sequencer. Reporter plasmidpG5luc for the one-hybrid assay was purchased fromPromega.

Transient Transfection AssayThe African green monkey fibroblast cell line CV-1was obtained from ATCC (Manassas, Va., USA) andmaintained in Dulbecco’s modified Eagle’s medium(DMEM) (Gibco-BRL, Gaithersburg, Md., USA) sup-plemented with penicillin, streptomycin, and 10%heat-inactivated fetal calf serum. Cells were seededat 2 × 105 per well of 6-well culture dishes and afterovernight culture transiently transfected with 1 µg eachof pGVPPRELuc luciferase reporter plasmid, PPARexpression plasmid, and RXRα expression plasmidtogether with the control Renilla luciferase expres-sion plasmid RL-TK (Promega) using Lipofectamine2000 (Gibco-BRL). Coactivator expression plasmidwas also included when a transfection was performedfor the PPARδ assay. For the one-hybrid assay, cellswere transfected with 1 µg each of pG5luc reporterplasmid and Gal4-PPARδ fusion expression plasmidwith RL-TK control plasmid. Cells were harvested 4 hafter transfection and plated again at 1.6 × 104 per wellonto 96-well plates. The drugs dissolved in dimethylsulfoxide were added to the culture and the cellswere incubated at 37oC for 24 h. After being washedwith PBS(-), cells were lysed with PLB (passive ly-sis buffer) (Promega) and the lysates were used forreporter assays. Expression of the reporter was mea-sured by the activity of firefly luciferase using thedual luciferase reporter assay system (Promega) andARVO HTS 1420 multilabel counter (Amersham Bio-sciences) as a luminometer. Firefly luciferase activitywas corrected for transfection efficiency based on theactivity of internal control Renilla luciferase.

Bishop-Bailey and Warner (2003) found thatPPARγ ligands induce prostaglandin production invascular smooth muscle cells and concluded that in-domethacin acts as a peroxisome proliferator-activatedreceptor-γ antagonist.

Fahmi et al. (2001) reported that peroxisome pro-liferator-activated receptor γ activators, such as 15-deoxy-�12,14-PGJ2, inhibit interleukin-1β-induced ni-tric oxide and metalloproteinase 13 production in hu-man chondrocytes.


Cheng et al. (2004) found that peroxisome prolif-erator-activated receptor γ , which is activated by lig-ands such as troglitazone or 15-deoxy-�12,14-PGJ2,inhibits interleukin-1β-induced membrane-associatedprostaglandin E2 synthase-1 expression in human syn-ovial fibroblasts by interfering with the early growthresponse protein Egr-1.

REFERENCES AND FURTHER READINGBishop-Bailey D, Warner TD (2003) PPARγ ligands induce

prostaglandin production in vascular smooth muscle cells:indomethacin acts as a peroxisome proliferator-activatedreceptor-γ antagonist. FASEB J 17:1925–1927

Blanquart C, Barbier O, Fruchart JC, Staels B, Glineur C(2003) Peroxisome proliferators-activated receptors: regu-lation of transcriptional activities and roles in inflamma-tion. J Steroid Biochem Mol Biol 85:267–273

Cabrero A, Laguna JC, Vázquez M (2002) Peroxisome prolif-erator-activated receptors and the control of inflammation.Curr Drug Targets Inflamm Allergy 1:243–248

Cheng S, Afif H, Martel-Pelletier J, Pelletier, Li X, Farrajota K,Lavigne M, Fahmi H (2004) Activation of peroxisome pro-liferator-activated receptor γ inhibits interleukin-1β in-duced membrane-associated prostaglandin E2 synthase-1expression in human synovial fibroblasts by interferingwith Egr-1. J Biol Chem 279:22057–22065

Clark RB (2002) The role of PPARs in inflammation and immu-nity. J Leukoc Biol 71:388–400

Delerive P, Fruchart JC, Staels B (2001) Peroxisome prolif-erators-activated receptors in inflammation control. J En-docrinol 169:453–459

Devchand PR, Keller H, Peters JM, Vasquez M, Gonzales FJ,Wahli W (1996) The PPARα-leukotriene B4 pathway in in-flammatory control. Nature 384:39–43

Diep QN, Benkirane K, Amiri F, Cohn JS, Endemann D,Schiffrin EL (2004) PPARγ activator fenofibrate inhibitsmyocardial inflammation and fibrosis in angiotensin II-infused rats. J Mol Cell Biol 36:295–304

Fahmi H, di Battista JA, Pelletier JP, Mineau F, Ranger P,Martell-Peletier J (2001) Peroxisome proliferator-activatedreceptor γ activators inhibit interleukin-1β induced nitricoxide and metalloproteinase 13 production in human chon-drocytes. Arthritis Rheum 44:595–607

Jiang C, Ting AT, Seed B (1998) PPAR-γ antagonists inhibitproduction of monocytes inflammatory cytokines. Nature391:82–86

Kojo H, Fukagawa M, Tajima K, Suzuki A, Fujimura T,Aramori I, Hayashi KI Nishimura S (2003) Evaluation ofhuman peroxisome-activated receptor (PPAR) subtype se-lectivity of a variety of anti-inflammatory drugs based on annovel assay for PPARδ (β). J Pharmacol Sci 93:347–355

Moller DE, Berger JP (2004) Role of PPARs in the regulationof obesity-related insulin sensitivity and inflammation. IntJ Obes 27:517–521

Nencioni A, Wesselborg S, Brossart P (2003) Role of peroxi-some proliferator-activated receptor γ and its ligands in thecontrol of immune responses. Crit Rev Immunol 23:1–13

Tai ES, Ali AB, Zhang Q, Loh LM, Tan CE, Retnam L, Oak-ley RME, Lim SK (2003) Hepatic expression of PPARγ ,a molecular target of fibrates, is regulated during inflamma-tion in a gender-specific manner. FEBS Lett 546:237–240

Woerly G, Honda K, Loyens M, Papin JP, Auwerx J, Staels B,Capron M, Dombrowicz D (2003) Peroxisome proliferator-activated receptors α and γ downregulate allergic inflam-mation and eosinophil activation. J Exp Med 198:411–421

H.3.1.12Binding to Histamine H4 Receptor

PURPOSE AND RATIONALEHistamine receptors have been classified on the ba-sis of pharmacological analysis (Hill et al. 1997).Histamine exerts its action via at least four receptorsubtypes. The H1 receptor couples mainly to Gq/11,thereby stimulating phospholipase C, whereas the H2receptor interacts with Gs to activate adenylyl cy-clase. The histamine H3 and H4 receptors couple to Giproteins to inhibit adenylyl cyclase, and to stimulateMAPK (Hough 2001).

The H4 receptor is highly expressed in peripheralleukocytes and intestinal tissue, making this receptoran interesting target in inflammatory diseases (Fung-Leung et al. 2004; De Esch et al. 2005; Lim et al. 2006;Zhang et al. 2006). The new receptor was cloned andcharacterized by Oda et al. (2000). Liu et al. (2001b)reported comparison of histamine H4 receptors in sev-eral species. Gbahou et al. (2006) compared the phar-macology of human histamine H3 and H4 receptorsand described structure–activity relationships of his-tamine derivatives. Thurmond et al. (2004) describeda potent and selective histamine H4 antagonist withanti-inflammatory properties. Lim et al. (2005) evalu-ated histamine H1-, H2-, and H3-receptor ligands at thehuman H4 receptor and identified 4-methylhistamineas the first potent and selective H4 receptor agonist.

PROCEDURECell CultureSK-N-MC cell lines, which stably express eitherthe human H3R (SK-N-MC/hH3) or H4R (SK-N-MC/hH4) as well as a cAMP-responsive-element-(CRE-) driven β-galactosidase reporter gene SK-N-MC/hH3 or SK-N-MC/hH4 cells (Lovenberg et al.1999; Liu et al. 2001a), were cultured in Eagle’s min-imum essential medium supplemented with 5% fetalcalf serum, 0.1 mg/ml streptomycin, 100 U/ml peni-cillin, and 600 µg/ml G418 at 37oC in 5% CO2 and95% humidity.

Radioligand Binding AssaysThe SK-N-MC/hH3 cell homogenates were incubatedfor 40 min at 25°C with approximately 1 nM [3H]N-methylhistamine in 25 mM KPO4 buffer and 140 mMNaCl (pH 7.4 at 25oC), with or without competingligands, whereas the SK-N-MC/hH4 cell homogenateswere incubated 1 h at 37°C in 10 nM [3H]histamineand 50 mM Tris-HCl (pH 7.4 at 37°C), with or with-out competing ligands. Bound radioligands were col-


lected on 0.3% polyethyleneimine-pretreated What-man GF/C, and washed three times with 3 ml of ice-cold washing buffer (4°C) containing 25 mM Tris-HCland 140 mM NaCl (pH 7.4 at 4°C) for the hH3R and50 mM Tris-HCl (pH 7.4 at 4°C) for the hH4R).

EVALUATIONBinding analysis of 10 nM [3H]JNJ 7777120 (testcompound) and 0.1 nM [125I]iodophenpropit to thehH4R was performed with the same conditions as de-scribed for [3H]histamine. In saturation binding anal-ysis, the non-specific binding of [3H]histamine or[3H]JNJ 7777120 was determined with 1 µM cloben-propit. The binding analysis of [3H]mepyramine and[125I]iodoaminopotentidine binding to human H1Rand human H2R, respectively, was performed accord-ing to Bakker et al. (2004). The binding data were ana-lyzed with Prism 4.0 (GraphPad Software, San Diego,Calif., USA), and data are presented as mean ±SEM.Mouse and rat H4R radioligand binding assays wereperformed according to Liu et al. (2001b).

REFERENCES AND FURTHER READINGBakker RA, Weiner DM, ter Laak T, Beuming T, Zuiderveld OP,

Edelbroek M, Hacksell U, Timmerman H, Brann MR,Leurs R (2004) 8R-lisuride is a potent stereospecifichistamine H1-receptor partial agonist. Mol Pharmacol65:538–549

De Esch IJ Thurmond RL, Jongejan A, Leurs R (2005) The his-tamine H4 receptor as a new therapeutic target for inflam-mation. Trends Pharmacol Sci 26:462–469

Fung-Leung WP, Thurmond RL, Ling P, Karlsson L (2004) His-tamine H4 receptor antagonists: the new antihistamines?Curr Opin Invest Drugs 5:1174–1183

Gbahou F, Vincent L, Humbert-Claude M, Tardivel-Lacombe J,Chabret C, Arrang JM (2006) Compared pharmacologyof human histamine H3 and H4 receptors: structure-activ-ity relationships of histamine derivatives. Br J Pharmacol147:744–754

Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP,Young JM, Schunack W, Levi R, Haas HL (1997) Interna-tional Union of Pharmacology. XIII. Classification of his-tamine receptors. Pharmacol Rev 49:253–278

Hough LB (2001) Genomics meets histamine receptors: newsubtype, new receptor. Mol Pharmacol 59:415–419

Lim HD, van Rijn RM, Ling P, Bakker RA, Thurmond RL,Leurs R (2005) Evaluation of histamine H1-, H2-, and H3-receptor ligands at the human H4 receptor: identification of4-methylhistamine as the first potent and selective H4 re-ceptor agonist. J Pharmacol Exp Ther 314:1310–1321

Lim HD, Smits RA, Leurs R, de Esch IJP (2006) The emerg-ing role of the histamine H4 receptor in anti-inflammatorytherapy. Curr Topics Med Chem 6:1365–1373

Liu C, Ma X-J, Jiang X, Wilson SJ, Hofstra CL, Blevitt J, Py-ati J, Li X, Chai W, Carruthers N, Lovenberg TW (2001a)Cloning and pharmacological characterization of a fourthhistamine receptor (H4) expressed in bone marrow. MolPharmacol 59:420–426

Liu C, Wilson SJ, Kuei C, Lovenberg TW (2001b) Comparisonof human, mouse, rat, and guinea pig histamine H4 recep-tors reveals substantial pharmacological species variation.J Pharmacol Exp Ther 299:121–130

Lovenberg TW, Roland BL, Wilson SJ, Jiang X, Pyati J, Hu-var A, Jackson MR, Erlander MG (1999) Cloning and func-tional expression of the human histamine H3 receptor. MolPharmacol 55:1101–1107

Oda T, Morikawa N, Saito Y, Masuho Y, Matsumoto SI (2000)Molecular cloning and characterization of a novel type ofhistamine receptor preferentially expressed in leukocytes.J Biol Chem 275:36781–36786

Thurmond RL, Desai PJ, Dunford PJ, Fung-Leung WP, Hof-stra CL, Jiang W, Nguyen S, Riley JP, Sun S, WilliamsKN, Edwards JP, Karlsson L (2004) A potent and selectivehistamine H4 antagonist with anti-inflammatory properties.J Pharmacol Exp Ther 309:404–413

Zhang M, Venable JD, Thurmond RI (2006) The histamine H4receptor in autoimmune disease. Expert Opin Invest drugs15:1443–1452

H.3.2In Vivo Methods for Anti-inflammatory Activity

H.3.2.1General considerations

The inflammatory process involves a series of eventsthat can be elicited by numerous stimuli, e. g., infec-tious agents, ischemia, antigen-antibody interactions,chemical, thermal or mechanical injury. The responseis accompanied by the clinical signs of erythema,edema, hyperalgesia and pain. Inflammatory responsesoccur in three distinct phases, each apparently medi-ated by different mechanisms:

• an acute, transient phase, characterized by local va-sodilatation and increased capillary permeability,

• a subacute phase, characterized by infiltration ofleukocytes and phagocytic cells,

• and a chronic proliferative phase, in which tissuedegeneration and fibrosis occur.

According to these phases, pharmacological meth-ods have been developed.

Methods for testing acute and subacute inflamma-tion are:

• UV-erythema in guinea pigs• Vascular permeability• Oxazolone-induced ear edema in mice• Croton-oil ear edema in rats and mice• Paw edema in rats (various modifications and vari-

ous irritants)• Pleurisy tests• Granuloma pouch technique (various modifications

and various irritants)


The proliferative phase is measured by methods fortesting granuloma formation, such as:

• Cotton wool granuloma• Glass rod granuloma• PVC sponge granuloma.

Furthermore, methods for testing immunologicalfactors have been developed, such as:

• Adjuvant arthritis in rats (various modifications)• Experimental allergic encephalomyelitis• Schultz-Dale-reaction• Passive cutaneous anaphylaxis• Arthus type immediate hypersensitivity• Delayed type hypersensitivity

(see Chapter I).

H.3.2.2Methods for Testing Acute and Subacute Inflammation

H.3.2.2.1Ultraviolet Erythema in Guinea Pigs

PURPOSE AND RATIONALEThe test was first described by Wilhelmi (1949) whowas able to delay the development of ultraviolet ery-thema on albino guinea pig skin by systemic pretreat-ment with clinically equivalent doses of phenylbuta-zone and other nonsteroidal anti-inflammatory agents.The test procedure was further developed by Winderet al. (1958) and since that time modified by variousinvestigators.

