MECHANISMS OF INTERACTION BETWEEN ANTICHOLINESTERASES

AND OPIOIDS UPON RODENT NOCICEPTION

by

Paul GREEN, BSc (Hons)., MSc

A thesis subm itted in accordance w ith the requirements o f the University o f Surrey fo r the degree of

Doctor o f Philosophy

Department of B iochemistry University o f Surrey Guildford, Surrey

November, 1985

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ACKNOWLEDGEMENTS

I am very g ra te fu l to my supervisor, Dr Ian K itchen, who has given invaluable

advice and encouragement throughout my doctoral studies.

I would like to thank my mum and dad, brothers and Julie fo r a ll th e ir help

and support.

I would also like to thank Mr Paul Bishop (Biochem istry Workshop) fo r the

skilled construction o f locom otor a c tiv ity m onitor and the hot-p la te , D r John

Shorter (Beecham Pharmaceuticals) fo r the locomotor a c tiv ity m onitor

software, and Dr M artin Crowder (Mathematics Department) fo r the

s ta tis tica l models used in the analysis o f the antinociceptive data.

This work has been carried out w ith the support o f the Procurem ent

Executive, M in is try o f Defence.

I am gra te fu l to Miss Tracey Bakall fo r her excellent typing o f th is thesis.

SUMMARY

The lite ra tu re describing the role o f the cholinergic system and opioids on

antinociception is reviewed. The e ffec ts o f anticholinesterase agents and drugs

used in the treatm ent o f anticholinesterase poisoning were studied fo r the ir

e ffects on opioid antinociception and locomotion. In both mice and rats, the

irreversib le anticholinesterase, DFP, produced apparent antinociception, but only

at doses which caused m otor incapacita tion. P re-trea tm ent w ith DFP

potentiated the antinociceptive potency of the opioid drug a lfen tan il but had

li t t le e ffe c t on morphine or fentanyl using the hot-p la te test in m ice. The

reversible anticholinesterases, neostigmine and pyridostigm ine, did not a ffe c t

a lfen tan il antinociception in this species. In rats, DFP potentiated a lfen tan il and

fentanyl antinociception using the paw pressure test, but had no e ffe c t on

morphine. In m ice, drugs used to trea t anticholinesterase poisoning (atropine,

pralidoxime and diazepam) did not a lte r the e ffe c t o f DFP on a lfen tan il

antinociception. DFP produced a decrease in locom otor a c tiv ity in m ice which

was reversed fo llow ing atropine and pralidoxime adm in istration. DFP decreased

the hyperlocomotory a c tiv ity o f a lfen tan il in m ice, probably by increasing

a lfen tan il catatonia, but had l i t t le e ffe c t on morphine or fentanyl. When

diazepam was administered as part o f the trea tm ent fo r anticholinesterase

poisoning, locomotor scores o f opioid treated mice were markedly reduced. In

mice radiolabelled d istribu tion studies showed that DFP trea tm ent enhanced the

entry of a lfen tan il into a ll brain regions, w hilst morphine and fen tanyl levels

were unaltered. Studies on plasma protein binding o f a lfen tan il showed this was,

in part, due to a displacement o f a lfen tan il bound to plasma proteins by DFP,

enabling free drug to enter the brain. These studies suggest tha t fentanyl would

least like ly produce in teraction in individuals poisoned by irrevers ib le

anticholinesterase agents. In addition, the new opioid drug, a lfen tan il, may be

liable to interactions when used clin icall^w ith other drugs by displacement from

plasma protein binding sites to a lte r its antinociceptive a c tiv ity .

CONTENTS

ACKNOWLEDGEMENTS . i

SUMMARY ii

LIST OF TABLES AND FIGURES x

PUBLICATIONS x iii

CHAPTER 1 - INTRODUCTION 1

1.1. General In troduction. 2

1.2. Choice of Drugs Used. 2

1.3. Uses o f Anticholinesterases. 4

1.3.1. Reversible Anticholinesterases: 4

C lin ica l Uses.

1.3.2. Reversible Anticholinesterases: 3

N on-clin ica l Uses.

1.3.3. Irreversib le Anticholinesterases: 5

C lin ica l Uses.

1.3.4. Irreversible Anticholinesterases: 6

N on-clin ica l Uses.

1.3.3. Anticholinesterases as Chemical 6

W arfare Agents.

1.4. Aim of the Study. 8

1.5. Term inology. 8

1.6. A Review o f the L ite ra tu re on the Role 11

of the Cholinergic System and Opioids

on Antinociception.

1.6.1. Introduction. 11

1.6.2. Nociceptive Models. 11

1.6.3. Anticholinsterases and Antinociception. 14

(a) Neostigmine 14

(b) Physostigmine 15

1.6.4. Irreversible Anticholinesterases 16

1.6.5. Other Anticholinesterase Agents. 17

1.6.6. Cholinom im etics and Antinociception. 19

(a) Muscarinic Agonists 19

(b) N ico tin ic Agonists 21

(c) Choline Esters 22

1.6.7. Cholinergic Antagonists and Antinociception 26

1.6.8. Miscellaneous Compounds. 29

1.6.9. Acetylcholine Histopharmacology and 30

Opioids

(a) Role o f Acetylcholine in Nociceptive 30

Pathways.

(b) Acetylcholine and Acupuncture- 32

Induced Antinociception

(c) Acetylcholine-O pioid Interaction 33

in Non-Nociceptive Systems.

1.6.10. Conclusions. 35

CHAPTER 2 - ANTINOCICEPTIVE ACTIVITY OF MORPHINE, 61

FENTANYL AND ALFENTANIL IN M ICE AND

RATS: EFFECTS OF DFP AND DRUGS USED

IN THE TREATMENT OF DFP POISONING

INTRODUCTION 62

2.1. An tinociceptive Models. 62

2.2. In it ia l Experiments. 64

MATERIALS AND METHODS 65

2.3. Animals and Experimental Conditions. 65

2.4. Drugs. 66

2.5. Safety Procedures for DFP. 66

2.6. Dosing Protocols in Mice. 67

2.7. Dosing Protocols in Rats. 69

2.8. Antinociceptive Testing in Mice. 71

2.9. Antinociceptive Testing in Rats. 71

2.10. S ta tis tica l Procedures. 73

2.10.1. H ot-P la te Data. 73

2.10.2. Paw Pressure Data. 74

RESULTS 73

2.11. P ilo t Studies. 73

2.12. E ffe c t of DFP on Nociception in M ice. 75

2.13. E ffe c t o f DFP and Drugs Reversing the 81

E ffects o f DFP on Opioid Antinociception

in Mice.

2.14. E ffe c t o f Atropine and Pralidoxime and 83

Diazepam on Nociception.

2.15. E ffe c t o f Neostigmine and Pyridostigm ine 83

on Opioid Antinociception.

2.16. Antagonism of A lfen tan il, Alone and W ith 88

DFP, on Naloxone.

2.17. E ffe c t of DFP Upon Nociceptive Responses 88

in the Rat.

2.18. E ffe c t of DFP Pre-Treatm ent on 89

Opioid Antinociception in Rats.

DISCUSSION 94

2.19. Drug Injection Times. 94

2.20. E ffe c t of DFP on Nociceptive Responses 94

in Mice and Rats.

2.21. E ffe c t of DFP on Morphine Antinociception. 96

2.22. E ffe c t o f DFP in Fentanyl and A lfe n ta n il 97

Antinociception.

- 2.23. Proposed Mechanisms fo r DFP Potentia tion 98

of A lfen tan il.

2.23.1. Absorption 98

2.23.2. D istribution 98

2.23.3. Receptor Action 99

2.23.4. Metabolism 99

2.23.3. Excretion 100

2.24. E ffe c t o f Atropine and Pralidoxime 100

on DFP Potentiation o f A lfe n ta n il Antinociception.

2.25. E ffe c t o f Atropine and Pralidoxime 101

on the Antinociceptive A c tiv ity o f Morphine.

2.26. E ffe c t o f Diazepam on the Antinociceptive 102

A c tiv ity o f Opioids in Mice Receiving DFP,

Atropine and Pralidoxime.

2.27. Variation in Antinociceptive Potency o f 102

A lfe n ta n il in Mice.

CHAPTER 3 - LOCOMOTOR ACTIVITY OF MICE: EFFECT OF DFP 104

AND DRUGS USED IN THE TREATMENT OF DFP

POISONING

INTRODUCTION 105

3.1. Assessment o f Drug E ffe c t on Spontaneous Locomotor 105

A c tiv ity .

MATERIALS AND METHODS 106

3.2. Animal and Experimental Conditions. 106

3.3. Drugs. 106

3.4. Dosing Protocols. 106

3.5. Locomotor A c tiv ity Testing. 106

3.6. S ta tis tica l Procedures. 107

3.7. E ffe c t of DFP on Locom otor A c tiv ity . 108

3.8. E ffe c t of Atropine and Pralidoxime on Locomotor 108

A c tiv ity .