PROCEDUREAlbino guinea pigs (Pirbright white strain) of bothsexes with an average weight of 350 g are used. Eigh-teen h prior testing, the animals are shaved on bothflanks and on the back. Then they are chemically de-pilated by a commercial depilation product or by a sus-pension of barium sulfide. Twenty minutes later, thedepilation paste and the fur are rinsed off in runningwarm water. On the next day, the test compound isdissolved (or suspended) in the vehicle and half thedose of the test compound is administered by gavage(at 10 ml/kg) 30 min before ultraviolet exposure. Con-trol animals are treated with the vehicle alone. Fouranimals are used for each treatment group and con-trol. The guinea pigs are placed in a leather cuff witha hole of 1.5 × 2.5 cm size punched in it, allowing theultraviolet radiation to reach only this area. An origi-nal Hanau ultraviolet burner Q 600 is warmed up forabout 30 min prior to use and placed at a constant dis-

tance (20 cm) above the animal. Following a 2 min ul-traviolet exposure, the remaining half of the test com-pound is administered. The investigator has to protecthimself/herself by gloves and ultraviolet glasses. Theerythema is scored 2 and 4 h after exposure.

EVALUATIONThe degree of erythema is evaluated visually by 2 dif-ferent investigators in a double-blinded manner. Thefollowings scores are given:

• 0 = no erythema,• 1 = weak erythema,• 2 = strong erythema,• 4 = very strong erythema.

Animals with a score of 0 or 1 are consideredto be protected. The scoring after 2 and after 4 hgives some indication of the duration of the effect.ED50 values can be calculated. Doses of 1.5 mg/kgindomethacin p.o., 4 mg/kg phenylbutazone p.o. and60 mg/kg acetylsalicylic acid p.o. have been found tobe effective.

CRITICAL ASSESSMENTThe test has the advantage of simplicity but needstraining of the investigators. Attempts to use reflec-tion photometers in order to eliminate subjective scor-ing were unsuccessful. Corticosteroids after systemicapplication are rather ineffective in this test, however,can be evaluated after topical administration. The testis not particularly useful to study the duration of theanti-inflammatory effect.

MODIFICATIONS OF THE TESTYawalkar et al. (1991) tested several steroids after localapplication in the ultraviolet-induced dermatitis inhi-bition in guinea pigs. Clobetasol propionate was moreeffective than hydrocortisone, halobetasol propionatewas superior to both corticosteroids.

Woodward and Owen (1979) used the albinoguinea-pig ear as the site of inflammation producedby UV radiation. Ear temperature, water content ofthe ear and vascular permeability were measured. In-domethacin, phenylbutazone and aspirin given subcu-taneously were active but paracetamol was not.

Warren et al. (1993) studied the role of nitric ox-ide synthase and cyclo-oxygenase in the skin bloodflow to UVB irradiation in the shaved dorsal skin ofanesthetized male Sprague Dawley rats with a laserDoppler flow probe. Topical application of clobetasol-


17-propionate immediately after irradiation inhibitedthe 18 h UVB response in a dose-dependent manner.

Gloxhuber (1976) measured skin thickness usingcalipers in hairless mice after UV-irradiation of theback and treatment with anti-inflammatory drugs.

Woodbury et al. (1994), Kligman (1994) describeda rapid assay of the anti-inflammatory activity of top-ical corticosteroids by inhibition of a UVA-inducedneutrophil infiltration in hairless mouse skin. Skh-hairless mice were irradiated with UVA light onan area of 2 × 2 cm square on the dorsal trunk fore200 min in anesthesia. Steroid treatment was oncedaily for 7 days. Irradiation was on the 8th day. Neu-trophils were counted microscopically in punch biop-sies.

REFERENCES AND FURTHER READINGGloxhuber Ch (1976) A new inflammation model. Arzneim

Forsch/Drug Res 26:43–45Kligman LH (1994) Rapid assay of the anti-inflammatory ac-

tivity of topical corticosteroids by inhibition of a UVA-induced neutrophil infiltration in hairless mouse skin. II.Assessment of name brand versus generic potency. ActaDerm Venereol (Stockh) 74:18–19

Selve N (1991) EM 405: a new substance with an uncom-mon profile of anti-inflammatory activity. Agents Actions32:59–61

Warren JB, Loi RK, Coughlan ML (1993) Involvement of ni-tric oxide synthase in delayed response to ultraviolet lightirradiation of rat skin in vitro. Br J Pharmacol 109:802–806

Wilhelmi G (1949) Ueber die pharmakologischen Eigenschaftenvon Irgapyrin, einem neuen Präparat aus der Pyrazolreihe.Schweiz Med Wschr 79:577–582

Wilhelmi G, Domenjoz H (1951) Vergleichende Untersuchun-gen über die Wirkung von Pyrazolen und Antihistaminenbei verschiedenen Arten der experimentellen Entzündung.Arch Int Pharmacodyn 85:129–143

Winder CV, Wax J, Burr V, Been M, Rosiere CE (1958) A studyof pharmacological influences on ultraviolet erythema inguinea pigs. Arch Int Pharmacodyn 116:261–292

Woodbury RA, Kligman LH, Woodbury MJ, Kligman AM(1994) Rapid assay of the anti-inflammatory activity of top-ical corticosteroids by inhibition of a UVA-induced neu-trophil infiltration in hairless mouse skin. I. The assay andits sensitivity. Acta Derm Venereol (Stockh) 74:15–17

Woodward DF, Owen DAA (1979) Quantitative measurementof the vascular changes produced by UV radiation and car-rageenin using the guinea-pig ear as the site of inflamma-tion. J Pharmacol Meth 2:5–42

Yawalkar S, Wiesenberg-Boettcher I, Gibson JR, Siskin SB,Pignat W (1991) Dermatopharmacologic investigations ofhalobetasol propionate in comparison with clobetasol 17-propionate. Am Acad Dermatol 25:1137–1144

H.3.2.2.2Vascular Permeability

PURPOSE AND RATIONALEThe test is used to evaluate the inhibitory activity ofdrugs against increased vascular permeability which

is induced by a phlogistic substance (Miles and Miles1992). Mediators of inflammation, such as histamine,prostaglandins and leucotrienes are released followingstimulation e. g. of mast cells. This leads to a dilationof arterioles and venules and to an increased vascularpermeability. As a consequence, fluid and plasma pro-teins are extravasated and edemas are formed. Theseeffects are counteracted by H1-antihistaminics, in-hibitors of arachidonic acid metabolism and by leu-cotriene receptor antagonists. In addition, membrane-stabilizing drugs are able to reduce capillary perme-ability. Vascular permeability is increased by intracu-taneous injection of the mast cell-degranulating com-pound 48/80. The increase of permeability can be rec-ognized by the infiltration of the injected sites of theskin with the vital dye Evan’s blue.

PROCEDUREMale Sprague-Dawley rats with a body weight be-tween 160 and 200 g are used. The ventral sides of theanimal are shaved. Five ml/kg of an 1% solution ofEvan’s blue are injected intravenously. One hour laterthe animals are dosed with the test compound orallyor intraperitoneally or with the vehicle. Ten animalsare used for each test group and the control. Thirtyminutes later, the animals are briefly anaesthetizedwith ether and 0.05 ml of an 0.01% solution of com-pound 48/80 are injected intracutaneously at 3 sitesboth at the left and ventral side. Ninety minutes af-ter the injection of compound 48/80 the animals aresacrificed by ether anesthesia. The abdominal skin isremoved and the dye-infiltrated areas of the skin aremeasured.

EVALUATIONThe diameter of the dye-infiltrated areas is measuredin millimeters in two perpendicular directions and themean values of all injection sites in one animal arecalculated. The percent inhibition in the treated ani-mals as compared to the control group is calculated.A treated animal which shows values less than 50%of controls can be considered as positive. ED50 valuescan be calculated in this way. Phenylephrine at a doseof 15 mg/kg has been found to be effective.

CRITICAL ASSESSMENT OF THE METHODThe test for vascular permeability is useful for charac-terization of a new anti-inflammatory compound.

Since compounds with sympathomimetic activityhave a pronounced effect this test cannot be regardedas a primary screening test for anti-inflammatory prod-ucts. Together with an observation of writhing or


“squirming” of mice, Whittle (1964) has proposed tobe able to distinguish between narcotic and non nar-cotic analgesics.

MODIFICATIONS OF THE METHODShionoya and Ohtake (1975) described a simplemethod for extraction of extravasated dye (Evan’sblue) in the skin.

Frimmer and Müller (1962) presented a critical sur-vey on the use of dye methods for quantitative deter-mination of increased capillary permeability followingintracutaneous injection of active substances.

McClure et al. (1992) used the Olympus CUE-2 Im-age Analyzer to quantify vascular permeability in theMiles assay in guinea pigs.

Zentel and Töpert (1994) used oxazolone-inducedEvans blue extravasation for preclinical evaluation oftopical corticosteroids. FemaleNMRI-mice were sen-sitized by topical application of 50 µl of 40% oxa-zolone in ethanol to 4 cm2 of the left flank. After13 days the animals were injected intravenously with0.2 ml of 0.5% Evan’s blue in water and 20 µl of 4%oxazolone in ethanol were topically applied to 6 cm2

of the right flank immediately after injection. Three hlater the challenged skin was treated with various cor-ticosteroids in ointment. The animals were sacrificed24 h after treatment and the challenged skin removed.Evans blue extravasation was measured spectrophoto-metrically at 623 nm.

Teixeira et al. (1993) studied acute inflammatory re-actions in guinea pig skin measuring infiltration of111In-labelled eosinophils and neutrophils and edemaformation by extravasation of 125I-human serum albu-min.

Fujii et al. (1996) quantified vascular permeabil-ity by the extravasation of pontamine sky blue in theskin of male ddY mice after subcutaneous injection oflipopolysaccharides.

Blackham and Woods (1986) measured extravasa-tion of pontamine sky blue in the mouse peritonealcavity.

Cambridge et al. (1996) investigated 6-hydroxy-dopamine-induced plasma extravasation in rat skin af-ter intravenous injection of 125I-human serum albuminand Evan’s blue.

Rouleau et al. (1997) measured the inhibitionof capsaicin-induced plasma extravasation by a his-tamine H3 receptor agonist prodrug by analysis of ex-travasated Evan’s blue in skin, eye conjunctiva, nasalmucosa, trachea, main bronchi, esophagus and urinarybladder of rats.

Watanabe et al. (1984) used fluorescein isothio-cyanate-labeled bovine serum albumin as tracer tomeasure vascular permeability in the carrageenin airpouch of rats.

Collins et al. (1993) studied the pro-inflammatoryproperties of the human recombinant vascular perme-ability factor containing 165 amino acids in rabbits.

Urinary bladder cystitis induced by cyclophos-phamide was used as model of intestinal inflammationand pain by several authors (Ahluwahlia et al. 1994;Bon et al. 1996; Boucher et al. 1997; Alfieri and Gard-ner 1997). Male Wistar rats weighing 300–400 g weretreated first with test drug or saline subcutaneouslyor intraperitoneally and then injected 5 min later with150 mg/kg i.p. cyclophosphamide. One h later, anaes-thesia was induced by 40 mg/kg i.p. pentobarbitoneand 50 mg/kg Evans blue were injected into the jugu-lar vein. Fifteen min later, the rat was exsanguinated byinfusion of 50 ml saline into the left cardiac ventricle.The urinary bladder, the left kidney, the superior lobeof the left lung and approximately 1-cm portions of theduodenum and jejunum were removed and blotted be-fore dry weighing. The content of Evens blue dye wasdetermined by spectrophotometry at 620 nm after ex-traction in known volumes of formamide at 60°C for60 h.

Ferrets were treated in the same way, but the doseof cyclophosphamide was 125 mg/kg and the volumeof exsanguination was 300 ml.

Hirota et al. (1995) induced chemical peritonitis inrats by applying 0.02 M HCl on the surface of the ce-cum or appendix and quantified the inflammation bymeasuring the extravasation of intravenously injectedEvan’s blue bound to albumin extracted from those tis-sues.

REFERENCES AND FURTHER READINGAhluwalia A, Maggi C, Santiccioli P, Lecci A, Giulani S (1994)

Characterisation of the capsaicin-sensitive component ofcyclophosphamide-induced inflammation in the rat urinarybladder. Br J Pharmacol 111:1017–1022

Alfieri A, Gardner C (1997) The NK1 antagonist GR203040inhibits cyclophosphamide-induced damage in the rat andferret bladder. Gen Pharmacol 29:245–250

Bennett AJ, West GB (1978) Measurement of the changesin vascular permeability in rat skin. J Pharmacol Meth1:105–108

Blackham A, Woods FAM (1986) Immune complex mediatedinflammation in the mouse peritoneal cavity. J PharmacolMeth 15:77–85

Bon K, Lantéri-Minet M, de Pommery J, Michiels JF,Menétrey D (1996) Cyclophosphamide cystitis as a modelof visceral pain in rats. A survey of hindbrain structuresinvolved in visceroception and nociception using the ex-pression of c-Fos and Krox-24 proteins. Exp Brain Res108:404–416


Boucher M, Meen M, Codron JP, Coudoré F, Kémény JL, Es-chalier A (1997) Cyclophosphamide cystitis in rats: A newbehavioral model of visceral pain. Fund Clin Pharmacol11:160

Cambridge H, Ajuebor MN, Brain SD (1996) Investigation of6-hydroxydopamine-induced plasma extravasation in ratskin. Eur J Pharmacol 301:151–157

Collins PD, Connolly DT, Williams TJ (1993) Characterizationof the increase in vascular permeability induced by vascularpermeability factor in vivo. Br J Pharmacol 109:195–199

Feldberg W, Miles A (1953) Regional variations of increasedpermeability of skin capillaries induced by a histamine lib-erator and their relation to the histamine content in skin.J Physiol 120:205–213

Frimmer M, Müller FW (1962) Brauchbarkeit und Grenzen derFarbstoffmethoden zur Bestimmung vermehrter Durchläs-sigkeit der Haut-Capillaren. Med Exp 6:327–330

Fujii E, Irie K, Ogawa A, Ohba K, Muraki T (1996) Role ofnitric oxide and prostaglandins in lipopolysaccharide-in-duced increase in vascular permeability in mouse skin. EurJ Pharmacol 297:257–263

Hirota K, Zsigmond EK, Matsuki A, Rabito SF (1995) Topicalketamine inhibits albumin extravasation in chemical peri-tonitis in rats. Acta Anaesthesiol Scand 39:174–178

Lembeck F, Holzer P (1979) Substance P as neurogenic me-diator of antidromic vasodilation and neurogenic plasmaextravasation. Naunyn Schmiedeberg’s Arch Pharmacol310:175–183

McClure N, Robertson DM, Heyward P, Healy DL (1994) Im-age analysis quantification of the Miles assay. J PharmacolToxicol Meth 32:49–52

Miles AA, Miles EM (1952) Vascular reactions to histamine,histamine-liberator and leukotaxine in the skin of guinea-pigs. J Physiol 118:228–257

Nagahisa A, Kanai Y, Suga O, Taniguchi K, Tsuchiya M, LoweIII JA, Hess HJ (1992) Antiinflammatory and analgesic ac-tivity of a non-peptide substance P receptor antagonist. EurJ Pharmacol 217:191–195

Pouleau A, Garbag M, Ligneau X, Mantion C, Lavie P,Advenier C, Lecomte JM, Krause M, Stark H, Schu-nack W, Schwartz JC (1997) Bioavailability, antinocicep-tive and antiinflammatory properties of BP 2–94, a his-tamine H3 receptor agonist prodrug. J Pharmacol Exp Ther281:1985–1094

Saria A, Lundberg JM, Skofitsch G, Lembeck F (1983) Vas-cular protein leakage in various tissues induced by sub-stance P, capsaicin, bradykinin, serotonin, histamine and byantigen challenge. Naunyn-Schmiedeberg’s Arch Pharma-col 324:212–218

Sensch KH, Zeiller P, Raake W (1979) Zur antiexsudativenund antioedematösen Wirkung von Sympathikomimetika.Arzneim Forsch/Drug Res 29:116–121

Shionoya H, Ohtake S (1975) A new simple method for extrac-tion of extravasated dye in the skin. Japan J Pharmacol 103,Suppl 25:103

Teixeira MM, Williams TJ, Hellewell PG (1993) Role ofprostaglandins and nitric oxide in acute inflammatory re-actions in guinea-pig skin. Br J Pharmacol 110:1515–1521

Watanabe K, Nakagawa H, Tsurufuji S (1984) A new sensitivefluorometric method for measurement of vascular perme-ability. J Pharmacol Meth 11:167–176

Whittle BA (1964) The use of changes in capillary permeabilityin mice to distinguish between narcotic and non narcoticanalgesics. Br J Pharmacol 22:246–253

Zentel HJ, Töpert M (1994) Preclinical evaluation of a newtopical corticosteroid methylprednisolone aceponate. J EurAcad Dermatol Venereol 3, Suppl 1:S32–S38

H.3.2.2.3Inhibition of Leukocyte Adhesionto Rat Mesenteric Venules In Vivo

PURPOSE AND RATIONALEReversible adherence of leukocytes to endothelium,basement membranes and other surfaces is an essen-tial event in the establishment of inflammation. Theirentry into tissues is controlled by the dynamic in-teraction between adhesion molecules expressed bythese cells and the endothelium. White cells circulat-ing in the blood have the tendency to adhere to thewalls of blood vessels and this tendency is greatlyincreased in states of inflammation. Normally, whenleukocytes collide with the vessel wall, the collisionbehaves elastically and the cells bounce off and backinto the lumen. However, biochemical changes in in-flamed tissues results in inelastic collisions of cellsand an increase in their adhesion, thus initiating rollingof leukocytes along the endothelial surface. As adhe-sion further increases, rolling is slowed and may befollowed by the cells coming to a complete stop andtheir migration out of the vessel. This can be observedby preparing a mesenteric venule of an anesthetizedrat and following the flow and rolling of leukocytes bymeans of a microscope, thus allowing in-vivo studies.In this test procedure, adhesion of leukocytes, to thevessel wall, is artificially induced by the application ofthe formyl-methionyl peptide fMet-Leu-Phe (FMLP).Formyl peptides are released from bacteria and mito-chondria of damaged tissue, so these peptides providea specific signal marking the presence of invading bac-teria or tissue damage. The density of FMLP recep-tors ranges from 104 to 105 per cell, depending on thecell type. Activation of leukocytes through this recep-tor results in rapid expression of preformed L-selectin(LECAM-1) on the cell surface which causes the cellsto roll along the endothelial surface. LECAM-1 arevery rapidly shed from the surface of leukocytes, how-ever, and integrins take over to maintain further adhe-sion and migration into the tissue.

PROCEDURESprague-Dawley rats are anesthetized by adminis-tration of Nembutal. The trachea, jugular vein andcarotid artery are prepared free, the abdominal cav-ity is opened and a section of ileum is pulled out anddraped over a heated microscope table. Prior to testcompound administration, the number of spontaneousadhering leukocytes is counted, every 5 min, in a de-fined section of a venule (covered with paraffin oil)during a 30-min period (control). Blood pressure, body


temperature and velocity of blood flow are also reg-istered. The test compound is administered via con-tinuous infusion during the entire test procedure be-ginning at t = –30 min. Following the determination ofcontrol values for spontaneous adhesion, FMLP (f-Met-Leu-Phe, 10−4 M) is dripped twice (t = –30 minand t = 0 min) on the preparation and the number ofadhering leukocytes is determined every 5 min overa 90 min period, beginning with the second applica-tion of FMLP (t = 0 min). Each test group consists ofat least 10 animals. The mean leukocyte count of everyrat prior to FMLP and/or test substance application istaken as the 100% value to obtain the baseline for fur-ther comparisons. The test compounds are dissolved in0.9% NaCl shortly before application.

EVALUATIONFollowing the second topical application of FMLP(10−4 M), the number of adhering leukocytes in themesenteric venule section is counted 30 min after thestimulus is given, and again at the end of the observa-tion period of 2 h. The influence of a continuous i.v.infusion of the test drug is compared with the positivecontrol group (FMLP stimulation, without drug).

MODIFICATIONS OF THE METHODA simple, rapid, in vitro assay for granulocyte adher-ence was developed by MacGregor et al. (1974). Hep-arinized whole blood is filtered through nylon fiberspacked in Pasteur pipettes, and the percentage of gran-ulocytes adhering was calculated.

Neutrophil adherence was tested in vitro by Burchet al. (1992). Human umbilical vein endothelial cellswere plated at 5 × 104 cells/well into collagen coatedculture plates and grown to confluence. Neutrophilswere labeled with 51Cr. Experimental agents wereadded to the neutrophils before their activation withFMLP. After 15 min incubation at 37°C, the non-adherent leukocytes were removed by gentle aspira-tion followed by a wash with saline. The adherent neu-trophils were lysed by 1 N NaOH and the radioactivitywas quantitated.

REFERENCES AND FURTHER READINGBurch RM, Connor JR, Bator JM, Weitzberg M, Laemont K,

Noronha-Blob L, Sullivan JP, Steranka LR (1992) NPC15669 inhibits the reverse passive Arthus reaction in ratsby blocking neutrophil recruitment. J Pharm Exp Ther263:933–937

Lawrence MB, Springer TA (1991) Leukocytes roll on a selectinat physiologic flow rates: Distinction from and prerequisitefor adhesion through integrins. Cell 65:859–873

MacGregor RR, Spagnuolo PJ, Lentnek AL (1974) Inhibitionof granulocyte adherence by ethanol, prednisone, and as-

pirin, measured with an assay system. New Engl J Med291:642–646

Stecher VJ, Chinea GL (1978) The neutrophil adherence assayas a method for detecting unique anti-inflammatory agents.Agents Actions 8:258 262

Zielinski T, Müller HJ, Schleyerbach R, Bartlett RR (1994) Dif-ferential effects of leflunomide on leukocytes: Inhibition ofrat in vivo adhesion and human in vitro oxidative burst with-out affecting surface marker modulation. Agents Actions41 Spec Conf Issue: C276–278

H.3.2.2.4Oxazolone-Induced Ear Edema in Mice

PURPOSE AND RATIONALEThe oxazolone-induced ear edema model as first de-scribed by Evans (1971) in mice is a model of de-layed contact hypersensitivity that permits the quan-titative evaluation of the topical and systemic anti-inflammatory activity of a compound following topicaladministration.

PROCEDUREMice of either sex with a weight of 25 g are used. Be-fore each use a fresh 2% solution of oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one) in ace-tone is prepared. The mice are sensitized by applica-tion of 0.1 ml on the shaved abdominal skin or 0.01 mlon the inside of both ears under halothane anesthesia.The mice are challenged 8 days later again under anes-thesia by applying 0.01 ml 2% oxazolone solution tothe inside of the right ear (control) or 0.01 ml of ox-azolone solution, in which the test compound or thestandard is solved. Special pipettes of 0.1 ml or 0.01 mlare used. Groups of 10 to 15 animals are treated withthe irritant alone or with the solution of the test com-pound. The left ear remains untreated. The maximumof inflammation occurs 24 h later. At this time the ani-mals are sacrificed under anesthesia and a disc of 8 mmdiameter is punched from both sides. The discs are im-mediately weighed on a balance. The weight differ-ence is an indicator of the inflammatory edema.

EVALUATIONAverage values of the increase of weight are calculatedfor each treated group and compared statistically withthe control group. A 0.003% solution of hydrocorti-sone and a 1% solution of indomethacin were found tobe active.

CRITICAL ASSESSMENT OF THE METHODThe method is suitable for both steroidal and non-steroidal compounds as well as for the evaluation ofvarious topical formulations.


MODIFICATIONS OF THE METHODGriswold et al. (1974) applied a 3% solution of oxa-zolone to the left paw of mice. The edema was assessedplethysmographically.

Various cutaneous models of inflammation for theevaluation of topical and systemic pharmacologicalagents have been discussed by Young and Young(1989).

Bailey et al. (1995) described a contact hypersensi-tivity model in mice for rank-ordering formulated cor-ticosteroids. Male Swiss Webster mice were sensitizedwith 20 µl of 2% oxazolone on the inner and outer as-pects of each ear (10 µl each side). Mice were chal-lenged 7 days later with 2% oxazolone in acetone:oliveoil (4:1) on both sides of the right ear. Animals weretopically treated with corticosteroids or non-steroidalantiinflammatory drugs or 20 mg formulated corticos-teroids immediately after challenge. The mice weresacrificed after 24 h and edema and myeloperoxidaseactivity were determined. Edema was measured bytaking the weight of 6 mm trephine punch biopsies ofthe right and left ears. Inhibition was calculated fromchange in ear weight of control or drug treated ears ver-sus placebo treated ears. Myeloperoxidase activity wasassessed spectrophotometrically (Williams et al. 1983)on tissue homogenates. In a delayed-type hypersensi-tivity model animals were treated as in the contact hy-persensitivity model, except the mice were sensitizedwith 40 µl of 2% oxazolone in acetone:olive oil (4:1)on the unshaved inguinal areas.

Meingassner et al. (1997) studied anti-inflammatoryactivity using allergic contact dermatitis im mice,rats and pigs. Mice were sensitized on the shavedabdomen with 50 µl of 2% oxazolone solution inacetone. After 7 days, they were challenged with10 µl of 2% (for topical testing) or 0.5% (for sys-temic testing) oxazolone on the inner surface ofthe right ears. Pinnal weight was taken as a mea-sure of inflammatory edema 24 h after challenge. Fe-male Sprague Dawley rats were sensitized by ap-plication of 80 µl of 2,4-dinitrofluorobenzene solu-tion applied in 20 µl volumes to the inner surfaceof both ear lobes and to both shaved inguinal re-gions on day 1. Allergic contact dermatitis was elicitedwith 30 µl of 0.5% 2,4-dinitrofluorobenzene appliedto the test sites of ≈ 15 mm in diameter on bothshaved flanks on day 12. Animals were treated by gav-age 2 h before and immediately after challenge. Der-matitis was evaluated by measuring the thickness ofthe lifted skin fold at the test sites with a spring-loaded micrometer. Domestic pigs were sensitizedwit 400 µl of 10% 2,4-dinitrofluorobenzene applied to

four areas on both ears and groins. Challenge reac-tions were elicited 12 days later with 15 µl of 2,4-dinitrofluorobenzene (1%) applied topically to testsites arranged in four craniocaudal lines on the dor-solateral shaved back (24 or 32 per pig). Test siteswere treated twice either with 20 µl solution of testcompound or with ≈ 50 mg of a cream formulationapplied topically 30 min and 6 h after challenge. Oneday after challenge the test sites were visually evalu-ated for intensity and extent of erythema and indura-tion.

REFERENCES AND FURTHER READINGAlpermann HG, Sandow J, Vogel HG (1982) Tierexperimentelle

Untersuchungen zur topischen und systemischen Wirk-samkeit von Prednisolon-17-ethylcarbonat-21-propionat.Arzneim Forsch/Drug Res 32:633–638

Bailey SC, Asghar F, Przekop PA, Kurtz ES (1995) A novel con-tact hypersensitivity model for rank-ordering formulatedcorticosteroids. Inflamm Res 44, Suppl 2:S162–163

Evans PD, Hossack M, Thomson DS (1971) Inhibition of con-tact sensitivity in the mouse by topical application of corti-costeroids. Br J Pharmacol 43:403

Griswold DE, DiLorenzo JA, Calabresi P (1974) Quantificationand pharmacological dissection of oxazolone-induced con-tact sensitivity in the mouse. Cell Immunol 11:198–204

Meingassner JG, Grassberger M, Fahrngruber H, Moore HD,Schuurman H, Stütz A (1997) A novel anti-inflammatorydrug, SDZ ASM 981, for the topical and oral treatmentof skin diseases. In vivo pharmacology. Br J Dermatol137:568–576

Williams RN, Paterson CA, Eakins KE, Bhattacherjee P(1983) Quantification of ocular inflammation: Evaluationof polymorphonuclear leukocyte infiltration by measuringmyeloperoxidase activity. Current Eye Res 2:465–470

Young JM, Young LM (1989) Cutaneous models of inflamma-tion for the evaluation of topical and systemic pharmaco-logical agents. In: Pharmacological Models in the Controlof Inflammation. Alan R. Liss, Inc., pp 215–231

H.3.2.2.5Croton-oil Ear Edema in Rats and Mice

PURPOSE AND RATIONALEThe method has been developed primarily as a bioas-say for the concomitant assessment of the antiphlo-gistic and thymolytic activities of topically appliedsteroids by Tonelli et al. (1965)

PROCEDUREFor tests in mice the irritant is composed as follows(v/v): 1 part Croton oil, 10 parts ethanol, 20 partspyridine, 69 parts ethyl ether. For tests in rats thefollowing mixture is prepared (v/v): 4 parts Crotonoil, 10 parts ethanol, 20 parts pyridine, 66 parts ethylether. The standards and the test compounds are dis-solved in this solution. For tests in mice male NMRI-mice with an weight of 22 g, for tests in rats male


Sprague-Dawley rats with a weight of 70 g are used.Ten animals are used for controls and each test group.The test compounds are dissolved in a concentra-tion of 0.03 mg/ml to 1 mg/ml for mice and in a 3to 10 times higher concentration for rats in the irri-tant solution. On both sides of the right ear 0.01 mlin mice or 0.02 ml in rats are applied. Controls re-ceive only the irritant solvent. The left ear remainsuntreated. The irritant is applied under ether anesthe-sia. Four hours after application the animals are sac-rificed under anesthesia. Both ears are removed anddiscs of 8 mm diameter are punched. The discs areweighed immediately and the weight difference be-tween the treated and untreated ear is recorded indi-cating the degree of inflammatory edema. In the origi-nally described method the ears are removed by sharp,straight scissors 6 h after application and weighedas total. The animals were sacrificed 48 h after top-ical administration and the thymus glands were re-moved, weighed and expressed as mg thymus/100 gbody weight.

EVALUATIONThe antiphlogistic effect can be determined by ex-pressing the increase in weight of the treated ear aspercentage of the weight of the contralateral controlear. The difference of both weights is divided by theweight of the contralateral ear times 100. Otherwise,the difference between both ears or excised discs iscalculated as the average values for treated and controlgroups and the effect is evaluated by statistical meth-ods. Concentration of 0.5 to 1 mg/ml hydrocortisonehave been proven to be effective.

CRITICAL ASSESSMENT OF THE METHODThe method is useful for evaluation of anti-inflammatory topical steroids especially in the modi-fication when thymus weight is determined simultane-ously. The method also can be used for topically ap-plied nonsteroidal antiphlogistics.

MODIFICATIONS OF THE METHODWilhelmi and Domenjoz (1951) tested various drugsusing Croton oil induced ear edema in mice and rab-bits.