3.9. E ffe c t o f Diazepam on Locomotor A c tiv ity . 108

3.10. E ffe c t o f Various Treatments on A lfe n ta n il- 108

Induced Hyperlocomotion.

3.11. E ffe c t of Various Treatments on Fentanyl- 112

Induced Hyperlocomotion.

3.12. E ffe c t o f Various Treatments on 112

Morphine-Induced Hyperlocomotion.

DISCUSSION 115

3.13. E ffe c t o f DFP on Locomotor A c tiv ity . 115

3.14. E ffe c t o f Atropine and Pralidoxime Alone and 116

W ith DFP on Locomotor A c tiv ity .

3.15. E ffe c t o f Diazepam Alone and W ith DFP on 116

Locomotor A c tiv ity .

3.16. E ffects o f DFP on Opioid-Induced Hyperlocom otion. 116

3.17. The E ffe c t o f Atropine and Pralidoxime on the 118

Locomotor A c t iv ity o f DFP and Opioid-Treated M ice.

3.18. E ffe c t o f Diazepam on Opioid-Induced Locomotion. 118

3.19. In te rpre ta tion o f Locomotor Data. 119

CHAPTER 4 - DISTRIBUTION OF 3H MORPHINE, FENTANYL 120

AND ALFENTANIL IN MOUSE BRAIN: EFFECT

OF DFP ADMINISTRATION

INTRODUCTION 121

4.1. D istribution Studies. 121

MATERIALS AND METHODS 122

4.2. Animals and Experimental Conditions. 122

4.3. Drugs. 122

4.4. Dosing Protocols. 123

4.5. D istribution Studies Protocol. 123

4.6. S c in tilla tion Counting. 123

RESULTS 125

4.7. E ffe c t o f DFP Pre-Treatm ent on A lfen tan il Brain 125

and Plasma Levels.

4.8. E ffe c t o f DFP Pre-Treatm ent on Morphine and 125

Fentanyl Brain and Plasma Levels.

DISCUSSION 131

4.9. Opioid L ipoph ilic ity and Access to the CNS. 131

4.10. E ffe c t o f DFP on Opioid Brain Levels. 131

4.11. Mechanisms of Increased Levels o f A lfen tan il 132

Produced by DFP.

CHAPTER 5 - IN VIVO AND IN VITRO PLASMA PROTEIN BINDING 135

OF 3H ALFENTANIL AND 3 H FENTANYL: EFFECT

OF DFP ADMINISTRATION

INTRODUCTION 136

5.1. Plasma Protein Bind o f Drugs. 136

5.2. Plasma Protein Binding o f A lfen tan il, Fentanyl and 136

DFP.

5.3. Assessment o f Drug Binding to Plasma Proteins. 137

MATERIALS AND METHODS 140

5.4. Anim al and Experimental Conditions. 140

5.5. Drugs. 140

5.6. In Vivo Protein Binding Protocol. 140

5.7. In V itro Protein Binding Protocol. 140

5.8. U ltracen trifuga tion Protocol. 141

5.9. Calculation o f Free and Bound Drug 141

Concentration.

5.10. Determ ination o f Drug Adsorption in 141

U itracen trifuga tion Apparatus.

5.11. Recovery Experiments. 144

5.12. Percentage o f Plasma Protein Binding of 144

Fentanyl and A lfen tan il.

5.13. E ffe c t o f DFP on Binding Fentanyl and 144

A lfe n ta n il to Plasma Proteins.

DISCUSSION 147

5.14. DFP Binding to Plasma Proteins. 147

5.15. Plasma Protein Binding o f A lfen tan il and 147

Fentanyl.

5.16. E ffe c t o f DFP on Plasma Protein Binding of 149

A lfen tan il.

5.17. Consequences o f A lfen tan il Displacement from 151

Plasma Protein Binding Sites.

5.18. Protein Binding and Relationship to Variation 152

in A lfen tan il Potency.

CHAPTER 6 - GENERAL DISCUSSION 153

REFERENCES 156

APPENDIX A 185

APPENDIX B 186

APPENDIX C 189

APPENDIX D (i) 190

APPENDIX D (ii) 191

LIST OF TABLES AND FIGURES

Fig. 1.1.

F ig .1.2.

Table 1.1.

Table 1.2.

Table 1.3.

Table 1.4.

F ig. 2.1.

F ig. 2.2.

Fig. 2.3.

Table 2.1.

Table 2.2.

Table 2.3.

Table 2.4.

Table 2.5.

Table 2.6.

Three Dimensional Structures and Formulae 3

of Morphine, Fentanyl and A lfen tan il.

B ritish troops w ill go to war on valium . 9

Methods o f Nociceptive Assessment. 12

Anticholinesterases and Antinociception. 37

Cholinom im etics and An tinociception. 46

Cholinergic Antagonists and Antinociception. 57

Dosing Protocols fo r A n tinociceptive Testing 68

in M ice.

Dosing and Testing Protocols in Rats 70

Use of the Ugo Basile Analgesy M eter. 72

E ffe c t of Repeated Exposure to Antinociceptive 76

Testing Procedures on H ot-P la te Times of

Naive Mice.

E ffe c t of M u ltip le Saline Injections on H ot- 77

Plate Response Times in M ice.

E ffe c t o f DFP on H ot-P la te Reaction Times 78

in the Mouse.

E ffe c t of D iffe re n t Vehicles on the Antinociceptive 79

A c t iv ity o f DFP.

E ffe c t of A tropine and Naloxone on 2mg/kg 80

DFP Induced Antinociception in M ice.

E ffe c t o f DFP (Img/kg) and o f Drugs Reversing 82

DFP Poisoning on Opioid-Induced Antinociception

in M ice.

Table 2.7.

Table 2.8.

Table 2.9.

Table 2.10

Fig. 2.3.

Fig 2.4.

Fig 2.5.

Fig 2.6.

F ig. 3.1.

Fig. 3.2.

F ig. 3.3.

Fig. 3.4.

F ig. 3.5.

E ffe c t o f Intram uscularly-Adm inistered

Diazepam and In jection Vehicle on

H ot-P la te Reaction Times in Mice.

E ffe c t of Various Doses of Neostigmine

on the Antinociceptive A c tiv ity o f A lfen tan il.

E ffe c t of Pyridostigm ine (lm g/kg) on

A n tinociceptive Potencies o f Morphine, Fentanyl

and A lfe n ta n il.

Naloxone Antagonism of Antinociception Induced

by A lfe n ta n il W ith and W ithout DFP.

Time Course fo r the E ffe c t o f DFP on the

Nociceptive Responses in the Rat Paw Pressure Test.

E ffe c t o f DFP on Morphine Antinociception in the

Rat Paw Pressure Test.

E ffe c t o f DFP on Fentanyl Antinociception in the

Rat Paw Pressure Test.

E ffe c t o f DFP on A lfen tan il Antinociception in the

Rat Paw Pressure Test.

Graph Showing the E ffe c t o f Various Drug

Treatments on Locomotor A c t iv ity in Mice.

Table Showing E ffe c t o f Various Drug Treatments

on the Locom otor A c t iv ity in Mice

E ffe c t o f DFP on A lfentanil-Induced Hyperlocom otion

Over 15 and 75 Minutes.

E ffe c t o f DFP on Fentanyl-Induced Hyperlocom otion

Over 15 and 75 Minutes.

E ffe c t o f DFP on Morphine-Induced Hyperlocom otion

Over 15 and 75 Minutes.

Fig. 4.1.

F ig. 4.2.

Fig. 4.3.

Table 4.1.

Table 4.2.

Fig. 4.4.

Fig. 5.1.

Fig. 5.2.

Table 5.1.

Table 5.2.

Fig. 5.3.

E ffe c t of DFP on Brain Levels o f (dpm g~^)

A lfen tan il

E ffe c t o f DFP on Brain Levels o f (dpm g~^)

Fentanyl.

E ffe c t of DFP on Brain Levels o f (dpm g~^)

Morphine.

E ffe c t o f DFP (lm g/kg” '*') on Plasma Levels o f

Morphine, Fentanyl and A lfe n ta n il.

E ffe c t o f DFP (Im g/kg- ^) on Brain Plasma Ratios fo r

Morphine, Fentanyl and A lfen tan il.

Scheme fo r Explanation o f Observed Increased

Brain A lfe n ta n il Levels o f DFP (lmg/kg)

Adm in istra tion.

Protocol fo r Plasma Protein Binding Experiments

In Vivo and In V itro .

The Amicon M icropartition MPS-1 System.

E ffe c t o f DFP Treatm ent on In Vivo and In V itro

Plasma Levels o f A lfen tan il.

E ffe c t o f DFP Treatm ent on Levels of In Vivo and

In V itro Free Plasma Levels o f A lfe n ta n il and

Fentanyl.

DFP Phosphorylation o f Protein by an A lky l

Phosphorylation Reaction.