Tubaro et al. (1985) tested various anti-inflam-matory drugs in the Croton oil test in mice. Granulo-cyte infiltration in plugs taken from the inflamed earswas assessed by measuring peroxidase activity.

Zentel and Töpert (1994) used Croton oil-inducedear edema in rats to evaluate topical corticosteroids.A plastic collar was fixed around the neck of Wistar

rats of either sex (160–200 g body weight) to excludeoral uptake of the compounds. Fifty µl of 5% Crotonoil in ethanol or ethanol alone were topically appliedto both ears. In the treatment groups drugs were coap-plied with Croton oil. Five h after treatment the ani-mals were sacrificed by CO2 gas and the ears removed.Edema formation was measured by the increase in wetweight.

Iwasaki et al. (1995) measured the inhibition ofCroton oil-induced ear edema in Wistar rats by locallyapplied clobetasol-17-propionate, a synthetic gluco-corticoid, and the influence of simultaneously appliedRU 486.

Weirich et al. (1977) measured skin temperature,ear thickness and weight of excised punches after Cro-ton oil induced edema in the ears of white rabbits andcalculated phlogostasis values as the products of thepercent reduction in skin temperature, auricular thick-ness and tissue weight in relation to controls. The au-thors recommended this model for the primary evalua-tion of topical anti-inflammatory agents.

Colorado et al. (1991) described an apparatus tomeasure Croton oil induced ear edema in mice usingprecisely reproducible pressure on the ear. The deviceallows to follow the time course of inflammation byrepeated measurements.

Akiyama et al. (1994) studied staphylococcus au-reus infection on experimental Croton oil-inflamedskin in mice. Staphylococcus aureus cells were inoc-ulated on the surface of skin inflamed by applicationof Croton oil in cyclophosphamide-treated mice. Skinspecimens were taken at 1, 3, 6, 12, and 24 h after in-oculation and examined by microscopy. The staphy-lococcus aureus cells which attached to the surfaceof the skin immediately after inoculation had invadedthe horny layer within 1 h. The cells gradually pene-trated deeper into the epidermis. Application of corti-costeroid ointments decreased the number of staphylo-coccus aureus cells in the lesions.

Anderson and Groth (1984) induced toxic con-tact reactions to Croton oil or dinitrochlorobenzene(DNCB), or allergic contact reactions to DNCB oroxazolone in guinea pig skin and tested the effect ofvarious locally applied corticosteroids by macroscopicassessment and microscopic evaluation of cellular in-filtrates.

Tarayre et al. (1984) used a 0.25% solution of can-tharidin in acetone and applied 0.025 ml to one mouseear. Two phases of inflammation were observed. Afterlocal application nonsteroidal drugs showed effects inthe first phase only, whereas steroids influenced bothphases.


De Young et al. (1987) induced ear inflammationin rats by intradermal injection of 10 ng recombinanthuman interleukin-1β in 10 µl of saline.

Maloff et al. (1989) injected 20 µl of interleukin-1 solution into the left ear of mice and found a dose-dependent increase of ear thickness and myeloperox-idase activity which reached the maximum after 24 h.These effects were reduced by high doses of gluco-corticoids but not by nonsteroidal anti-inflammatorydrugs.

Chang et al. (1987) applied 4 µg tetradecanoylphorbol acetate and test drugs dissolved in acetoneto the right ear of mice. Ear edema was calculated bysubtracting the thickness of the left ear (vehicle con-trol) from the right ear (treated ear).

De Young et al. (1989) examined the temporal pat-terns of edema and accumulation of the polymorphicnuclear cell marker enzyme myeloperoxidase follow-ing application of tetradecanoyl phorbol acetate tomouse ears. Topical and oral corticosteroids inhib-ited both edema and myeloperoxidase accumulation,whereby clobetasol propionate was more effective thanfluocinolone and dexamethasone. Cyclo-oxygenaseand lipoxygenase inhibitors were very effective againstmyeloperoxidase accumulation but were inactive ormoderately active vs. edema.

Murakawa et al. (2006) studied the involvementof tumor necrosis factor-α in phorbol ester 12-O-tetradecynoylphorbol-13-acetate- (TPA-) induced skinedema in mice.

Topical application of arachidonic acid to mouseear has become a widely used test (Young et al. 1983,1984; Opas et al. 1985; Crummey et al. 1987; Hensbyet al. 1987; Tomchek et al. 1991) One mg arachidonicacid is applied to the right ear of mice and vehicleto the left ear of each animal. Drugs are topically ap-plied in acetone to the ear 30 min prior to the arachi-donic acid application. Ear swelling was measured us-ing a caliper one hour after arachidonic acid.

Griswold et al. (1995) induced inflammation inmice by local application of arachidonic acid or phor-bol ester. Besides ear thickness, myeloperoxidase andDNA content was measured.

REFERENCES AND FURTHER READINGAkiyama H, Kanzaki H, Abe Y, Tada H, Arata J (1994)

Staphylococcus aureus infection on experimental crotonoil-inflamed skin in mice. J Dermatol Sci 8:1–10

Anderson CD, Groth O (1984) The influence on the dermal cel-lular infiltrate of topical steroid applications and vehicles inguinea pig skin: normal skin, allergic and toxic reactions.Contact Dermatitis 10:193–200

Alpermann HG, Sandow J, Vogel HG (1982) TierexperimentelleUntersuchungen zur topischen und systemischen Wirk-samkeit von Prednisolon-17-ethylcarbonat-21-propionat.Arzneim Forsch / Drug Res 32:633–638

Chang J, Blazek E, Skowronek M, Marinari L, Carlson RP(1987) The antiinflammatory action of guanabenz is me-diated through 5-lipoxygenase and cyclooxygenase inhibi-tion. Eur J Pharm 142:197–205

Colorado A, Slama JT, Stavinoha WB (1991) A new method formeasuring auricular inflammation in the mouse. J Pharma-col Meth 26:73–77

Crummey A, Harper GP, Boyle EA, Mangan FR (1987) Inhi-bition of arachidonic acid-induced ear oedema as a modelfor assessing topical anti-inflammatory compounds. AgentsActions 20:69–72

De Young LM, Spires DA, Kheifets J, Terrell TG (1987) Biologyand pharmacology of recombinant interleukin-1β-inducedrat ear inflammation. Agents Actions 21:325–327

De Young LM, Kheifets JB, Ballaron SJ, Young JM (1989)Edema and cell infiltration in the phorbol ester-treatedmouse ear are temporally separate and can be differen-tially modulated by pharmacologic agents. Agents Actions26:335–341

Griswold DE, Chabot-Fletcher M, Webb EF, Martin L, Hille-gass L (1995) Antiinflammatory activity of topical aura-nofin in arachidonic acid- and phorbol ester-induced in-flammation in mice. Drug Dev Res 34:369–375

Hensby CN, Eustache J, Shroot B, Bouclier M, Chatelus A, Lug-inbuhl B (1987) Antiinflammatory aspects of systemic andtopically applied retinoids. Agents Actions 21:238–240

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Murakawa M, Yamaoka K, Tanaba Y, Fukuda Y (2006) Involve-ment of tumor necrosis factor (TNF)-α in phorbol ester12-O-tetradecynoylphorbol-13-acetate (TPA)-induced skinedema in mice. Biochem Pharmacol 71:1331–1336

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Tarayre JP, Aliaga M, Barbara M, Villanova G, Caillol V,Lauressergues H (1984) Pharmacological study of can-tharidin-induced ear inflammation in mice. J PharmacolMeth 11:271–277

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H.3.2.2.6Paw Edema

PURPOSE AND RATIONALEAmong the many methods used for screening of anti-inflammatory drugs, one of the most commonly em-ployed techniques is based upon the ability of suchagents to inhibit the edema produced in the hindpaw of the rat after injection of a phlogistic agent.Many phlogistic agents (irritants) have been used, suchas brewer’s yeast, formaldehyde, dextran, egg albu-min, kaolin, Aerosil, sulfated polysaccharides like car-rageenin or naphthoylheparamine. The effect can bemeasured in several ways. The hind limb can be dis-sected at the talocrural joint and weighed. Usually, thevolume of the injected paw is measured before and af-ter application of the irritant and the paw volume ofthe treated animals is compared to the controls. Manymethods have been described how to measure the pawvolume by simple and less accurate and by more so-phisticated electronically devised methods. The valueof the assessment is less dependent on the apparatusbut much more on the irritant being chosen. Some irri-tants induce only a short lasting inflammation whereasother irritants cause the paw edema to continue overmore than 24 h.

PROCEDUREMale or female Sprague-Dawley rats with a bodyweight between 100 and 150 g are used. The animalsare starved overnight. To insure uniform hydration, therats receive 5 ml of water by stomach tube (controls)or the test drug dissolved or suspended in the samevolume. Thirty minutes later, the rats are challengedby a subcutaneous injection of 0.05 ml of 1% solutionof carrageenan into the plantar side of the left hindpaw. The paw is marked with ink at the level of thelateral malleolus and immersed in mercury up to thismark. The paw volume is measured plethysmographi-cally immediately after injection, again 3 and 6 h, andeventually 24 h after challenge.

ApparatusVarious devices have been developed for plethysmog-raphy of the paw. Winter et al. (1963) used mercury forimmersion of the paw. A more sophisticated apparatushas been described by Hofrichter et al. (1969). Alper-mann and Magerkurth (1972) described an apparatusbased on the principle of transforming the volume be-ing increased by immersion of the paw into a pro-portional voltage using a pressure transducer. Webband Griswold (1984) reported a sensitive method ofmeasuring mouse paw volume by interfacing a Met-tler DeltaRange top-loading balance with a micro-computer. Several authors used a commercially avail-able plethysmometer from Ugo Basile, Varese, Italy(Damas and Remacle-Volon 1992; Braga da Mottaet al. 1994; Legat et al. 1994; Griesbacher et al. 1994).

EVALUATIONThe increase of paw volume after 3 or 6 h is calculatedas percentage compared with the volume measured im-mediately after injection of the irritant for each animal.Effectively treated animals show much less edema.The difference of average values between treated an-imals and control groups is calculated for each timeinterval and statistically evaluated. The difference atthe various time intervals give some hints for the dura-tion of the anti-inflammatory effect. A dose-responsecurve is run for active drugs and ED50 values can bedetermined.

MODIFICATIONSMany agents can be used as irritants to induce pawedema in rats or mice. These are:

0.05 ml undiluted fresh egg white (Randall andBaruth 1976)

0.1 ml of 1% ovalbumin solution (Turner 1965)

0.1 ml of 1% formalin (Turner 1965)

0.1 ml of 0.2% carrageenan solution (Schönhöfer1967) Introduction

0.1 ml of 1% carrageenan solution plus 100 ng PGE2or PGI2 (Higgs et al. 1978; Portanova et al.1996)

0.1 ml of 1 to 3% dextran solution (Turner 1965)

0.1 ml of 2.5% brewer’s yeast powder suspension(Tsumuri et al. 1986)

0.1 ml of 0.5% β-naphthoylheparamine solution(Peterfalvi et al. 1966)

0.1 ml of 0.1% trypsin solution (Kalbhen and Smalla1977)


0.1 ml of 0.1% collagenase solution (Souza Pintoet al. 1995)

0.1 ml of 0.1% solution of collagenase fromClostridium histolyticum (Legat et al. 1994)

0.1 ml of solution of 100 IU hyaluronidase (Dewes1955, Kalbhen and Smalla 1977)

0.1 ml of complete Freund’s adjuvant

0.05 ml of 0.02% serotonin solution (Kalbhen andSmalla 1977)

0.1 ml of 0.005% bradykinin solution (Damas andRemacle-Volon 1992)

0.1 ml of 0.1 mg/ml prostaglandin E2 (Nikolov et al.1978)

0.1 ml of 2.0 µg/ml prostaglandin E2 (repeated injec-tions, Willis and Cornelsen 1973)

0.1 ml of 1% concanavalin A solution (Lewis et al.1976)

0.1 ml of 2.5% suspension of Aerosil

0.1 ml of 5% suspension of kaolin (Lorenz 1961;Wagner-Jauregg et al. 1962)

0.05 ml of bentonite gel (Marek 1980)

0.1 ml of nystatin 15000 units (Schiatti et al. 1970,Arrigoni-Martelli et al. 1971)

0.1 ml of 1% phytohaemagglutinin-P solution(Lewis et al. 1976)

0.01 ml of 0.5% adriamycin (mouse paw) (Siegelet al. 1980)

0.1 ml of 0.001–0.1% solutions of various phospho-lipases A2 (Cirino et al. 1989)

0.1 ml of 0.1% Zymosan solution (Gemmell et al.1979)

0.1 ml of 0.05% anti−IgG solution (Gemmell et al.1979)

0.1 ml of 2.5% mustard powder suspension(Tsumuri et al. 1986)

0.1 ml of solution containing 1 unit of cobra venomfactor(Leyck and Parnham 1990)

0.05 ml of 0.02–0.2% sonic extract from Porphy-romonas gingivalis (Griesbacher et al. 1994)

0.1 ml of 0.25% suspension of papaya latex (Guptaet al. 1994)

The edema induced by the various irritants lasts for dif-ferent times such as a few hours after serotonin and upto 2 days after Aerosil or after kaolin. These irritantstherefore are suitable to study not only the degree butalso the duration of the anti-inflammatory action.

StandardsDepending on the irritant steroidal and nonsteroidalanti-inflammatory drugs have a pronounced effect inthe paw edema test. With carrageenan as irritant dosesof 50 to 100 mg/kg phenylbutazone p.o. have beenfound to be effective.

CRITICAL ASSESSMENT OF THE METHODThe paw edema method has been used by many inves-tigators and has been proven to be suitable for screen-ing purposes as well as for more in depth evaluations.Dependent on the irritant steroidal and nonsteroidalanti-inflammatory drugs, antihistaminics and also, toa lesser degree, serotonin antagonists are active in thepaw edema tests. Since so many different irritants havebeen used by the various investigators the results areoften difficult to compare.

FURTHER MODIFICATIONS OF THE METHODBesides paw volume Shirota et al. (1984) determinedthe surface temperature of the inflamed paw in rats us-ing a special cage with rolling rods.

Kunz et al. (2004) assessed protein patterns in lum-bar cord during a zymosan-induced paw inflammationin rats employing a two-dimensional gel electrophore-sis revealing a time-dependent breakdown of scaffold-ing proteins such as neurofilament light chain protein.A calpain inhibitor prevented inflammation-inducedneurofilament light chain breakdown in the spinal cordand reduced hyperalgesia.

Brooks et al. (1991) used anesthetized dogs anddemonstrated that a significant inflammatory responsecan be elicited in the dog paw by subcutaneous in-jection of carrageenan. The increase in paw volumecan be quantitatively measured as a pressure changerecorded via a water-filled balloon fixed against thepaw with nonexpandable tape. Effective doses of non-steroidal antiinflammatory drugs were closer to humantherapeutic doses in dogs than in rats.

Oyanagui and Sato (1991) described an ischemicpaw assay in mice. A commercial rubber ring(1 × 1 mm, d = 42 mm) was bound 14 times to the righthind leg of mice just above the articulation. After20 min of ischemia, the rubber was cut off with scis-sors. Paw swelling was measured after an other 20 minof natural blood recirculation.

Wirth et al. (1992) described a thermic edemawhich was induced in anesthetized Sprague-Daw-ley rats by immersing paws of the right and lefthindlimb into water of 55°C. Immediately thereafter,the rats received the test drug (the bradykinin antag-


onist Hoe 140) intravenously. Paw volume was mea-sured at regular intervals by plethysmography.

Braga da Motta et al. (1994) described drug modu-lation of antigen-induced paw edema in guinea-pigs.Male short-haired guinea pigs weighing 250–350 g re-ceived on day 0 a single dorsal s.c. injection of 1 mlof phosphate buffered saline containing 20 µg of oval-bumin, dispersed in 1 mg Al(OH)3. The animals wereboosted with a similar injection of antigen on days 14,21, and 28. Thirty five days after the first injectionof antigen or Al(OH)3, the animals received an intra-plantar injection of 0.5, 5, 50, or 200 µg ovalbumin,diluted in 100 µl of phosphate buffered saline. Edemawas measured 2, 4, 6, 8, 24, and 48 h after the chal-lenge.

REFERENCES AND FURTHER READINGAlpermann HG, Magerkurth KO (1972) Messanordnung zur

Bestimmung der Wirkung von Antiphlogistika. ArzneimForsch/Drug Res 22:1078–1088

Alpermann HG, Sandow J, Vogel HG (1982) TierexperimentelleUntersuchungen zur topischen und systemischen Wirk-samkeit von Prednisolon-17-ethylcarbonat-21-propionat.Arzneim Forsch/Drug Res 32:633–638

Arrigoni-Martelli E, Schatti P, Selva D (1971) The influence ofanti-inflammatory and immunosuppressant drugs on nys-tatin induced oedema. Pharmacology 5:215–224

Braga da Motta JI, Cinha FQ, Vargaftig BB, Ferreira SH(1994) Drug modulation of antigen-induced paw oedemain guinea-pigs: effects of lipopolysaccharide, tumor necro-sis factor and leucocyte depletion. Br J Pharmacol112:111–116

Branceni D, Azadian-Boulanger A, Jequier R (1964) L’inf-lammation expérimentale par un analogue de l’héparine.Un test d’activité antiinflammatoire. Arch Int Pharmacodyn152:15–24

Brooks RR, Carpenter JF, Jones SM, Ziegler TC, Pong SF(1991) Canine carrageenin-induced acute paw inflamma-tion model and its response to nonsteroidal antiinflamma-tory drugs. J Pharmacol Meth 25:275–283

Burch RM, DeHaas Ch (1990) A bradykinin antagonist inhibitscarrageenan edema in rats. Naunyn-Schmiedeberg’s ArchPharmacol 342:189–193

Cirino G, Peers SH, Wallace JL, Flower RJ (1989) A study ofphospholipase A2-induced oedema in rat paw. Eur J Phar-macol 166:505–510

Damas J, Remacle-Volon G (1992) Influence of a long-actingbradykinin antagonist, Hoe 140, on some acute inflamma-tory reactions in the rat. Eur J Pharmacol 211:81–86

Dewes R (1955) Auswertung antiphlogistischer Substanzen mitHilfe des Hyaluronidase-Ödems. Arch Int Pharmacodyn104:19–28

Gemmel DK, Cottney J, Lewis AJ (1979) Comparative effectsof drugs on four paw oedema models. Agents and Actions9:107–116

Griesbacher T, Sutliff RL, Lembeck F (1994) Anti-inflammatoryand analgesic activity of the bradykinin antagonist, icat-ibant (Hoe 140), against an extract from Porphyromonasgingivalis. Br J Pharmacol 112:1004–1006

Gupta OP, Sharma N, Chand D (1994) Application of papaya-latex-induced rat paw inflammation: model for evaluation

of slowly acting antiarthritic drugs. J Pharmacol ToxicolMeth 31:95–98

Higgs EA, Moncada S, Vane JR (1978) Inflammatory effectsof prostacyclin (PGI2) and 6-oxo-PGF1α in the rat paw.Prostaglandins 16:153–161

Hofrichter G, Liehn HD, Hampel H (1969) Eine plethys-mometrische Messanordnung zur Bestimmung desRattenpfotenvolumens. Arzneim Forsch / Drug Res19:2016–2017

Kalbhen DA, Smalla HD (1977) Pharmakologische Studienzur antiphlogistischen Wirkung von Pentosanpolysulfat inKombination mit Metamizol. Arzneim Forsch/Drug Res27:1050–1057

Kunz S, Niederberger E, Ehnert C, Coste O, Pfenninger A, KruipJ, Wendrich TM, Schmidtko A, Tegeder I, Geisslinger G(2004) The calpain inhibitor MDL 28170 prevents inflam-mation-induced neurofilament light chain breakdown in thespinal cord and reduces hyperalgesia. Pain 110:409–418

Legat FJ, Griesbacher T, Lembeck F (1994) Mediation bybradykinin of rat paw oedema induced by collagenase fromClostridium histolyticum. Br J Pharmacol 112:433–460

Lewis AJ, Cottney J, Nelson DJ (1976) Mechanisms ofphytohaemagglutinin-P, concanavalin-A and kaolin-in-duced oedemas in the rat. Eur J Pharmacol 40:1–8

Leyck S, Parnham MJ (1990) Acute antiinflammatory andgastric effects of the seleno-organic compound ebselen.Agents Actions 30:426–431

Lorenz D (1961) Die Wirkung von Phenylbutazon auf dasPfotenoedem der Ratte nach oraler Applikation. Naunyn-Schmiedeberg’s Arch exp Path Pharm 241:516–517

Marek J (1980) Bentonite-induced paw edema as a tool for si-multaneous testing of prophylactic and therapeutic effectsof anti-inflammatory and other drugs. Pharmazie 36:46–49

Moore E, Trottier RW (1974) Comparison of various types ofcarrageenin in promoting pedal edema in the rat. Res Com-mun Chem Pathol Pharmacol 7:625–628

Nikolov R, Nikolova M, Peneva M (1978) Study of dipy-rone (Analgin) antagonism toward certain pharmacologi-cal effects of prostaglandins E2 and F2a. In: Ovtcharov R,Pola W (eds) Proceedings Dipyrone. Moscow Symposium,Schattauer-Verlag, Stuttgart New York, pp 81–89

Oyanagui Y, Sato S (1991) Inhibition by nilvadipine of is-chemic and carrageenan paw edema as well as of super-oxide radical production from neutrophils and xanthine ox-idase. Arzneim Forsch / Drug Res 41:469–474

Peterfalvi M, Branceni D, Azadian-Boulanger G, Chiflot L,Jequier R (1966) Etude pharmacologique d’un nouveaucomposé analgésique antiiflammatoire, la Glaphénine. MedPharmacol Exp 15:254–266

Portanova JP, Zhang Y, Anderson GD, Hauser SD, Masferrer JL,Seibert K, Gregory SA, Isakson PC (1996) Selective neu-tralization of prostaglandin E2 blocks inflammation, hyper-algesia, and interleukin 6 production in vivo. J Exp Med184:883–891

Randall LO, Baruth H (1976) Analgesic and anti-inflammatoryactivity of 6-chloro-alpha-methyl-carbazole-2-acetic acid(C-5720). Arch Int Pharmacodyn 220:94–114

Schiatti P, Selva D, Arrigoni-Martelli E (1970) L’edema localiz-zato da nystatin come modello di inflammazione sperimet-ale. Boll Chim Farm 109:33–38

Schönhöfer P (1967) Eine kritische Bemerkung zur Vergle-ichbarkeit der Wirkung entzündungshemmender Pharmakaauf die Glucosamin-6-phosphat-Synthese in vitro und amRattenpfotenödem in vivo. Med Pharmacol Exp 16:66–74

Shirota H, Kobayashi S, Shiojiri H, Igarashi T (1984) Determi-nation of inflamed paw surface temperature in rats. J Phar-macol Meth 12:35–43


Siegel DM, Giri SN, Scheinholtz RM, Schwartz LW (1980)Characteristics and effect of antiinflammatory drugs onadriamycin-induced inflammation in the mouse paw. In-flammation 4:233–248

Souza Pinto JC, Remacle-Volon G, Sampaio CAM, Damas J(1995) Collagenase-induced oedema in the rat paw and thekinin system. Eur J Pharmacol 274:101–107

Tsumuri K, Kyuki K, Niwa M, Kokuba S, Fujimura H(1986) Pharmacological investigations of the newantiinflammatory agent 2-(10,11-dihydro-10-oxodiben-zo(b,f)thiepin-2-yl) propionic acid. Arzneim Forsch/DrugRes 36:1796–1800

Wagner-Jauregg Th, Jahn U, Buech O (1962) Die antiphlogis-tische Prüfung bekannter Antirheumatika am Rattenpfoten-Kaolinödem. Arzneim Forsch / Drug Res 12:1160–1162

Webb EF, Griswold DE (1984) Microprocessor-assisted plethys-mograph for the measurement of mouse paw volume.J Pharmacol Meth 12:149–153

Willis AL, Cornelsen M (1973) Repeated injection ofprostaglandin E2 in rat paws induces chronic swellingand a marked decrease in pain threshold. Prostaglandins3:353–357

Winter CA, Risley EA, Nuss GW (1962) Carrageenin-inducedoedema in hind paw of the rat as an assay for antiinflam-matory drugs. Proc Soc Exp Biol Med 111:544–547

Winter CA, Risley EA, Nuss GW (1963) Antiinflamma-tory and antipyretic activities of indomethacin, (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole-3-acetic acid.J Pharmacol Exp Ther 141:369–376

Wirth KJ, Alpermann HG, Satoh R, Inazu M (1992) Thebradykinin antagonist HOE 140 inhibits carrageenan- andthermically induced paw edema in rats. Recent Progress onKinins, Birkhäuser, Basel, pp 428–431

H.3.2.2.7Pleurisy Test

PURPOSE AND RATIONALEPleurisy is a well known phenomenon of exudative in-flammation in man. In experimental animals pleurisycan be induced by several irritants, such as histamine,bradykinin, prostaglandins, mast cell degranulators,dextran, enzymes, antigens, microbes, and nonspecificirritants, like turpentine and carrageenan (Survey byDeBrito 1989). Carrageenan-induced pleurisy in ratsis considered to be an excellent acute inflammatorymodel in which fluid extravasation, leukocyte migra-tion and the various biochemical parameters involvedin the inflammatory response can be measured easilyin the exudate.

PROCEDUREMale Sprague-Dawley rats weighing 220–260 g areused. The animal is lightly anaesthetized with ether,placed on its back and the hair from skin over the ribsof the right side is removed using animal clippers. Theregion is swabbed with alcohol. A small incision ismade into the skin under the right arm between the sev-enth and eighth rib. The wound is opened and a further

shallow incision is made into the exposed intercostalmuscle. 0.1 ml of 2% carrageenin solution is injectedinto the pleural cavity through this incision. The in-jection needs to be made swiftly to avoid the risk ofinjuring the lung. The wound is closed with a Michelclip.

One hour before carrageenan injection and 24 and48 h thereafter, groups of 10 rats are treated withthe standard or the test compound subcutaneously ororally. A control group receives only the vehicle ofmedication. The animals are sacrificed 72 h after car-rageenin injection by ether inhalation. The animal ispinned on a dissection board with the forelimbs fullyextended. An incision in the skin over the xiphosternalcartilage is made to free the cartilage from overlyingconnective tissue. The cartilage is lifted with a forcepsand a small cut is made with scissors in the body wallbelow to gain access into the pleural cavity. One ml ofheparinized Hank’s solution is injected into the pleuralcavity through this cut. The cavity is gently massagedto mix its contents. The fluid is aspirated out of the cav-ity using a pipette. This is made easier if the dissectionboard is raised to an angle of 45–60°; the contents thenpool in the corners of the cavity. The aspirated exudateis collected in a graduated plastic tube.

EVALUATIONOne ml (the added Hank’s solution) is subtracted fromthe measured volume. The values of each experimen-tal group are averaged and compared with the controlgroup. ED50 values can be calculated using variousdoses. Several other parameters can be used:

• Measuring the white blood cell number in the exu-date using a Coulter counter or a hematocytometer,

• Determination of lysosomal enzyme activities,• Determination of fibronectin,• Determination of PgE2.

CRITICAL ASSESSMENT OF THE METHODThe pleurisy model has been accepted as a reliablemethod to study acute and subacute inflammation al-lowing the determination of several parameters simul-taneously or successively. The activity of steroids aswell as of non-steroidal drugs can be measured (Tom-linson et al. 1994; Harada et al. 1996).

MODIFICATIONS OF THE METHODThe Evans blue-carrageenan-induced pleural effusionmodel has been proposed by Sancilio (1969, 1973) forscreening of compounds with anti-inflammatory activ-ity.


Meyers et al. (1993) tested the effect of treatmentwith interleukin-1 receptor antagonist on the devel-opment of carrageenan-induced pleurisy in intact andadrenalectomized rats.

Fröde et al. (2001, 2002) tested the effects of TNF-α and IL-1β, IL-6, and IL-10 and their specific anti-bodies in the acute inflammatory response induced bycarrageenan in a mouse model of pleurisy. Adult Swissmice received a single intrapleural injection of 0.1 mlof sterile saline containing 1% carrageenan. As theinflammatory response caused by carrageenan in thepleural space of the mice exhibits a biphasic response,peaking at 4 h, characterized primarily by neutrophils,and at 48 h due mainly to mononuclear cells, both in-terval points were studied. On the day of the experi-ment, different doses of TNF-α, IL-1β, IL-6 or IL-10and their antibodies were injected into the pleural cav-ity of anesthetized mice. After 4 h or 48 h, the animalswere sacrificed, the thorax was opened and the pleuralcavity was washed with 1 ml of sterile PBS contain-ing 20 IU/ml heparin. All animals had been injected60 min previously with a solution of Evans blue dye(25 mg/kg, 0.2 ml, i.v.) in order to evaluate the degreeof exudation into the pleural space. Leukocytes werecounted and evaluated microscopically. The amount ofdye was estimated by colorimetry.