126

127

128

129

130

133

139

143

145

146

148

APPENDIX A 185

APPENDIX B 186

APPENDIX C 189

APPENDIX D(i) 190

APPENDIX D (ii) 191

PUBLICATIONS

Publications resulting from work in this thesis are listed below:

KITCHEN, I. and GREEN, P.G. (1983)

D iffe re n tia l e ffec ts o f diisopropylfluorophosphate poisoning and its

trea tm ent on opioid antinociception in the mouse.

L ife Sciences 33: 669-679.

GREEN, P.G. and KITCHEN, I. (1984)

Diisopropylfluorophosphate enhances entry o f a lfen tan il in to mouse brain.

B ritish Journal o f Pharmacology. 82: 297P.

GREEN, P.G. and KITCHEN, I. (1985)

D iffe re n t e ffec ts o f diisopropylfluorophosphate on the entry o f opioids in to

mouse brain.

B ritish Journal o f Pharmacology. 84: 657-661.

GREEN, P.G. and KITCHEN, I. (1985)

Displacement of ^H a lfen tan il from plasma protein binding sites by

diisopropylfluorophosphate in the mouse B ritish Pharmacological Society

Communication, September 1985. (in press).

GREEN, P.G. and KITCHEN, I. (1986)

Antinociception, opioids and the cholinerg ic system.

Progress in Neurobiology (in press)

CHAPTER 1

INTRODUCTION

1.1 General In troduction

Opioids have been used by humans fo r many thousands o f years. Opium was

f irs t used around 4000 BC when i t was mixed w ith wine by the Sumerians. Its

popularity as a drug remained, and is re flec ted by Sydenham who, in 1680,

w rote 'among the remedies which i t has pleased A lm ighty God to give to man

to relieve his suffering, none is so universal and efficacious as opium1. The

main active constituent o f opium, morphine, is s t i l l used therapeu tica lly as

the standard analgesic against which new agents are compared.

Despite Sydenham's praise, the therapeutic use o f opioids have a number o f

drawbacks, the most im portant o f which are respiratory depression and a

dependence lia b ility . Even though there has been extensive research in the

development of novel opioids, these problems have not been com plete ly

overcome. Another approach has involved the investigation o f non-opioid

drugs which either possess inherent analgesic a c tiv ity or are able to

potentia te the desirable (analgesic) but not the undesirable action o f opioids.

Cholinergic drugs, such as anticholinesterase agents, have received a lo t o f

a ttention in this area and the ir in te raction w ith opioids and nociceptive

systems is considered in Section 1.6.

1.2 Choice o f Drugs Used

Three opioids were used in the studies. Morphine as the standard analgesic

agent, and two synthetic opioids, fentanyl and a lfen tan il. These la tte r two

are of a new generation of opioids which are very potent and are extrem ely

rapid ly acting. These factors make them suitable fo r use as intravenous (i.v .)

analgesics for use in surgery. The structure o f these opioids is given in

Figure 1.1.

FENTANYL, , h n , o i co-oh » i oh ■ o i c h . c o o n ;. . c f h . : *•

ALFENTANIL l?4h3?n?0;'CHr°-CH,N - C - CH - CH,

Fig. 1.1. Three-dimensional Structures and Formulae of Morphine, Fentanyl

and Alfentanil

Three anticholinesterases were used though the compound d i­

isopropylfluorophosphate (DFP) was investigated most thoroughly.

Anticholinesterases are considered fu rthe r in the next Section.

1.3. Uses of Anticholinesterases

Anticholinesterases inh ib it the cholinesterase enzyme which results in a

build-up of excessive acetylcholine. The therapeutic and tox ic e ffec ts o f

these compounds are due to the accumulation of acetylcholine a t muscarinic

and n ico tin ic cholinergic receptors (Karczmar, 1967).

Anticholinesterases are norm ally divided into two groups, reversible and

irreversib le , which correspond to carbamyl esters and organo-phosphorous

compounds respectively. However both classes o f drugs react w ith the

cholinesterase enzyme in the same way, and so this c lassification re fle c ts

only quantita tive differences. There are, in fac t, very few tru ly reversible

compounds and even neostigmine is not considered to be in this class (Usdin,

1970). Nevertheless, fo r the sake of c la r ity this classification w ill be used in

the fo llow ing discussion.

1.3.1. Reversible Anticholinesterases: C lin ica l Uses

Therapeutically, reversible anticholinesterases are indicated in certa in

neuromuscular abnormalities such as myasthenia gravis, where neuromuscular

transmission is to be enhanced (see B ritish National Form ulary, 1983).

Neostigmine, pyridostigm ine, ambenonium, physostigmine and distigm ine are

the anticholinesterases prescribed fo r myasthenia gravis, pyridostigm ine

reported to have the fewest side-effects.

These drugs are also used to trea t urinary re tention, and because they

increase in testina l m o tility , they may be used as stim ulant laxatives

pa rticu la rly post-operative ly in case o f para ly tic ileus.

Probably the most common use fo r this class o f drug is in surgery. Non-

depolarising muscle relaxants which are used as a premedication in surgery,

compete fo r the acetylcholine receptor at the neuromuscular junction. The ir

action may be reversed by increasing the synaptic concentration o f

acetylcholine w ith anticholinesterases. The usual drugs used to reverse post

operative muscle re laxation are neostigmine and pyridostigm ine.

Anticholinesterases are also used in the trea tm ent o f glaucoma and indeed

the f irs t therapeutic use fo r this class o f drug was when physostigmine was

used fo r glaucoma (Laquer, 1877). Physostigmine is s t il l used fo r this purpose.

Reversible anticholinesterases are also used in the trea tm en t o f

anticholinesterase poisoning (see Section 1.3.3.).

1.3.2. Reversible Anticholinesterases: N on-clin ica l Uses

The major non-clin ica l use fo r reversible anticholinesterases is as

insecticides. Since the ir development in the 1950's, compounds such as

carbamyl and propoxur have been used extensively fo r the ir highly selective

insectic idal action.

1.3.3. Irreversib le Anticholinesterases: C lin ica l Uses

The f irs t organophosphorous anticholinesterase agent was synthesised in the

1850's (De Clerm ont, 1854). The f ir s t therapeutic use, however, was only

made some hundred years la te r when DFP was used to tre a t glaucoma

(Quillam , 1947). DFP became the drug o f choice in the trea tm ent o f

glaucoma as i t had the advantage over the carbamate drugs in tha t i t was

very long acting and could therefore be applied in frequently. However, i t

was established some years la te r that continued trea tm ent w ith long acting

anticholinesterase agents carried a high risk o f a specific type o f ca ta ract

(Axelsson and Holmberg, 1966). C urrently in the UK the only

organophosphorous compound used therapeutica lly, fo r the trea tm ent o f

glaucoma, is ecjhiopiate.

1.3.4. Irreversible Anticholinesterases: N on-clin ica l Uses

As fo r reversible anticholinesterases, the irreversib le organophosphorous

agents are extensively used as agricu ltu ra l insecticides. The f irs t

organophosphorous compound synthesised, te trae thy l pyrophosphate (TEPP)

(De C lerm ont, 1854), has been used as an insecticide though in recent years,

more selective agents have been developed. One of the most w idely used

insecticides is parathion, and this compound also probably accounts fo r more

accidental poisonings and fa ta lit ie s than any other organophosphorous

compound (Koelle, 1985). Even more selective compounds were developed to

reduce the toxico logica l risk to humans. For example, malathion has a UD^g

mouse:LD^g housefly ra tio o f 68 compared to only 6 fo r parathion (Usdin,

1970).

1.3.5. Anticholinesterases as Chemical Warfare Agents

The potentia l fo r anticholinesterases as chemical warfare agents was realized

in it ia lly in Germany by Lange in 1935 (Karczmar, 1970). This was fo llowed by

the synthesis and investigation o f new compounds (tabun and sarin) developed

specifica lly fo r the ir to x ic ity . Though these compunds were synthesised on a

large scale, they were never actually deployed due to the fear of re ta llia tio n

by A llied forces using the ir own organophosphates.

Work on chem ical warfare agents continued in post-war years, though

research proceeded at a low level, and B rita in stopped its development o f

new compounds in the 1950's. However, the USA, USSR and France s t il l

manufacture these compounds so the development o f e ffe c tive antidotes is an

im portant area of reseach.

P rotection against organophosphorous poisoning may be afforded by the

adm in istration o f several agents. The prophylactic e ffe c t o f carbamates has

been known fo r some tim e (Koster, 1946). Research in subsequent years has

shown tha t many carbamates pro tect against poisoning e ffe c tive ly , and tha t

a c tiv ity is dependent on many factors such as absorption, d is tribu tion and

metabolism (Gordon et al, 1978). The mechanism fo r the pro tective action o f

carbamates is dependent p rim arily on the form ation o f a semi-stable

carbamylated acetylcholinesterase; the enzyme in th is state cannot be

phosphorylated by an organophosphate. The carbamylated enzyme can

spontaneously break down to release the active enzyme (Wilson e t al, 1960)

and as the organophosphate is broken down re la tive ly rapid ly, su ffic ien t

released enzyme is present to maintain life (Berry and Davis, 1970).