REFERENCES AND FURTHER READINGAckerman N, Tomolonis A, Miram L, Kheifets J, Martinez S,

Carter A (1980) Three day pleural inflammation: A newmodel to detect drug effects on macrophage accumulation.J Pharmacol Exp Ther 215:588–595

De Brito FB (1989) Pleurisy and pouch models of acute inflam-mation. In: Pharmacological Methods in the Control of In-flammation. Alan R. Liss, Inc. pp 173–194

Dunn CJ, Doyle DV, Willoughby DA (1993) Investigation of theacute and chronic anti-inflammatory properties of diphos-phonates using a broad spectrum of immune and non-immune inflammatory reactions. Drug Dev Res 28:47–55

Fröde TS, Souza GEP, Calixto JB (2001) The modulatory roleplayed by TNF-α and IL-1β in the inflammation responsesinduced by carrageenan in the mouse model of pleurisy.Cytokine 13:162–168

Fröde TS, Souza GEP, Calixto JB (2002) The effects of IL-6 andIL-10 and their specific antibodies in the acute inflamma-tory response induced by carrageenan in the mouse modelof pleurisy. Cytokine 17:149–156

Harada Y, Hatanaka K, Kawamura M, Saito M, Ogino M, Ma-jima M, Ohno T, Ogino K, Yamamoto K, Taketani Y,Yamamoto S, Katori M (1996) Role of prostaglandinsynthase-2 in prostaglandin E2 formation in rat car-rageenin-induced pleurisy. Prostaglandins 51:19–33

Meyers KP, Czachowski CL, Coffey JW (1993) Effect of treat-ment with interleukin-1 receptor antagonist on the develop-ment of carrageenan-induced pleurisy in the rat. Inflamma-tion 17:121–134

Mielens ZE, Connolly K, Stecher VJ (1985) Effects of dis-ease modifying antirheumatic drugs and nonsteroidal anti-

inflammatory drugs upon cellular and fibronectin responsesin a pleurisy model. J Rheumatol 12:1083–1087

Mikami T, Miyasaka K (1983) Effects of several anti-inflammatory drugs on the various parameters involvedin the inflammation response in rat carrageenin-inducedpleurisy. Eur J Pharmacol 95:1–12

Sancilio L (1969) Evans blue-carrageenan pleural effusion asa model for the assay of nonsteroidal antirheumatic drugs.J Pharmacol Exp Ther 168:199–204

Sancilio LF, Fishman A (1973) Application of sequential anal-ysis to Evans blue-carrageenan-induced pleural effusionfor screening of compounds for anti-inflammatory activity.Toxicol Appl Pharmacol 26:575–584

Tomlinson A, Appleton I, Moore AR, Gilroy DW, Willis D,Mitchell JA, Willoughby DA (1994) Cyclo-oxygenase andnitric oxide synthase isoforms in rat carrageenin-inducedpleurisy. Br J Pharmacol 113:693–698

Tsurumi K, Mibu H, Okada K, Hasegawa J, Fujimura H(1986) Pharmacological investigations of the newantiinflammatory agent 2-(10,11-dihydro-10-oxodiben-zo[b,f]thiepin-2-yl) propionic acid. Arzneim Forsch/DrugRes 36:1806–1809

Ushida Y, Oh-Ishi S, Tanaka K, Harada Y, Ueno A, Katori M(1982) Activation of plasma kallikrein-kinin system and itssignificant role in the pleural fluid accumulation of rat car-rageenin-induced pleurisy. In: Fritz H (ed) Recent Progresson Kinins. Agents and Actions Suppl Vol 9:379–383

H.3.2.2.8Granuloma Pouch Technique

PURPOSE AND RATIONALEThe method originally invented by Selye has been de-veloped for screening by Robert and Nezamis (1957)using croton oil as irritant. An aseptic inflammationresulting in large volumes of hemorrhage exudate iselicited which resembles the subacute type of inflam-mation. Instead of croton oil carrageenan can be usedas irritant.

PROCEDUREMale or female Sprague-Dawley rats with a bodyweight between 150 and 200 g are used. Ten animalsare taken for controls and for test groups. The backof the animals is shaved and disinfected. With a verythin needle a pneumoderma is made in the middle ofthe dorsal skin by injection of 20 ml of air under etheranesthesia. Into the resulting oval airpouch 0.5 ml ofa 1% solution of Croton oil in sesame oil is injectedavoiding any leakage of air. Forty-eight hours later theair is withdrawn from the pouch and 72 h later any re-sulting adhesions are broken. Instead of croton oil 1 mlof a 20% suspension of carrageenan in sesame oil canbe used as irritant. Starting with the formation of thepouch, the animals are treated every day either orallyor subcutaneously with the test compound or the stan-dard. For testing local activity, the test compound isinjected directly into the air sac at the same time as the


irritant. On the 4th or the 5th day the animals are sac-rificed under anesthesia. The pouch is opened and theexudate is collected in glass cylinders. Controls havean exudate volume between 6 and 12 ml, which is re-duced dose dependent in the treated animals.

EVALUATIONThe average value of the exudate of the controls andthe test groups is calculated. Comparison is made bystatistical means. A clear dose response curve couldbe found by s.c. injection of 0.5, 1.0 and 2.0 mg hy-drocortisone acetate/rat. Also doses of 1.5 mg/kg in-domethacin were found to be active.

CRITICAL ASSESSMENT OF THE METHODThe method has been very useful to estimate the po-tency of anti-inflammatory corticosteroids both afterlocal and after systemic application. By injection ofa depot-preparation and induction of the granulomapouch after various time intervals up to 4 weeks theduration of action can also be determined (Vogel 1963,1965).

MODIFICATIONS OF THE METHODCarrageenin was used to induce exudate formation(Boris and Stevenson 1965).

Bobalik and Bastian (1967) developed a modifiedgranuloma pouch technique in which Mycobacteriumbutyricum (adjuvant) was used as phlogistic agent.

Moreno (1993) sensitized rats by subcutaneous in-jection of methylated bovine serum albumin emulsi-fied in complete Freund’s adjuvant one week prior tothe preparation of air pouches which were reinflated4 days later. Seven days after formation of the airpouches inflammation was induced in the pouches byinjection of 1 mg methylated bovine serum albumin.

Martin et al. (1994) described an air pouch model inthe 6-day-old rat by injection of carrageenan. Besidesthe usual parameters, leukocyte influx and the level ofprostaglandin E2 in the pouch exudate were measured.

In order to measure the effects of different classes ofproteinase inhibitors, Karran and Harper (1995) stud-ied collagen degradation in subcutaneous air pouchesin rats. The air pouches were formed in the dorsal re-gion and were inflamed 6 to 8 days later by injectingλ-type carrageenan. Degradation of 14C-collagen wasfollowed in the inflammatory exudate fluid of the airpouches.

Sugio and Tsurufuji S (1981) re-evaluated the vas-cular constriction hypothesis as the mechanism of anti-inflammatory action of glucocorticoids. Rats were in-jected with 8 ml of air subcutaneously on the dorsal

surface under light ether anesthesia to make an ovalair sac. After 24 h, 4.0 ml of 2% heat-sterilized solu-tion of carrageenin in 0.9% NaCl solution was injectedinto the air sac (day 0). Drug effects were tested onday 7. Vascular permeability in the granuloma pouchwas measured using 125I-HSA and 131I-HSA. About1 µCi of purified 125I-HSA in 0.2 ml saline was in-jected into the femoral vein. After 30 min, 1.0 ml of thepouch fluid was withdrawn to measure the leakage of125I-HSA into the pouch fluid. After administration ofthe drug, about 1 µCi of purified 131I-HSA was injectedinto the femoral vein. After 30 min, 1.0 ml of the pouchfluid was again withdrawn to measure the concentra-tion of 131I-HSA. The ratio of 125I-HSA/131I-HSA wastaken as an index of vascular permeability change in-duced by drug treatment.

Atkinson et al. (1962) implanted compressed pelletsof carrageenin subcutaneously to rats and measuredthe effects of some anti-inflammatory substances onwet weight of the pellets.

Bowers et al. (1985) described a method to in-duced a granuloma in the rat lung by instillation ofa 2% carrageenan solution into one lower lobe of thelung via the trachea. No respiratory impairment wasnoticed during this procedure.

Further phlogistic agents inducing specific inflam-matory cascades such as zymosan (complement acti-vation) or lipopolysaccharide (cytokine release) havebeen used for pharmacological evaluation of anti-inflammatory agents (Erdö 1994; Miller 1997).

REFERENCES AND FURTHER READINGAtkinson RM, Jenkins L, Tomich EG, Woollett EA (1962)

The effects of some anti-inflammatory substances on car-rageenin-induced granulomata. J Endocrinol 25:87–93

Bobalik GR, Bastian JW (1967) Effects of various antiphlogisticagents on adjuvant-induced exudate formation in rats. ArchInt Pharmacodyn 166:466–472

Boris A, Stevenson RH (1965) The effects of some non-steroidalanti-inflammatory agents on carrageenin-induced exudateformation. Arch Int Pharmacodyn 153:205–210

Bowers RR, Birch ML, Thomas DW (1985) A biochemicalstudy of the carrageenan-induced granuloma in the rat lung.Conn Tiss Res 13:191–206

De Brito FB (1989) Pleurisy and pouch models of acute inflam-mation. In: Pharmacological Methods in the Control of In-flammation. Alan R. Liss, Inc. pp 173–194

Erdö F, Török K, Szekely JI (1994) Measurement of interleukin-1 in zymosan air-pouch exudate in mice. Agents and Ac-tions 41:93–95

Karran EH, Harper GP (1995) Collagen degradation within sub-cutaneous air pouches in vivo: the effects proteinase in-hibitors. J Pharmacol Toxicol Meth 34:97–102

Martin SW, Stevens AJ, Brennan BS, Davies D, Rowd-land M, Houston JB (1994) The six-day-old rat air pouchmodel of inflammation: characterization of the inflamma-tory response to carrageenan. J Pharmacol Toxicol Meth32:139–147


Miller AJ, Hopkins SJ, Luheshi GN (1997) Sites of action of IL-1 in the development of fever and cytokine response to tis-sue inflammation in the rat. Br J Pharmacol 120:1274–1279

Moreno JJ (1993) Time course of phopsholipase A2, eikosanoidrelease and cellular accumulation in rat immunological airpouch inflammation. Int J Immunopharmacol 15:597–603

Robert A, Nezamis JE (1957) The granuloma pouch as a routineassay for antiphlogistic compounds. Acta Endocr (Kbh)25:105–112

Selye H (1953) On the mechanism through which hydrocorti-sone affects the resistance of tissues to injury. An experi-mental study with the granuloma pouch technique. J AmMed Ass 152:1207–1213

Ueno H, Maruyama A, Miyake M, Nakao E, Nakao K,Umezu K, Nitta I (1991) Synthesis and evaluation ofantiinflammatory activities of a series of corticosteroid17α-esters containing a functional group. J Med Chem34:2468–2473

Sugio K, Tsurufuji S (1981) Mechanisms of anti-inflammatoryaction of glucocorticoids: re-evaluation of vascular con-striction hypothesis. Br J Pharmacol 73:605–608

Vogel HG (1963) Intensität und Dauer der antiinflamma-torischen und glykoneogenetischen Wirkung von Pred-nisolon und Prednisolonazetat nach oraler und subcutanerApplikation an der Ratte. Acta Endocr (Kbh) 42:85–96

Vogel HG (1965) Intensität und Dauer der Wirkung von 6-Methylprednisolon und seinen Estern an der Ratte. ActaEndocr (Kbh.) 50:621–642

H.3.2.2.9Urate-Induced Synovitis

PURPOSE AND RATIONALEThe importance of urate in gout and the deposition ofsodium urate in gouty tophi is well known. Faires andMcCarty (1962) reported that they themselves werethe subjects for a study injecting 20 mg sodium uratecrystal suspension in their own knee-joint. They expe-rienced severe pain and prostration which resembledan acute gouty attack. Based on this experience theydeveloped an experimental model in dogs for testinganti-inflammatory compounds (McCarty et al. 1963,1966).

PROCEDUREPreparation of Sodium Urate Crystals0.4 g (0.01 Mol) sodium hydroxide pellets are dis-solved in 400 ml distilled water in a glass beaker;1.68 g (0.01 Mol) uric acid is added. The resultantopaque preparation is allowed to remain overnight atroom temperature. The next morning, the crystals areharvested by decanting the supernatant solution andare then washed 3 times in cold saline, resuspendedin saline and sterilized in an autoclave. Suspensionsfor injections are kept in rubber-stoppered, multi-dosevials containing 15 to 24 mg of urate per ml.

Unanesthetized healthy dogs weighing between18 and 25 kg are used. They are trained to lie quietlyon their backs in a dog cradle under light restraint. The

skin above one knee is shaved, disinfected and a sterile21-gauge needle inserted into the joint. Slight aspira-tion produces a small amount of clear, viscous synovialfluid, indicating entry into the joint. The needle is leftin place, a syringe containing the urate suspension isattached and volumes from 0.1 to 0.5 ml are injectedinto the joint (approximately 2–10 mg urate).

One hour before the injection of urate crystals theanimals are treated with the test compound or the stan-dard. Experiments are designed so that a pair of dogs istested on each of 2 days. On the first day, only one dogreceives the drug. One week later the opposite knee ofeach dog is injected, but the other dog is treated.

EVALUATIONA scoring system is adopted in which inflammatorysymptoms ranging from tenderness, limping, occa-sional 3-legged gait to complete 3-legged gait arescored from 1+ to 4+.

CRITICAL ASSESSMENT OF THE METHODIn spite of the fact that the experiment originallyhas been performed in human volunteers and thatthe method closely resembles pathological conditionsin man, due to animal protection law conditions themethod can be recommended only for special investi-gations.

MODIFICATIONS OF THE METHODCarlson et al. (1986) developed an automatedmicrocomputer-based system for determining caninepaw pressure quantitatively in the dog synovitis model.

According to Phelps et al. (1967) dogs are anes-thetized and placed on their sides with the hind legfirmly fixed with tape so that the femur and tibiaform a 90-degree angle. The knee is punctured witha needle. When a few drops of synovial fluid can bewithdrawn indicating a correct puncture of the joint,6–10 ml of saline are injected to distend the jointand a polyethylene catheter is inserted through theneedle, which is then withdrawn. 0.5 ml of a 0.02%sodium urate suspension are injected into the joint.The catheter is attached to a pressure transducer. Con-stant pressure recordings can be taken during theacute phase of inflammation. Pressure changes areplotted against time, whereby each dog is comparedwith his own control. Treatment with nonsteroidalanti-inflammatory drugs, such as indomethacin, showa considerable reduction of intraarticular pressure.

Fujihira et al. (1971) injected the urate suspensioninto the knee joint of a hind leg of well trained Bea-gle dogs. They were placed on three weighing ma-


chines whereby both forelegs rested on one balance,and the hindlegs individually on other balances. In thisway, the relative change of weight on each hindleg af-ter intra-articular injection of urate suspension can bemeasured, indicating a decrease of weight in the in-jected leg, counterbalanced to the other leg. Time re-sponse curves could be found after non-steroidal anti-inflammatory drugs.

Rosenthale et al. (1972) found a long-lasting in-flammatory effect of prostaglandins PGE1 and PGE2after injection in the knee joint of dogs.

Schaible and Schmidt (1985) induced an acute ex-perimental arthritis in the knee joint of anesthetizedcats by intraarticular injection of a 4% kaolin suspen-sion and recorded the activity of single fine afferentunits from filaments of the saphenous nerve.

Perkins and Campell (1992) injected either sodiumurate crystals or Freund’s complete adjuvant into oneknee of rats. The maximum tolerated pressure was de-termined with or without treatment by analgesic drugsafter 18–24 h (urate injections) or 64–70 h (Freund’scomplete adjuvant).

Daniel and Jouvin (1984) induced inflammation ofthe guinea pig palatal mucosa by injection of a mi-crocrystalline suspension of monosodium urate.

Botrel et al. (1994) induced chronic inflammationin the knee joint of Beagle dogs by intra-articular in-jection of Freund’s complete adjuvant. Besides bodytemperature, differences in skin temperature, differ-ence in stifle diameter, the vertical force exerted by thearthritic hind limb measured by a force plate was cho-sen as parameter.

Schött et al. (1994) induced monoarthritis in rats byinjection of 300 µg carrageenan in 50 µl saline into theright tibio-tarsal joint. Weight bearing was found to bean objective measure of arthritic pain.

Carleson et al. (1996) induced acute inflammationin the temporomandibular joint of rats by intraarticularinjection of substance P and measured neurokinin A,calcitonin gene-related peptide and neuropeptide Y inthe perfusate.

REFERENCES AND FURTHER READINGBotrel MA, Haak T, Legrand C, Concordet D, Chevalier R,

Toutain PL (1994) Quantitative evaluation of an experi-mental inflammation induced with Freund’s complete ad-juvant in dogs. J Pharmacol Toxicol Meth 32:63–71

Carleson J, Alstergren P, Appelgren A, Appelgren B, Kopp S,Theodorsson E, Lundeberg T (1996) A model for experi-mental induction of acute temporomandibular joint inflam-mation in rats: Effects of substance P (SP) in neuropeptide-like immunoreactivity. Life Sci 59:1193–1201

Carlson RP, Datko LJ, Welch TM, Purvis WF, Shaw GW,Thompson JL, Brunner TR (1986) An automatedmicrocomputer-based system for determining canine paw

pressure quantitatively in the dog synovitis model. J Phar-macol Meth 15:95–104

Chau TT (1989) Analgesic testing in animal models. In: Phar-macological models in the control of inflammation. AlanR. Liss, Inc., pp 195–212

Daniel A, Jouvin JL (1984) Experimentally induced inflamma-tion of the guinea pig palatal mucosa by injection of a mi-crocrystalline suspension of monosodium urate. J Pharma-col Meth 12:155–166

Dubinsky B, Gebre-Mariam S, Capetola RJ, Rosenthale ME(1987) The antialgesic drugs: Human therapeutic correlatesof their potency in laboratory animal models of hyperalge-sia. Agents and Actions 20:50–60

Faires JS, McCarty DJ (1962) Acute arthritis in man and dog af-ter intrasynovial injection of sodium urate crystals. Lancet2:682–685

Fujihira E, Mori T, Nakazawa M, Ozawa H (1971) A simplemethod for evaluating analgesic efficacy of non-steroidalanti-inflammatory drugs. Chem Pharm Bull 19:1506–1508

McCarty DJ, Faires JS (1963) A comparison of the durationof local anti-inflammatory effects of several adrenocorti-costeroid esters – a bioassay technique. Curr Ther Res5:284–290

McCarty DJ, Phelps P, Pyenson J (1966) Crystal-induced in-flammation in canine joints. I. An experimental model withquantification of the host response. J Exp Med 124:99–114

Perkins MN, Campell EA (1992) Capsazepine reversal of theantinociceptive action of capsaicin in vivo. Br J Pharmacol107:329–333

Phelps P, McCarty DJ (1967) Animal techniques for evaluatinganti-inflammatory drugs. In: Siegler PE. Moyer JH (eds)Animal and pharmacological techniques in drug evalua-tion. Vol 2. Year Book Medical Publishers, Inc., Chicago,pp 742–747

Rosenthale ME, Kassarich J, Schneider F (1966) Effect of anti-inflammatory agents on acute experimental synovitis indogs. Proc Soc Exp Biol Med 122:693–696

Rosenthale ME, Dervinis A, Kassarich J, Singer S (1972)Prostaglandins and anti-inflammatory drugs in the dog kneejoint. J Pharm Pharmacol 24:149–150

Schaible HG, Schmidt RF (1985) Effects of an experimentalarthritis on the sensory properties of fine articular afferentunits. J Neurophysiol 54:1109–1122

Schött E, Berge OG, Ängeby-Möller K, Hammerström G, Dals-gaard CJ, Brodin E (1994) Weight bearing as an objectivemeasure of arthritic pain in the rat. J Pharmacol ToxicolMeth 31:79–83

Tanaka K, Shimotori T, Makino S, Aikawa Y, Inaba T,Yoshida C, Takano S (1992) Pharmacological studiesof the new anti-inflammatory agent 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one.1st Communication: anti-inflammatory, analgesic andother related properties. Arzneim Forsch/Drug Res42:935–944

H.3.2.3Methods for Testing the Proliferative Phase(Granuloma Formation)

H.3.2.3.1Cotton Wool Granuloma

PURPOSE AND RATIONALEThe method has been described first by Meier et al.(1950) who showed that foreign body granulomas


were provoked in rats by subcutaneous implantationof pellets of compressed cotton. After several days,histologically giant cells and undifferentiated connec-tive tissue can be observed besides the fluid infiltra-tion. The amount of newly formed connective tissuecan be measured by weighing the dried pellets after re-moval. More intensive granuloma formation has beenobserved if the cotton pellets have been impregnatedwith carrageenin.

PROCEDUREMale Wistar rats with an average weight of 200 g areanaesthetized with ether. The back skin is shaved anddisinfected with 70% ethanol. An incision is made inthe lumbar region. By a blunted forceps subcutaneoustunnels are formed and a sterilized cotton pellet isplaced on both sides in the scapular region. The pelletsare either standardized for use in dentistry weighing20 mg or pellets formed from raw cotton which pro-duce a more pronounced inflammation than bleachedcotton. The animals are treated for 7 days subcuta-neously or orally. Than, the animals are sacrificed, thepellets prepared and dried until the weight remainsconstant. The net dry weight, i. e. after subtracting theweight of the cotton pellet is determined.

EVALUATIONThe average weight of the pellets of the control groupas well as of the test group is calculated. The percentchange of granuloma weight relative to vehicle controlgroup is determined.

CRITICAL ASSESSMENT OF THE METHODThe method has been useful for evaluation of steroidaland nonsteroidal anti-inflammatory drugs. For testingcorticosteroids, the test can be performed in adrenalec-tomized rats.

MODIFICATIONS OF THE METHODBush and Alexander (1960) produced granulomata inrats by means of cotton-wool pellets which have beenimpregnated with carrageenin.

Tanaka et al. (1960) implanted filter paper pelletssoaked with 7% formalin solution in rats.

Hicks (1969) implanted pellets impregnated with ir-ritant substances, such as capsicum oleoresin.

Instead of cotton pellets, paper disks have been im-planted (Tsurumi et al. 1986).

Roszkowski et al. (1971) immersed the cotton pel-lets in a 1% carrageenan solution, dried overnight andsoaked in a 0.25 oxytetracycline solution before im-plantation.

D’Arcy and Howard (1967) induced a localized in-flammatory reaction in the chorio-allantoic membraneof the chick embryo by the implantation of a sterilefilter paper disc, followed by re-incubation in situ for4 days.

Rudas (1960) described a method for quantitativeevaluation of the granulation tissue formed in exper-imental wounds. Plastic rings were incorporated intothe wounds on the back of rats inhibiting contractionof the wound edges and epithelialization of the wound.The growth of granulation tissue inside the rings wasmeasured.

REFERENCES AND FURTHER READINGAlpermann HG, Sandow J, Vogel HG (1982) Tierexperimentelle

Untersuchungen zur topischen und systemischen Wirk-samkeit von Prednisolon-17-ethylcarbonat-21-propionat.Arzneim Forsch/Drug Res 32:633–638

Bush IE, Alexander RW (1960) An improved method for theassay of antiinflammatory substances in rats. Acta Endocr(Kbh) 35:268–276

Hicks R (1969) The evaluation of inflammation induced by ma-terial implanted subcutaneously in the rat. J Pharm Phar-macol 21:581–588

Meier R, Schuler W, Desaulles P (1950) Zur Frage des Mech-anismus der Hemmung des Bindegewebswachstums durchCortisone. Experientia 6:469–471

Penn GB, Ashford A (1963) The inflammatory response to im-plantation of cotton pellets in the rat. J Pharm Pharmacol15:798–803

Roszkowski AP, Rooks WH, Tomolonis AJ, Miller LM(1971) Anti-inflammatory and analgesic properties of d-2-(6′-methoxy-2′-naphthyl)-propionic acid (NAPROXEN).J Pharmacol Exper Ther 179:114–123

Rudas B (1960) Zur quantitativen Bestimmung von Granula-tionsgewebe in experimentell erzeugten Wunden. ArzneimForsch 10:226–229

Tanaka A, Kobayashi F, Miyake T (1960) A new anti-inflammatory activity test for corticosteroids. The forma-lin-filter paper pellet method. Endocrinol Japon 7:357–364

Tsurumi K, Mibu H, Okada K, Hasegawa J, Fujimura H(1986) Pharmacological investigations of the newantiinflammatory agent 2-(10,11-dihydro-10-oxodiben-zo[b,f]thiepin-2-yl) propionic acid. Arzneim Forsch/DrugRes 36:1806–1809

H.3.2.3.2Sponge Implantation Technique

PURPOSE AND RATIONALEThe sponge implantation technique was described firstby Saxena (1960) for short term experiments but wasused subsequently to study the formation of granulo-mata using long-term implantation.

PROCEDURESponges used for implantation are prepared frompolyvinyl foam sheets (thickness 5 mm). Discs arepunched out to a standard size and weight (10.0±


0.02 mg) using a 13 mm cork borer. The sponges arethen soaked in 70% v/v ethanol for 30 min, rinsed fourtimes with distilled water and heated at 80°C for 2 h.Prior to implantation in the animal, the sponges aresoaked in sterile 0.9% saline in which either drugs,antigens or irritants have been suspended. Typical ex-amples include 1% carrageenan, 1% yeast, 1% Zy-mosan A, 6% dextran, heat killed Bordetella pertussis(4 × 109 to 5 × 1010 organisms/ml) or 0.5% heat killedmycobacterium tuberculosis.

Sponges are implanted in female Wistar rats weigh-ing 150–200 g under ether anesthesia. A 20 mm dorsalincision is made and the dermis separated from the un-derlying muscle layer by insertion of blunt forceps toform separate cavities into which sponges are inserted.Up to 8 sponges may be implanted per rat. The dorsalincision is closed with Michel clips and the animals aremaintained at a constant temperature of 24°C.

For short term experiments, the animals are treatedwith test drug or standard once before implantationorally or subcutaneously. For long term experiments,the rats are treated daily up to 3 weeks.

EVALUATIONFor estimation of the fluid phase of sponge exudates,e. g. protein content, enzyme levels and biological me-diators such as prostaglandins as well as for leukocytemigration, the sponges are removed already after 9 h.

For studying the chronic phase of inflammation be-sides dry weight DNA, indicating cell content, hex-osamine, indicating glycosaminoglycane content, andhydroxyproline, indicating collagen content, can bedetermined.

CRITICAL ASSESSMENT OF THE METHODThe sponge implantation technique has been proven tobe a versatile method which was used and modified bymany investigators.

MODIFICATIONS OF THE METHODBoucek and Noble (1955) implanted polyvinylsponges in rats, hamsters, rabbits and humans.

Holm-Pedersen and Zederfeldt (1971) implanted2 cubes 10 × 10 × 10 mm of cellulose sponge con-nected with a silk suture. After various implanta-tion periods, the sponges were dissected free and thestrength of the connection between the two parts of thesponge was determined after removal of the connect-ing suture.

Paulini et al. (1974, 1976) implanted polyester-polyurethane sponges which were inserted at both ends

of a 15 mm long PVC tube separated by a cotton woolplug.

Bonta et al. (1979) used polyether sponges measur-ing 4 × 1.5 × 0.5 cm. A thin polyethylene cannula is in-serted into a hole of the sponge and fixed with twostitches. After implantation of the sponge the cannulais pulled through a subdermal tunnel to a neck incisionwhere about 1.5 cm is exteriorized and closed witha tube sealer. One ml of a 2% carrageenin solution isinjected into the sponge via the cannula. To study thelocal effect of drugs, the test compounds can be in-jected together with the carrageenin. The drugs can beadministered repeatedly at any time.

The cannulated sponge method was further modi-fied by Bragt et al. (1980) using a subdermally im-planted Teflon cylinder. This cylinder is provided withholes to ensure contact and exchange between theinner chamber and the surrounding tissue and withtwo cannulae allowing injection of material and with-drawal of exudate at any given stage of granuloma de-velopment.

Damas and Remacle-Volon (1992) implanted in ratssterilized polyester sponges which were removed after4 h and weighed.

REFERENCES AND FURTHER READINGBonta IL, Adolfs MJP, Parnham MJ (1979) Cannulated sponge

implants in rats for the study of time-dependent pharmaco-logical influences on inflammatory granulomata. J Pharma-col Meth 2:1–11

Boucek RJ, Noble NL (1955) Connective tissue. A technique forits isolation and study. AMA Arch Pathol 59:553–558

Bragt PC, Bonta IL, Adolfs MJP (1980) Cannulated Teflonchamber implant in the rat: A new model for continuousstudies on granulomatous inflammation. J Pharmacol Meth.3:51–61

Damas J, Remacle-Volon G (1992) Influence of a long-actingbradykinin antagonist, Hoe 140, on some acute inflamma-tory reactions in the rat. Eur J Pharmacol 211:81–86

Ford-Hutchinson AW, Walker JR, Smith MJH (1978) Assess-ment of anti-inflammatory activity by sponge implantationtechniques. J Pharmacol Meth 1:3–7

Higgs GA (1989) Use of implanted sponges to study the acuteinflammatory response. In: Pharmacological Methods inthe Control of Inflammation. Alan R. Liss, Inc., pp 151–171

Holm-Pedersen P, Zederfeldt B (1971) Granulation tissue for-mation in subcutaneously implanted cellulose sponges inyoung and adult rats. Scand J Plast Reconstr Surg 5:13–16

Paulini K, Körner B, Beneke G, Endres R (1974) A quantitativestudy of the growth of connective tissue: Investigation onimplanted polyester-polyurethane sponges. Conn Tiss Res2:257–264

Paulini K, Körner B, Mohr W, Sonntag W (1976) The ef-fect of complete Freund – adjuvant on chronic proliferat-ing inflammation in an experimental granuloma model. ZRheumatol 35:123–131

Saxena PN (1960) Effects of drugs on early inflammation reac-tion. Arch Int Pharmacodyn Ther 126:228–237

H.4 · Antipyretic Activity 1113

H.3.2.3.3Glass Rod Granuloma

PURPOSE AND RATIONALEThe glass rod granuloma as first described by Vogel(1970) reflects the chronic proliferative inflammation.Of the newly formed connective tissue not only wetand dry weight, but also chemical composition and me-chanical properties can be measured.

PROCEDUREGlass rods with a diameter of 6 mm are cut to a lengthof 40 mm and the ends rounded off by flame melt-ing. They are sterilized before implantation by boil-ing in water. Male Sprague-Dawley rats with an initialweight of 130 g are anaesthetized with ether, the backskin shaved and disinfected. From an incision in thecaudal region a subcutaneous tunnel is formed in cra-nial direction with a closed blunted forceps. One glassrod is introduced into this tunnel finally lying on theback of the animal. The incision wound is closed bysutures. The animals are kept in separate cages. Therods remain in situ for 20 or 40 days. Treatment withdrugs is either during the whole period or only duringthe last 10 or 2 days. At the end the animals are sac-rificed under CO2 anesthesia. The glass rods are pre-pared together with the surrounding connective tissuewhich forms a tube around the glass rod. By incisionat one end the glass rod is extracted and the granu-loma sac inverted forming a plain piece of pure con-nective tissue. Wet weight of the granuloma tissue isrecorded. The specimens are kept in a humid cham-ber until further analysis. For measurement of the me-chanical properties the specimens are fixed into theclamps of the Instron(R) instrument allowing a gaugelength of 30 mm. The load until break is recordedwith a crosshead speed of 50 mm/min. In order to cal-culate tensile strength (N/mm2), the value of load atrupture (N) is divided by cross sectional area (mea-sured as volume = wet weight divided by length). Fi-nally, the granuloma tissue is dried and the dry weightis recorded. In addition, biochemical analyses, suchas determination of collagen and glycosaminoglycans,can be performed.