Further protection from organophosphate poisoning may be achieved by

pharmacologically antagonising the e ffects o f excess acetylcholine, by

adm inistering an anticholinergic such as atropine. In addition, the inh ib ited

cholinesterase enzymes may be reactivated by the adm in istra tion o f oxime

such as pralidoxime mesylate. This is achieved by accelerating the

spontaneous reactiva tion o f the inhib ited cholinesterase by prom oting

hydrolysis o f the inhib itor-enzym e complex (Wills, 1970). This additional

trea tm ent w ith an oxime enhances the pro tective potency of the carbamate

and anticholinergic regimen (Gordon et al, 1978).

The most recent addition to the ba tte ry o f drugs used to tre a t

organophosphate poisoning is diazepam. I t has been shown to greatly enhance

the pro tective a c tiv ity of atropine against physostigmine le th a lity in mice

(Klemm, 1983). I t is believed to have this e ffe c t by v irtue o f its a n ti­

convulsant a c tiv ity . I t does have the disadvantage, however, o f im pairing

m otor function and performance (Gall, 1981; Fig. 1.2.).

1.4. Aim of the Study

The in it ia l objective o f the research described in this thesis was to study

potentia l in teractions between irreversib le anticholinesterase agents and

opioid analgesics, in an a ttem pt to h ighlight drug in teraction problems which

m ight occur in individuals poisoned w ith anticholinesterase agents, trea ted

fo r such poisoning and given opioid drugs fo r the re lie f o f pain. This s ituation

is c learly most pertinent in a m ilita ry context, i f in the fie ld , personnel

exposed to organophosphorous compounds and receiving antidotes also sustain

in juries requiring surgery. In a broader sense, the aim of this thesis was to

study the nature and a ttem pt to analyse the mechanisms of in te rac tion

between opioids and anticholinesterase agents.

1.5. T erminoloqy

In this thesis, in describing the e ffects o f opioids on pain responses in

animals, the term 'antinociception1 is used, as d is tinc t from the word

'analgesia' used in the human context.

Analgesia is defined as 'absence of pain' and pain as 'suffering distress o f body

or mind' (Oxford English D ictionary). As suffering and distress are subjective

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phenomena, animals therefore cannot be described as perceiving pain per se.

However, they are able to sense and respond to noxious stim u li, so the terms

'nociception' and 'antinociception ' are used.

The term 'opioid' has been used throughout this thesis to describe analgesic

drugs. This is d is tinc t from the term 'opiate', the use o f which should be

confined to those products o f the opium poppy and re lated synthetic

alkaloids. 'Opioid' describes a ll drugs which are opiate-like in th e ir action

(see K itchen, 1984).

1.6. A Review of the Literature on the Role of the Cholinergic

System and Opioids on Antinociception

1.6.1 Introduction

The potentia ting e ffe c t o f anticholinesterase agents on the a c tiv ity o f opioid

analgesics has been known fo r several years (Flodmark and Wramner, 1945;

Slaughter, 1950) and there is now a large body o f lite ra tu re describing the

in te raction between cholinergic agents and opioids. This review summarizes

the present lite ra tu re and suggests mechanisms underlying in teractions.

Most o f the available lite ra tu re concerns work using animal models o f pain

perception though there are reports fo r human models and fo r c lin ica l pain,

and i t is these studies which indicate a potentia l therapeutic ro le fo r

cholinergic agents in enhancing the desirable analgesic e ffects o f opioids,

w h ilst reducing the undesirable components such as respiratory depression.

Furthermore, these studies have gone someway in elucidating the physiology

o f pain perception and have indicated possible fu tu re developments in the

pharmacology o f pain contro l. .

1.6.2. Nociceptive Models

The assessment o f the potency o f analgesic drugs is usually in it ia lly carried

out in experimental animals. A varie ty o f tests have been developed, using

several animal species to determine the antinociceptive a c tiv ity o f drugs and

a b rie f description of the tests described in this review is given in Table 1.1.

Even though a ll the models described are responsive to opioids, i t is

im portant to consider that differences in the nociceptive s tim u li (heat,

pressure, e lec trica l) may well involve d is tinc t pathways u tiliz ing d iffe re n t

neurotransm itters and that processing o f nociceptive in form ation may vary

TABLE 1.1. METHODS OF NOCICEPTIVE ASSESSMENT

Test Species Method of nociceptive stim ulation

End-point or quantifica tion

Reference

Hot plate RatMouse

Animals placed on heated m etal plate (48 to 55°C)

L ift in g , shaking or lick ing paw, or jumping

Woolfe andMacDonald,1944.

Tail f lic k RatMouseCat

Tail exposed to localized heat source e.g. focussed ligh t

F licking o f ta il away from heat

D'Amour and Smith,1941.

Tail immersion RatMouse

Term inal part ofta il immersed in water(50-55°C)

F lick ing or lif t in g ta il out o f water

Janssen et al., 1963.

Hot wire Rat Tail heated by hot w ire

F lick ing o f ta il away from heat

Davies et al., 1946.

Tail c lip RatMouseRabbitCat

Application of pressure by a c lip on base o f ta il

A ttem p t to b ite or remove clamp. Quantalresponse

H affner,1929.

W rithing Mouse Intraperitoneal in jection o f ir r ita n t substance (e.g. acetic acid, phenoxybenzo- quinone)

Number o f abdominal constrictions observed in set perioid

HendershotandForsaith,1939.

Form alin test RatCat

Subcutaneous in jection o f 5% form alin into forepaw pad

Scoring of in tensity and duration o f lif t in g , lick ing , shaking o f paw

O'Keefe,1964.

Paw pressure Rat Pressure applied to normal or inflamed hind paw. Pressure increasing w ith tim e

W ithdrawal o f foo t, struggling or vocalization

Randall andS e litto ,1957.

Ear clamp RabbitDog

Varying pressure applied by clip on ear

A ttem p t to w ithdraw or vocalization

Weinstock,1980.

Toe pinch Guineapig

CatPrim ate

Varying pressure applied by clip on toe

A ttem p t to w ithdraw or vocalization

C o llie r,1962.

Test Species Method of End-point or Referencenociceptive stim ulation quantification

Electroshock

Shocktitra t io n

Taile lec trica lstim ulation

Tooth pulp e lec trica l stim ulation

Radiant heat

Note:

Rat Application o f e lec tricMouse current, usually to

fee t

Rat E lec tric current toMouse feet w ith stepwisePrim ate increase in current

w ith tim e

Rat E lec tric current viaMouse electrodes in

base o f ta il

Rabbit E lec tric stim ulationto upper incisors

Many Part o f skin (maySpecies be blackened) exposed (eg. dog, to intense focussedhuman) ligh t or laser

Vocalization, Evans,vocalization 1961.a fte r dischargeor w ithdrawalo f paws

Time spent at Weiss andeach shock level Laties,before 1970.responding bypressing leverto reduce current

As fo r ta il Carol andclamp or L im ,electroshock 1960.

Threshold to H o ffm e is te rjaw opening or 1968.lick ing response

Varies w ith Hardyspecies: escape et al., 1940.response (Human(rodent), skin testing)tw itc h (dog),verbal report(human)

References re fe r to the f irs t report o f a particu la r method, or to a w idely used adaptation o f a method. There are o f course, numerous variations and refinem ents o f many of the tests given in this table.

between species. Modulation of nociception by some drugs may, therefore,

be test and species dependent. In addition some opioid receptor selective

agonists exhib it varying degrees o f antinociception depending on the test

used. For example, k-op io id agonists are more e ffec tive against pressure

than therm al s tim u li (Tyers, 1980).

1.6.3. Anticholinesterases and Antinociception

a) Neostigmine

The f irs t studies o f the antinociceptive a c tiv ity o f anticholinesterase in man

were carried out by Flodmark and Wramner (1945). They showed

neostigmine, a quaternary reversible anticholinesterase, had antinociceptive

a c tiv ity and also potentiated morphine antinociception. A s im ila r

potentia tion was observed fo r other opioids (Slaughter, 1950). In contrast to

man, animal studies have fa iled to show antinociceptive a c tiv ity o f

neostigmine (Saxena and Gupta, 1957). However, there are reports of

potentia tion by neostigmine o f morphine antinociception (Knoll and Komlos,

1952; Saxena and Gupta, 1957). These observations have not been confirm ed

by other workers except when neostigmine is administered i.c .v . (Pedigo e t

al., 1975). By this route the antinociception is antagonised by naloxone and

atropine but not mecamylamine indicating tha t both opioid and muscarinic

systems mediate the e ffe c t (Pedigo et al., 1975).

As the quaternary compound neostigmine does not readily cross the blood-

brain barrier i t exerts its action peripherally fo llow ing systemic in jection . I t

is possible that the potentia tion o f opioids may be due to im paired

metabolism or altered d istribution. The observed antinociceptive e ffe c t in

humans (Flodmark and Wramner, 1945) may be due to the test used (radiant

heat). As the authors suggest, neostigmine may increase cutaneous blood

flow and so dissipate the radiated heat more rapid ly. A lte rna tive ly , there

may be an e ffe c t on peripheral pain sensitive neurons.

b) Physostiqmine

Table II shows tha t physostigmine is reported to possess antinociceptive

a c tiv ity in most o f the tests described. Most o f the reports showing no

antinociceptive a c tiv ity involved centra l adm in istration o f physostigmine.