EVALUATIONSeveral parameters can be determined by this method.Granuloma weight was reduced by corticosteroidsdepending on dose and time of administration andwas also diminished after treatment with nons-teroidal anti-inflammatory agents and lathyrogeniccompounds. Furthermore, antiproliferative terpenoidsreduced the granuloma weight. The mechanical pa-

rameters showed different results after these drugs in-dicating a different mode of action. Treatment withcorticosteroids increased tensile strength. Only afterlong term treatment with toxic doses a decrease wasfound. Anti-inflammatory compounds, such as acetyl-salicylic acid or indomethacin and antiproliferative ter-penoids showed an increase of strength at medium andhigh doses.

CRITICAL ASSESSMENT OF THE METHODIn contrast to most other granuloma methods, the glassrod granuloma measures the late proliferative phase ofinflammation. Since the newly formed connective tis-sue is not contaminated with the irritant biochemicalanalyses can be performed. The peculiar feature is thepossibility to study the mechanical properties of newlyformed proliferative connective tissue.

REFERENCES AND FURTHER READINGVogel HG (1970) Das Glasstabgranulom, eine Methode zur Un-

tersuchung der Wirkung von Corticosteroiden auf Gewicht,Festigkeit und chemische Zusammensetzung des Gran-ulationsgewebes an Ratten. Arzneim Forsch/Drug Res20:1911–1918

Vogel HG (1975) Collagen and mechanical strength in variousorgans of rats treated with d-penicillamine or amino-aceto-nitrile. Conn Tiss Res 3:237–244

Vogel HG (1977) Mechanical and chemical properties of con-nective tissue organs in rats as influenced by non-steroidalanti-rheumatic drugs. Conn Tiss Res 5:91–95

Vogel HG, De Souza NJ, D’s A (1990) Effect of terpenoids iso-lated from Centella asiatica on granuloma tissue. Acta ther-apeut 16:285–298

H.3.3Side Effects of Anti-inflammatory Compounds

See Vogel (2006).

REFERENCES AND FURTHER READINGVogel SM (2006) Safety Pharmacology of Antiinflammatory

Drugs. In: Vogel HG, Hock FJ, Maas J, Mayer D (eds)Drug Discovery and Evaluation – Safety and Pharmacoki-netic Assays, Chapter I.K. Springer-Verlag, Berlin Heidel-berg New York

H.4Antipyretic Activity


Treatment with antipyretics has been very importantin the pre-antibiotic era. Nevertheless, for treatment ofacute viral diseases and for treatment of protozoal in-fections like malaria reduction of elevated body tem-perature by antipyretics is still necessary. For anti-


inflammatory compounds, an antipyretic activity is re-garded as a positive side effect. To evaluate these prop-erties, fever is induced in rabbits or rats by injection oflipopolysaccharides or Brewer’s yeast.

H.4.0.2Antipyretic Testing in Rats

PURPOSE AND RATIONALEThe subcutaneous injection of Brewer’s yeast suspen-sion is known to produce fever in rats. A decrease intemperature can be achieved by administration of com-pounds with antipyretic activity.

PROCEDUREA 15% suspension of Brewer’s yeast in 0.9% salineis prepared. Groups of 6 male or female Wistar ratswith a body weight of 150 g are used. By insertionof a thermocouple to a depth of 2 cm into the rectumthe initial rectal temperatures are recorded. The ani-mals are fevered by injection of 10 ml/kg of Brewer’syeast suspension subcutaneously in the back below thenape of the neck. The site of injection is massaged inorder to spread the suspension beneath the skin. Theroom temperature is kept at 22–24°C. Immediately af-ter yeast administration, food is withdrawn. 18 h postchallenge, the rise in rectal temperature is recorded.The measurement is repeated after 30 min. Only ani-mals with a body temperature of at least 38°C are takeninto the test. The animals receive the test compound orthe standard drug by oral administration. Rectal tem-peratures are recorded again 30, 60, 120 and 180 minpost dosing.

EVALUATIONThe differences between the actual values and thestarting values are registered for each time interval.The maximum reduction in rectal temperature in com-parison to the control group is calculated. The re-sults are compared with the effect of standard drugs,e. g. aminophenazone 100 mg/kg p.o. or phenacetin100 mg/kg p.o.

CRITICAL ASSESSMENT OF THE METHODThe antipyresis test in rats in can be regarded as a clas-sical method in pharmacology.

MODIFICATIONS OF THE METHODStitt and Shimada (1991), Shimada et al. (1994) in-duced fever in rats by microinjecting 20 ng PGE1 di-rectly into one of the brain’s circumventricular organs

of the rat known as the organum vasculosum laminaeterminalis.

Luheshi et al. (1996) induced fever by intraperi-toneal injection of 100 µ g/kg lipopolysaccharide intorats and measured the inhibition of fever by inter-leukin-1 receptor antagonist.

Telemetry has been used to record body tempera-ture in animals (Riley et al. 1978; Gallaher et al. 1985;Clement et al. 1989; Guillet et al. 1990; Kluger et al.1990; Bejanian 1991; Watkinson et al. 1996; Milleret al. 1997).

REFERENCES AND FURTHER READINGBejanian M, Jones BL, Syapin PJ, Finn DA, Alkana RJ

(1991) Brain temperature and ethanol sensitivity inmice: A radiotelemetric study. Pharmacol Biochem Behav39:457–463

Brune K, Alpermann H (1983) Non-acidic pyrazoles: inhibitionof prostaglandin production, carrageenan oedema and yeastfever. Agents Actions 13:360–363

Burn JH, Finney DJ, Goodwin LG (1950) Chapter XIV: An-tipyretics and analgesics. In: Biological Standardisation,Oxford University Press, London, New York, pp 312–319

Clement JG, Mills P, Brockway B (1989) Use of telemetry torecord body temperature and activity in mice. J PharmacolMeth 21:129–140

Gallaher EJ, Egner DA, Swen J (1985) Automated re-mote temperature measurement in small animals usinga telemetry/microcomputer interface. Comput Biol Med15:103–110

Guillet MC, Molinié B, Laduron PM, Terlain B (1990) Effects ofketoprofen in adjuvant-induced arthritis measured in a newtelemetric model test. Eur J Pharmacol 183:2266–2267

Inoue K, Fujisawa H, Sasaki Y, Nishimura T, Nishimura I, In-oue Y, Yokota M, Masuda T, Ueda F, Shibata Y, Kimura K,Inoue K, Komiya Y, Nishioka J (1991) Pharmacologicalproperties of the new non-steroidal anti-inflammatory agentEtodolac. Arzneim Forsch/Drug Res 41:228–235

Kluger MJ, Carole AC, Franklin B, Freter R, Abrams BD (1990)Effect of gastrointestinal flora on body temperature of ratsand mice. Am J Physiol 258:R552–R557

Loux JJ, DePalma PD, Yankell SL (1972) Antipyretic testingof aspirin in rats. Toxicol Appl Pharmacol 22:672–675Ri-ley JL, Thursten JR, Egemo CL, Elliot HL (1978) A ra-diotelemetry transmitter for transmitting temperatures fromsmall animals. J Appl Physiol 45:1016–1018

Luheshi G, Miller AJ, Brouwer S, Dascombe MJ, RothwellNJ, Hopkins SJ (1996) Interleukin-1 receptor antagonistinhibits endotoxin fever and systemic interleukin-6 induc-tion in the rat. Am J Physiol, Endocrinol Metab 270/133–1:E91–E95

Miller AJ, Hopkins SJ, Luheshi GN (1997) Sites of action of IL-1 in the development of fever and cytokine response to tis-sue inflammation in the rat. Br J Pharmacol 120:1274–1279

Roszkowski AP, Rooks WH, Tomolonis AJ, Miller LM(1971) Anti-inflammatory and analgesic properties of d-2-(6′-methoxy-2′-naphthyl)-propionic acid (NAPROXEN).J Pharmacol Exper Ther 179:114–123

Shimada SG, Otterness IG, Stitt JT (1994) A study of the mech-anism of action of the mild analgesic dipyrone. Agents Ac-tions 41:188–192

Smith PK, Hambourger WE (1935) The ratio of the toxicity ofacetanilide to its antipyretic activity in rats. J PharmacolExp Ther 54:346–351

H.4 · Antipyretic Activity 1115

Stitt JT, Shimada SG (1991) Calcium channel blockers inhibitendogenous pyrogen fever in rats and rabbits. J Appl Phys-iol 71:951–955

Tanaka K, Shimotori T, Makino S, Aikawa Y, Inaba T,Yoshida C, Takano S (1992) Pharmacological studiesof the new anti-inflammatory agent 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one.1st Communication: anti-inflammatory, analgesic andother related properties. Arzneim Forsch/Drug Res42:935–944

Watkinson WP, Highfill JW, Slade R, Hatch GE (1996) Ozonetoxicity in the mouse: comparison and modeling of re-sponses in susceptible and resistant strains. J Appl Physiol80:2134–2142

H.4.0.3Antipyretic Testing in Rabbits

PURPOSE AND RATIONALELipopolysaccharides from Gram-negative bacteria,e. g. E. coli, induce fever in rabbits after intravenousinjection. Only lipopolysaccharide fractions are suit-able, which cause after 60 min an increase of bodytemperature of 1°C or more at a dose between 0.1 and0.2 µg/kg. In the rabbit, two maxima of temperature in-creases are observed. The first maximum occurs after70 min, the second after 3 h.

PROCEDURERabbits of both sexes and of various strains witha body weight between 3 and 5 kg can be used. The an-imals are placed into suitable cages and thermocouplesconnected with an automatic recorder are introducedinto the rectum. The animals are allowed to adapt tothe cages for 60 min. Then 0.2 ml/kg containing 0.2 µglipopolysaccharide are injected intravenously into therabbit ear. Sixty min later the test compound is admin-istered either subcutaneously or orally. Body tempera-ture is monitored for at least 3 h.

EVALUATIONA decrease of body temperature for at least 0.5°C formore than 30 min as compared with the temperaturevalue before administration of the test compound is re-garded as positive effect. This result has been foundafter 45 mg/kg phenylbutazone s.c. or 2.5 mg/kg in-domethacin s.c.

CRITICAL ASSESSMENT OF THE METHODMeasurement of body temperature in rabbits withpolysaccharide induced fever is a more sensitive testthan the yeast fever in rats. Furthermore, the methodis used as a decisive test for the absence of pyrogensin parenteral drugs by several pharmacopoeias such asUSP 23 (1955).

MODIFICATIONS OF THE METHODCashin and Heading (1968) described a simple and re-liable assay for antipyretic drugs in mice, using intrac-erebral injection of pyrogens.

Davidson et al. (1991) tested the effect of humanrecombinant lipocortin on the pyrogenic action of thesynthetic polyribonucleotide polyinosini:polycytidylicacid in rabbits.

Yeast-induced pyrexia in rats has been used forantipyretic efficacy testing by Loux et al. (1982) andCashin et al. (1977).

van Miert et al. (1977) studied the effects of an-tipyretic agents on fever and ruminal stasis induced byendotoxins in conscious goats.

Petrova et al. (1978) used turpentine-induced feverin rabbits to study antipyretic effects of dipyrone andacetylsalicylic acid.

Lee et al. (1985) studied the antipyretic effect ofdipyrone on endotoxin fever of macaque monkeys.

Loza Garcia et al. (1993) studied the potentiationof chlorpromazine-induced hypothermia by the an-tipyretic drug dipyrone in anesthetized rats.

Shimada et al. (1994) studied the mechanism of ac-tion of the mild analgesic dipyrone preventing fever in-duced by injection of prostaglandin E1 or interleukin-1β into the organum vasculosum terminalis of ratbrain.

REFERENCES AND FURTHER READINGCashin CH, Heading CE (1968) The assay for anti-pyretic drugs

in mice, using intracerebral injection of pyretogenins. Br JPharmacol 34:148–158

Cashin CH, Dawson W, Kitchen EA (1977) The pharma-cology of benoxaprofen (2-[4-chlorophenyl]-α-methyl-5-benzoxazole acetic acid), LRCL 3794, a new com-pound with anti-inflammatory activity apparently unrelatedto prostaglandin synthesis. J Pharm Pharmacol 29:330–336

Davidson J, Flower RJ, Milton AS, Peers SH, Rotondo D (1991)Antipyretic actions of human recombinant lipocortin-1. BrJ Pharmacol 102:7–9

Deeter LB, Martin LW, Lipton JM (1989) Antipyretic effect ofcentral α-MSH summates with that of acetaminophen oribuprofen. Brain Res Bull 23:573–575

Lee TF, Mora F, Myers RD (1985) Effect of intracerebroventric-ular vasopressin on body temperature and endotoxin feverof macaque monkey. Am J Physiol 248:R674–R678

Loza Garcia MI, Baamonde Arbaiza A, Hidalgo Balsera A, An-dres-Trelles F (1993) Potenciación por dipirona (metami-zol) magnésica y dipirona sódica de la hipotermia pro-ducida por chlorpromazina en rata anestesiada. An RealAcad Farm 59:181–190

Matuszek M, Szreder Z, Korolkiewicz Z (1990) The antipyreticeffect of some newer alpha-1 antagonists. Eur J Pharmacol183:2279–2280

Petrova L, Nikolova M, Nikolov R, Stefanova D (1978) Dipy-rone and acetylsalicylic acid comparative pharmacologi-cal research. Antipyretic, anti-inflammatory and analgesic


action. In: Ovtcharov R, Pola W (eds) Proceedings Dipy-rone. Moscow Symposium, Schattauer-Verlag, StuttgartNew York, pp 99–107

Shimada SG, Otterness IG, Stitt JT (1994) A study of the mech-anism of action of the mild analgesic dipyrone. Agents Ac-tions 41:188–192

Szeder Z (1990) Comparison of the effect of prazosin with thatof dihydrobenzperidol and nifedipine on thermoregulatoryresponses produced by pyrogen in rabbits. Gen Pharmacol21:833–838

Szreder Z, Korolkiewicz Z (1991) Inhibition of pyrogen Es-cherichia coli fever with intracerebral administration ofprazosin, dihydrobenzperidol and nifedipin in the rabbits.Gen Pharmacol 22:381–388

USP 23 (1995) Pyrogen test. The United States Pharmacopeia23, p 1718

van Miert ASJPAM, van Essen JA, Tromp GA (1972) The an-tipyretic effect of pyrazolone derivates and salicylates onfever induced with leukocytic or bacterial pyrogen. Archint Pharmacodyn 197:388–391

van Miert ASJPAM, van der Wal-Komproe, van Duin CTM(1977) Effects of antipyretic agents on fever and ruminalstasis induced by endotoxins in conscious goats. Arch IntPharmacodyn 225:39–50

Zimecki M, Schnaper HW, Wieczorek Z, Webb DR, Pierce CW(1990) Inhibition of interleukin 1 (IL-1)-elicited leukocy-tosis and LPS-induced fever by soluble immune responsesuppressor (SIRS). Immunopharmacol 19:39–46

drug discovery and evaluation || analgesic, anti-inflammatory, and anti-pyretic activity

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