Since physostigmine readily crosses the blood-brain barrier, the lack o f e ffe c t

o f centra l adm in istra tion suggests tha t physostigmine may be m etabolized in

the periphery to the active compound. Indeed, FUrst et al. (1982) studied the

antinociceptive a c tiv ity o f eseroline, the prim ary m etabolite o f

physostigmine, and showed i t to be a naloxone reversible analgesic when

administered either peripherally or cen tra lly . The structure o f eseroline is

s im ilar to morphine and the antinociceptive e ffe c t appears independent o f its

anticholinesterase a c tiv ity .

Studies w ith atropine and hyoscine (Harris et al., 1969; Wong and Bentley,

1978; Dayton and G arret, 1973) and naloxone (Harris e t al., 1969; Romano

and King, 1981) indicate tha t physostigmine exerts its action through e ither

the cholinergic or opioid system or both. The a b ility o f naloxone to

antagonise physostigmine was not found in some studies (Pleuvrey and Tobias,

1971; Lipman and Spencer, 1980; Conzantis e t al., 1983) and has even been

shown to potentia te physostigmine (Romano and King, 1981). O ther evidence

suggesting tha t physostigmine’s e ffe c t may be independent of the opioid

system are that tolerance may be induced fo llow ing m ultip le adm inistrations

o f physostigmine, w hilst morphine antinociception is not a ffected by this

regimen (Dayton and G arret, 1973; L it t le and Rees, 1974). In studies in the

rhesus monkey, physostigmine appears to increase morphine antinociception

(Pert, 1975). However, this e ffe c t is observed only at doses tha t also

produced general behavioural depression.

The antinociceptive e ffects of physostigmine may be inhib ited by

pre trea tm ent w ith compounds depleting centra l 5-HT (Bhattacharya and

Nayak, 1978). 5-HT has been im plicated in antinociception (Messing and

Ly tle , 1977), and may be part o f a pathway also involving cholinergic neurons.

I t is worth considering tha t the neurotoxins used to deplete 5-HT do not have

absolute spec ific ity and may w e ll disrupt other systems.

Widman et al. (1978) described the e ffe c t o f cen tra lly administered calciumry

chloride on physostigmine antinociception. They observed tha t Ca + ionsn

reduced antinociception, and conclude that Ca + appeared to exert its

e ffec ts intraneuronally and postsynaptically. D ivalent ions are also involved

in acetylcholine induced antinociception (Section 1.6.6.(c)) and are also

discussed in Section 1.6.8.

1.6.4 Irreversible Anticholinesterases

Diisopropylfluorophosphate (DFP) has been investigated by several workers.

In studies w ith m ice, DFP was not found to be antinociceptive by one group

up to 2 mg/kg (Cox and Tha, 1972). Morphine and fentanyl antinociception

appear unaffected by DFP, but a lfen tam il antinociception is potentia ted

(K itchen and Green, 1983). Bhargava and Way (1972), however, did conclude

that DFP does potentia te morphine antinociception, though the e ffe c t was

not s ta tis tica lly s ign ificant.

The e ffe c t of DFP in rats has been studied thoroughly by one group (Koehn

and Karczm ar, 1977; Koehn and Karczm ar, 1978; Koehn et al., 1980). They

have determined that DFP has antinociceptive a c tiv ity in hot plate and ta il

f lic k tests from 0.5 mg/kg. Antagonist studies indicate that this e ffe c t is

mediated through both cholinergic and opioid systems. DFP's antinociceptive

action was stereoseiectively inhib ited by (-)benzomophan antagonists. The

(+)isomers were e ffe c tive only to a much lesser degree. The role o f this

stereose lectiv ity is considered fu rthe r in section 1.6.6. It is worthy o f note,

however, that in animals made to leran t to morphine, DFP's e ffe c t was

unaltered (Koehn et al., 1980). Saxena (1958) fa iled to show an

antinociceptive e ffe c t o f DFP in rats, or any morphine potentia ting a c tiv ity .

This may be due e ither to the u tiliza tio n o f pressure as the nociceptive

stimulus, or the lower dose (200 ug/kg) employed.

Clement and Copeman (1984) have studied the very potent anticholinesterase

agents, soman and sarin. Both these agents produce antinociception that

persists fo r up to 96 hours a fte r adm in istration. Further studies w ith soman

showed only an additive e ffe c t w ith morphine, and tha t naloxone was only a

pa rtia l antagonist up to 10 mg/kg, whereas atropine com plete ly antagonised

soman's e ffe c t at 2.2 mg/kg. This in form ation suggests tha t a t least some of

soman's antinociceptive a c tiv ity is exerted through non-opioid systems.

1.6.5. Other Anticholinesterase Agents

There are a few reports concerning antinociceptive ac tiv itie s o f some less

fam ilia r anticholinesterase compounds, some of which are natura lly occurring

plant products. Galanthamine is a natural alkaloid, isolated from

Am aryllidaceae, which possesses anticholinesterase a c tiv ity . I t also has a

chemical structure s im ilar to codeine. Its antinociceptive a c tiv ity has been

studied in the ra t hot plate and the mouse w rith ing tests (Cozantis et al.,

1983). In both these models galanthamine was antinociceptive, and was

shown to be able to potentia te morphine antinociception in the ra t. Its mode

o f action appears to be species dependent: its antinociceptive a c t iv ity is

antagonized by naloxone (0.1 mg/kg) in the ra t, but is re frac to ry to naloxone

up to 0.5 mg/kg in the mouse. I t is also possible that the nociceptive stimulus

may be a determ ining fac to r as to whether the action of galanthamine is

opioid mediated.

Tetrahydroam inoacridine (THA) was found to be inactive up to 3 mg/kg in the

mouse hot plate antinociceptive test (Wooten and Klemm, 1980) though i t

appears to prolong the antinociceptive e ffects o f morphine. Using a higher

dose o f 7.5 mg/kg and a d iffe re n t test model, Wong and Bentley (1978)

observed antinociceptive a c tiv ity fo r THA which was pa rtia lly antagonized by

atropine at 2 mg/kg. THA also potentiated the action o f morphine; th is

action was also antagonized by atropine, and in addition the antagonistic

a c tiv ity o f naloxone was enhanced w ith THA pretreatm ent.

A n tinociceptive e ffects of dibromopyruvic' acid have been reported (M artin et

al., 1958). This compound has weak anticholinesterase a c tiv ity (1/400 tha t o f

neostigmine) and may also possess muscarinic agonist a c tiv ity . However, its

antinociceptive a c tiv ity was not blocked by atropine at 2 mg/kg. Ibogaine is

an indole alkaloid from Tubernanthe iboqa and is able to in h ib it

cholinesterase (Vincent and Sero, 1942). Though ibogaine its e lf is not

antinociceptive up to 40 mg/kg, lower doses are able to potentia te morphine

antinociception and le th a lity (Schneider and M cArthur, 1956).

Recently, the organophosphorus insecticide tr io rth o to ly l phosphate has been

reported to produce a s ix-fo ld potentia tion o f heroin's potency in the mouse

w rith ing test (Cohen, 1984).

1.6.6. Cholinom im etics and Antinociception

a) Muscarinic Agonists

The data fo r oxotremorine shown in Table 1.3. clearly shows that i t is able to

induce antinociception in four species using many forms o f nociceptive

stim ula tion. Only the ra t paw pressure test fa iled to show clear

antinociceptive a c tiv ity as the e ffec ts were small and variable (B ill e t al.,

1983). Oxotremorine has been shown to be very potent on a molar basis in a ll

the studies shown, w ith an ED5Q as low as 11 ug/kg being reported by Metys

et al., 1969. I t appears to exert its action through both cholinergic and opioid

systems as atropine, hyoscine and other anticholinergics are e ffe c tive

antagonists (B ill et al., 1983; Harris e t al., 1969; Leslie, 1969; Ireson, 1970;

Paalzow and Paalzow, 1973; Wong and Bentley, 1979; Lewis et al., 1983) as

are naloxone, naltrexone and benzomorphans (Harris et al., 1969; B a rto lin i e t

al., 1979; Ben-Sreti and Sewell, 1982; Lewis et al., 1983). I t is possible tha t

catacholamines may also be involved as Pleuvrey and Tobias (1971) reported

potentia tion by treatm ents which lower noradrenaline. Slater (1981)

however, reports that neuronal lesioning w ith 6-hydroxydopamine, which

destroys catecholam inergic neurons, acts through the noradrenergic system

to antagonize oxotrem orine’s e ffe c t. Other reports also indicate a role fo r

noradrenaline in the mediation o f oxotremorines antinociceptive actions

(Sethy et al., 1971; Barar and Madan, 1976). Oxotremorine is believed to be

the active m etabolite o f the compound trem orine (Cho et al., 1961), and

trem orine itse lf has been shown to be antinociceptive in mice (van Eick and

Bock, 1971; Lenke, 1938).

A recent study using stereospecific opioid antagonists has yielded im portan t

im form ation on the mode of action of oxotremorine (Ben-Sreti and Sewell,

1982). They report a reciprocal stereospecific sensitiv ity fo r oxotrem orine

and opioid analgesics toward opioid antagonists. The (-)benzomorphan

isomers, M rl452 and Mr2266, antagonized opioid induced antinociception but

had no e ffe c t on oxotremorine-induced antinociception. Conversely the

(+)benzomorphans, M rl453 and Mr2267 antagonized oxotremorine but not

opioid antinociception. Furtherm ore (+)benzomorphans antagonized

oxotremorine antinociception, but not tremorogenic or hypotherm ic actions

(Ben-Sreti et al, 1982), suggesting muscarinic receptor subpopulations

mediating these physiological e ffects, Koehn et al (1980) have shown the

e ffec ts of (+) and (-) opioid antagonists on DFP antinociception in the ra t to

be opposite to tha t observed by Ben-Sreti and Sewell (1982) in m ice. This

species d ifference may be due to the involvement, only in the mouse, o f

noradrenaline in oxotremorine's antinociceptive action. A lte rn a tive ly , DFP

may. act through the endogenous opioid system (Koehn et al 1980) w h ils t

oxotremorine may not as i t is re frac to ry to (-)benzomorphan antagonism

(Bensreti and Sewell, 1982).

Other muscarinic agonists, arecoline, RS 86, and pilocarpine have also been

reported to be antinociceptive in a number o f species and tests, though

potentia tion o f opioids has only been reported fo r pilocarpine (Knoll and

Komlos, 1952; Wong and Bentley, 1979) and this e ffe c t is not universal

(Kaakkola and Ahtee, 1977; Malec and Langwinski, 1982). An im portant

exception in the antinociceptive a c tiv ity of these muscarinic agonists is in

studies in non-human primates. Using the rhesus monkey, Pert (1975) fa iled

to show any antinociception fo r arecoline or pilocarpine. S im ila rly the

nociceptive response of the squirrel monkey was unaffected by pilocarpine

(Houser and Houser, 1973). This does not appear to be due to the nociceptive

test employed (e lec tric shock titra tio n ), since pilocarpine has been shown to

be active in e lectro foo t shock in rats (Houser and van H art, 1973; Kaakkola

and Ahtee, 1977) and arecoline was e ffe c tive in ta il e lec trica l s tim ula tion

(Metys et al., 1969). In addition, arecoline has antinociceptive a c tiv ity , as

observed fo llow ing e lec trica l s tim ula tion of rabb it tooth pulp (Metys et al.,

1969). The d iffe ring results, therefore, lie in species differences. Pert (1975)

suggests tha t the responses o f rodents may involve polysynaptic spinal

reflexes which may be sensitive to cholinergic agents. A lte rn a tive ly the

neurochemical coding o f pain may be d iffe ren t in primates. These results

indicate the possible p itfa lls when extrapolating data obtained in rodents to

primates, including humans.

b) N ico tin ic Agonists

In the preceding sections most o f the papers describing antinociceptive

actions of cholinom im etics and the e ffects o f antagonists, have indicated

tha t cholinom im etics exert the ir action through CNS muscarinic receptors.

Indeed, the use o f n ico tin ic antagonists has been shown to be in e ffec tive in

antagonizing cholinergic-induced antinociception (Pedigo et al., 1975; Dewey

et al., 1975). However, there have been some reports describing potent

antinociceptive actions o f nicotine and dimethylphenylpiperazine. N icotine

has been shown to be antinociceptive in m ice, rats and dogs using a va rie ty o f

nociceptive stim u li. I t is very rapid ly acting, exerting an e ffe c t 30 seconds

a fte r s.c. adm in istra tion (T ripa th i et al., 1982). I t appears to be acting only

through n ico tin ic receptors as atopine and hyoscine were in e ffec tive in

antagonizing nicotines antinociceptive e ffe c t, (Phan et al., 1973; T ripa th i et

al., 1982), w h ils t mecamylamine was e ffe c tive (Phan et al., 1973; Sahley and

Berntson, 1979; T ripa th i e t al., 1982). However, Sahley and Berntson (1979)

observed that hyoscine did antagonise nicotine and proposed tha t nicotines

antinociceptive a c tiv ity was mediated via release o f acetylcholine (see

Section 1.6.6.(c)).

Species differences in nicotines action have been observed. Very close

corre lations between nicotine brain levels and antinociception is seen in the

mouse, but there is a poor corre lation in the ra t (T ripath i et al., 1982).

Further studies indicated tha t th is finding is probably mainly due to the rapid

development o f tachyphylaxis only in the ra t. This same group also observed

tha t naloxone had very l i t t le e ffe c t in the ra t, producing a maximum o f 23%

inh ib ition of nicotine induced analgesia at a high dose o f 10 mg/kg. In mice,

however, naloxone was e ffe c tive from 0.1 mg/kg, though complete

anatagonism could not be obtained. Sahley and Berntson (1979) also showed

tha t naloxone was ine ffec tive in the ra t.

The specific ganglionic stim ulant, dimethylphenylpiperazine has been shown

to be antinociceptive in mice (Phan et al., 1973) and this appears to be a

n ico tin ic e ffe c t as mecamylamine is an e ffe c tive antagonist.

These data, therefore, indicate that stim ulation o f the n ico tin ic receptor in

the CNS produces potent antinociception and on a molar basis equivalent to

morphine (T ripath i et al., 1982). Again species differences are apparent as in

the mouse nicotine’s action has an opioid component and does not exh ib it

tolerance. In the ra t, antinociception is mediated through the n ico tin ic

receptor and rapid tolerance occurs.

c) Choline Esters

The in traven tricu la r adm in istration o f acetylcholine has been shown to induce

antinociception in mice. This e ffe c t can be blocked w ith atropine, though not

w ith the quaternary derivative atropine m ethy ln itra te or the n ico tin ic

antagonist mecamylamine, indicating tha t acetylcholine exerts its

antinociceptive e ffe c t through centra l muscarinic receptors (Pedigo e t al.,

1975). Acetylcholine has also been reported to inh ib it morphine

antinociception (M udgill et al., 1974). However, the e ffe c t is small and

occurs 30 minutes a fte r i.c.v. in jection. As peak antinociception o f

acetylcholine occurs around 10 minutes post in jection, its antagonist e ffe c t

on morphine antinociception probably acts through a mechanism d is tinc t from

its antinocicpetive e ffe c t. There is also evidence tha t a component o f

acetylcholine's antinociceptive action is via opioid receptors. Its

antinociceptive actions are antagonised by naloxone (Pedigo e t al., 1975;

Pedigo and Dewey, 1981) and the rank order o f potency o f five narco tic

antagonists is the same fo r both acetylcholine and morphine (Pedigo e t al.,

1975). Furtherm ore, the divalent ions Mg^+, Mn^+ and Ca^+ but not Sr^+ or

Ba^+ also inh ib it both acetylcholine (Widman et al., 1978) and morphine

(Harris et al., 1977) antinociception.

I t has also been reported tha t i.c.v. adm in istration o f 40 pg acetylcholine

d iffe re n tia lly altered the level o f four peptide and non-peptide brain

fractions which had opio id-like a c tiv ity (Chan et al., 1982). I t is possible tha t

these opio id-like m ateria ls mediate the antinociceptive e ffe c t o f

acetylcholine.

There are, however, indications tha t acetylcholine exerts its antinociceptive

e ffe c t through mechanisms d is tinct from that fo r morphine. For example,

Pedigo et a i. (1975) found that the 1-isomers o f pentazocine and cyclazocine

blocked morphine w h ils t the d-isomers were w ithout e ffe c t; the converse is

true fo r acetylcholine. Pedigo and Dewey (1981) have observed this reverse

stereose lectiv ity w ith the (+) and (-) isomers o f naloxone (cf. oxotremorine).

In addition, studies w ith naloxone have yielded d iffe ren t apparent pA2 values

fo r naloxone versus morphine and acetylcholine (6.86 and 6.09 respectively)

and d iffe re n t slopes on the Schild p lot (-0.944 fo r morphine, -1.46 fo r

acetylcholine) (Pedigo and Dewey, 1981). These marked differences indicate

tha t naloxone inh ib its acetylcholine-induced antinociception non-

com petatively. They also showed tha t there is an assym etrical cross

tolerance between morphine and acetylcholine. The rapid tolerance to the

antinociceptive e ffe c t o f acetylcholine only s ligh tly decreased morphine

antinociception, however, tolerance to morphine produced a 12-fo ld increase

in the ED^g of acetylcholine.

W ith respect to in te raction and involvement o f amine systems, though

reserpine and tetrabenazine pretreatm ent appeared to inh ib it acetylcholine

antinociception, these compounds lack spec ific ity , and more specific

depletors o f dopamine, noradrenaline or 5-HT were w ithout e ffe c t (Pedigo

and Dewey, 1981).

In summary, the evidence indicates tha t acetylcholine exerts its

antinociceptive through muscarinic receptors (Dewey et al., 1975) and tha t

the mediation is independent o f opioid pathways.

Choline adm inistration has been shown to increase brain turnover and levels

of acetylcholine (Cohen and Wurtman, 1975). Knoll and Komlos (1952) found

tha t choline appeared to potentia te morphine antinociception. However, the

dose o f 200 mg/kg used is probably tox ic. A t this dose o f choline morphine

LD^g is reduced from 705 mg/kg to 66 mg/kg (Ho et al., 1979). A

non-specific behavioural depression may therefore be responsible fo r an

increased hot-p la te latency. The opposite e ffe c t o f choline was observed by

B o ttic e lli et al. (1972) who found a dose-dependent attenuation o f morphine

antinociception using 30 and 60 mg/kg. A t 60 mg/kg, choline alone did not

a ffe c t nociception. The authors Concluded tha t morphine's action may be due

to its a b ility to decrease acetylcholine release (Jhamandas ^ t al., 1971) and

so choline, which has been shown to increase acetylcholine levels (Cohen and

Wurtman, 1975) may therefore attenuate the antinociceptive e ffec ts of

morphine. This hypothesis however, is not easy to reconcile w ith the

lite ra tu re concerning i.c.v. adm in istration o f acetylcholine (vide supra) and

the lite ra tu re concerning the e ffects o f opioid analgesics on brain

acetycholine levels is contrad ictory (see Dewey et al., 1976). I t is possible

tha t choline may be acting on the post-synaptic cholinergic receptor; th is has

been proposed by Fredrickson and Pinsky (1975).

Carbachol does not cross the blood-brain barrier but several studies using

i.c.v. and in tracerebra l (i.e.) adm in istration have demonstrated

antinociceptive a c tiv ity o f carbachol. Furtherm ore, i.e. application studies

have gone someway in identify ing the particu la r groups o f cells w ith in the

brain tha t are responsive to carbachol. Metys et al, co-workers (1969)

showed tha t i.c.v. carbachol (at 1.25 gg) was antinociceptive in both ra t and

rabb it. Local i.e. application indicated tha t the septal areas and m idbrain

re ticu la r form ation - centra l grey region were both sensitive to 0.3 gg

carbachol, whereas cudate putamen and dosal hippocampal adm in istra tion

only showed effects between 5 and 10 gg, doses at which transventricu la r

d iffusion may w e ll occur.

Brodie and P roud fit (1984) examined the role o f the cholinergic system on the

nucleus raphe magnus (NRM). This structure in the caudal medulla has been

shown to a ffe c t nociceptive processing (see Fields and Basbaum, 1978), and

also to possess cholinergic term inal markers. For example, cholinerg ic

receptors (Kobayashi et al., 1978) acetylcholinesterase (Palkovitz and

Jacobowitz, 1974) and choline acetyltransferase (Kobayashi et al., 1975).

Brodie and P roud fit (1984) showed that carbachol (i.e.) produced

antinociceptive responses, which could be reversed by atropine or prevented

by atropine pre trea tm ent. The authors suggest that carbachol acts on the

NRM to activa te descending raphe-spinal neurons, which impinge on spinal

cord dosal horn neurons. It is these neurons which are sensitive to noxious

s tim ulation (see Brodie and P roud fit, 1984 fo r references).

In the cat, Katayama et al. (1984a,b) found a group o f cells in the

parabrachial region (PBR) very sensitive to the antinociceptive e ffec ts o f

carbachol. This appears to be solely a muscarinic response as i t is inhib ited

by atropine, but not mecamylamine or naloxone. Morphine at a dose o f 25 gg

had no e ffe c t when in jected into the same site . Although a role fo r these

groups of cells which appear to mediate nociceptive in form ation via

muscarinic receptors is not clear, i t is possible that they are the substrate fo r

environm entally induced antinociception. This form of antinociception is

discussed in Section 5.

1.6.7. Cholinergic Antagonists and Antinociception

The previous sections have described the lite ra tu re concerning

cholinom im etic agents and give a strong indication that antinociception is at

least in part mediated by acetylcholine in the CNS. The use o f antagonists in

nociceptive studies is shown in Table 4 and provides fu rthe r support fo r the.

role of acetylcholine in nociception and opioid function.

Hemicholinium decreases acetylcholine levels in the cerebral cortex

(Rodriguez de Lores Arnaiz et al., 1970), by in te rfe ring w ith choline transport

(Marchbanks, 1968) and pretreatm ent w ith this compound s ligh tly antagonises

the antinociceptive action o f morphine (Bhargava et al., 1974).

Hem icholinium itse lf has no antinociceptive a c tiv ity . This would suggest tha t

part o f morphine's action involves cholinergic neurones. Hem icholinium has

also been shown to inh ib it acupuncture antinociception fo llow ing i.c .v .

adm in istra tion (Ren et al., 1980). The role o f the cholinergic system in

acupuncture antinociception is discussed in Section 1.6.7.(b).

The muscarinic antagonist, hyoscine, administered by the peritoneal route is

not antinociceptive in mice (B ill e t al., 1983) or rats (Houser and van H art,

1973; B ill et a l., 1983; Lewis et a l., 1983) and indeed causes s ign ifican t

hyperalgesia in ra t ta il f lic k tests at 1 mg/kg (Watkins et al., 1984).

However, s.c. adm in istration has been shown to produce potent

antinociception using the mouse w rith ing test, which may re fle c t route o f

adm in istra tion or perhaps a peripheral in teraction w ith the w rith ing agent

phenylbenzoquinone.

Data from prim ate studies (Houser and Houser, 1973; Pert, 1975) indicate

tha t hyoscine is antinociceptive at low doses. Though con flic ting w ith the

rodent data these results are consistant w ith the findings described in

Section 1.6.6.(a), on the study o f muscarinic agonists in primates, indicating

again tha t pain pathways may be species dependent. When hyosine was

administered into discreet brain regions that had been shown to be responsive

to morphine, no antinociception was observed. Ventricu la rly administered

hyoscine was e ffe c tive however, thus strongly suggesting a cholinerg ic

pathway tha t modulates nociception at least in part d is tinct from opioid

sensitive systems (Pert and Maxey, 1975).

Opioid induced antinociception has been shown to be inhib ited in two species

by hyoscine (B ili et al., 1983) though not in the w rith ing test, but this could

be re lated to hyoscine's own antinociceptive a c tiv ity in this test. Malec and

Langwinski (1982) found tha t hyosine enhanced codeine's effectiveness whilst

leaving morphine, fentanyl and pentazocine unaffected.

The use o f cholinergic antagonists has indicated possible transm itte r

substrates fo r endogenous environm entally induced antinociception.

Endogenous antinociception may be induced by stressful s tim u li, such as cold

water swimming (Bodnar et al., 1978), e lec tric shocks (Jackson, 1979), and

figh ting (Rodgers and Hendrie, 1983). In many cases the antinociception is

naloxone reversible im plying a role fo r endogenous opioid systems. However,

some forms of environm entally induced antinociception are re frac to ry to

narcotic antagonists, fo r example b rie f continuous, as opposed to prolonged

in te rm itta n t, foo t shock (Lewis et al., 1980), and hind paw, as opposed to

fro n t paw, foo t shock (Watkins and Mayer, 1982) appear to u tilize non-opioid

mechanisms. The cholinergic system has been im plicated by some workers in

environm entally induced antinociception, as hind paw foo t shock induced

antinociception and antinociception induced by classical conditioning are

blocked by hyosine (1 mg/kg), but not the quaternary deriva tive ,

methylhyoscine (Watkins et al., 1984). Prolonged in te rm itta n t shock was dose

dependently antagonised by hyoscine from 0.1 mg/kg (Lewis e t al., 1983),

s im ila rly long term opioid antinociception induced by inescapable foo t shock

followed by m ild re instating shocks 24 hours la te r was also antagonized by

hyoscine at 2.5 mg/kg. The indications are tha t some form s of

environm entally induced antinociception are mediated via centra l muscarinic

system, whilst others act through endogenous opioid systems which employ

in term ediary muscarinic cholinergic pathways.

1.6.8 Miscellaneous Compounds

The role of the cholinergic system in nociception has been im plicated by

pharmacological treatm ents not d irec tly involving the use o f cholinergic

drugs. There are several neurotransm itters which have been reported to be

involved in nociception, one o f which is the inh ib ito ry amino acid

Y -am inobutyric acid (GABA). The role o f GABA as an analgesic has been

suggested from studies w ith an analogue, baclofen (Saelens et al., 1980). The

lack o f antagonism by naloxone indicates that baclofen acts through an

opioid-independent system.

Kendall et al. (1982) investigated the role o f GABA in nociception using

d irec tly acting agonist (kojic amine and THIP), a GABA-degradation inh ib ito r

(y-v iny lG A B A ) and an inh ib ito r o f high a ff in ity transport (n ipecotic acid

e thyl ester). As w ith previous studies, naloxone (5 mg/kg) fa iled to a lte r the

antinociceptive a c tiv ity o f these compounds in mice using hot plate and ta il

immersion tests. In contrast atropine (5 mg/kg) inhib ited the antinociceptive

a c tiv ity of a ll treatm ents w hilst mecamylamine (5 mg/kg) was in e ffec tive .

Those drugs which m odify GABAergic transmission probably do not exert

the ir e ffe c t by acting d irec tly on muscarinic receptors as only n ipecotic acid

showed sign ificant a ff in ity fo r the receptor in binding studies. The results

indicate then, that GABA is exerting its antinociceptive e ffe c t via

in term ediary cholinergic neurons.

2+ 2 The divalent cations barium (Ba +) and strontium (Sr +) are believed to be

able to substitute fo r calcium ions to induce release o f neurotransmitter.s

2+(Rubin, 1970) and Ba has been shown to release acetylcholine (Silinsky,

1977). Using the mouse ta il- f l ic k model Welch et al. (1983) demonstrated

2+ 2+that in traven tricu la r administered Ba and Sr induced antinociception.

This e ffe c t was reversed by atropine but naloxone antagonism was not dose

2 2+related. These results suggest that Ba +'s and Sr +,s inherent antinociceptive

a c tiv ity is mediated via acetylcholine release.

The involvement o f the cholinergic system in antinociception may be o f

c lin ica l significance w ith the recent development o f the analgesic drug

meptazinol. This drug appears to possess both opioid and cholinerg ic

antinociceptive components as B ill e t al. (1983) demonstrated in several

rodent antinociceptive tests (see Table 1.4.) that m eptazinol’s e ffec ts were

inhib ited by naloxone, atropine and hysocine. It has been reported recently

that meptazinol possesses cholinergic a c tiv ity by v irtue o f its marked

anticholinesterase a c tiv ity (G alli, 1983). The possibility o f u tilis ing the

cholinergic component c lin ica lly is o f great in terest as the drug seeking

behaviour and addictive lia b ility o f cholinergic or opio id-cholinergic

analgesics may be less than that observed w ith opioids.

1.6.9 Acetylcholine Histopharmacology and Opioids

a) Role o f Acetylcholine in Nociceptive Pathways

The previous sections have described how the cholinergic system is involved

in nociceptive processing. The question arises as to whether there is an

anatom ical corre late of these pharmacological observations.

Morphine has been shown to inh ib it acetylcholine release from the guinea-pig

ileum (Paton, 1957; Schaumann, 1957) and to increase levels in the brain (e.g.

Herken et al., 1957). These studies demonstrating morphine action in

increasing brain acetylcholine levels was observed only at doses above tha t

required fo r antinociception. However, these were measurements in whole

brain, and more recent studies have measured regional changes in

acetylcholine, w ith antinociceptive doses. Green et al. (1976) showed tha t in

mice, 10 mg/kg morphine increased acetylcholine levels s ign ifican tly in the

stria tum and at higher doses (300 mg/kg) in the hippocampus. In rats

increases in s tria ta l acetylcholine was observed a fte r 30 mg/kg, and a fte r

90 mg/kg increased levels were measured in the hippocampus; these e ffec ts

were shown to be naloxone reversible. There is m v itro evidence tha t this

e ffe c t is due to morphine's action on h ig h -a ffin ity Na+-dependent choline

transport (HANDCU), an ind ica tor o f cholinergic neuronal a c tiv ity m vivo

(Kuhar and M urrin, 1978). I t has been shown tha t at antinociceptive doses

morphine stim ulates HANDCU in mouse s tria ta l (Vallano et al., 1982) and ra t

hippocampal (Vallano and McIntosh, 1980) synaptosomes. In addition,

enkephalin at 40 nM was shown to decrease neocortical HANDCU (Wenk,

1984). I t is also possible tha t opioids increase acetylcholine levels by

inh ib iting its release, i.e. decreasing cholinergic neuronal a c tiv ity . Indeed,

release o f co rtica l acetylcholine may be evoked by e lec trica l s tim u la tion o f

nerves in the forepaw o f anaesthetized rats (Jhamandas and Sutak, 1983) and

this is enhanced fo llow ing systemic adm in istration o f the narcotic antagonists

naloxone or naltrexone. This suggests tha t there is an inh ib ito ry ro le fo r

endogenous opioids on acetylcholine release. Opioid receptor regulation

appears to be lim ite d to certa in pathways (Wood et al., 1984) viz, septal-

hippocampal and substantia innom inata-cortica l pathways where agonists a t

p, 6 and e receptors exert inh ib ito ry regulatory influences, w h ils t k-agonists

appear to be ine ffec tive . In addition, there is a co-loca liza tion o f enkephalin

and acetylcholine in the la te ra l system of olivocochlear cells (A ltschu ler et

al., 1983) and several preganglionic parasympathetic neurons (Glazer and

Basbaum, 1980).

This co-existence, and possibly co-release, may also occur in other neuronal

systems, and the lite ra tu re describing the relationship between opioid and

acetylcholine turnover and release suggest a close relationship between these

two systems. I t is quite plausible, tha t acetylcholine plays a key ro le in the

actions o f endogenous and exogenous analgesia.

b) Acetylcholine and Acupuncture-Induced Antinociception

In China, acupuncture is used routine ly as a method to induce surgical

analgesia. The mechanism of this e ffe c t is s t il l poorly understood. I t is only

in recent years tha t th is technique has, at least in the West, received any

credence and undergone sc ie n tific enquiry. The possible mechanisms involved

\d acupuncture-induced analgesia have been reviewed recently (Han and

Terenius, 1982). There are several reports describing the role o f the

cholinergic system in acupuncture antinociception. Acupuncture causes an

increase in acetylcholine in the CSF and the increase correlates w ith increase

analgesia (He et al., 1979). In rats, acetylcholine levels were found to

increase during electroacupuncture-induced antinociception in the locus

coeruleus and dorsal raphe (Ge, 1979) hypothalamus and nucleus caudatus

(Wang et al., 1979). In addition, increased acetylcholinesterase a c tiv ity was

observed in the thalamus fo llow ing electroacupuncture and locus coeruleus

though a c tiv ity was reduced in the substantia gelatinosa (A i et al., 1979).

Pharmacological manipulation of the cholinergic system has added to the

evidence fo r a role o f acetylcholine in acupuncture-antinociception.

Inh ib ition o f acetylcholine synthesis w ith hemicholinium (i.c.v.) was shown to

inh ib it electroacupuncture in rats (Han, 1979; Guan, 1979), and this could

pa rtia lly be reversed w ith the acetylcholine precursor choline chloride (Han,

1979). Blockade of muscarinic receptors w ith atropine in rats and rabbits

(Han, 1979) and w ith hyoscine in rabbits (He et al., 1979) was found to block

analgesia induced by acupuncture, w h ils t the anticholinesterase

physostigmine has been shown to potentia te acupuncture analgesia in rats

(Han, 1979; G u a n e ta l., 1979; A i, 1979).

There is also evidence fo r the involvement o f other transm itte r systems (e.g.

endogenous opioid and 5-HT) suggesting a m ultip le neurtransm itter system

mediating the e ffec ts o f acupuncture (See, Han and Terenius, 1982, fo r

review).

c) Acetylcholine-Q pio id In te raction in Non-Nociceptive Systems

There are a number o f reports in the lite ra tu re describing in teractions

between centra l cholinergic and opioid systems not involved in nociception,

but having potentia l c lin ica l applications. For example, anaesthesia has been

shown to be potentiated or prolonged fo llow ing anticholinesterases (Pellanda,

1933; Green and Davis, 1956). Another potentia l benefic ia l e ffe c t o f the

opioid-cholinergic in teraction has been investigated by Weinstock and her

colleagues. The respiratory depressant e ffe c t o f morphine, or other opioids

tha t are routine ly given as part of pre-operative medication is usually not

im portant, however, in patients w ith lung diseases or breathing problems such

an e ffe c t is po ten tia lly fa ta l. However, physostigmine pretreatm ent has been

shown to antagonize respiratory depression in experimental animals

(Weinstock et al., 1980) and humans (Snir-Mor et a l., 1983). This property o f

physostigmine has been exploited successfully in the trea tm ent o f severe

resp ira tory depression resulting from overdose in heroin addicts (Rupreht et

al., 1984). The authors point out tha t physostigmine trea tm ent is preferable

to the usual procedure of adm in istration o f naloxone, as acute w ithdraw al is

not precip ita ted.

Treatm ent of opioid addiction is an im portant area of research in opioid

pharmacology, and there is evidence that the cholinergic system modulates

the processes of tolerance and dependence to opioids. The lite ra tu re has

been extensively reviewed (Wahlstrbm, 1978); short-term morphine

trea tm ent ( 3 days) appear to increase acetylcholine turnover in it ia lly

(Bhargava and Way, 1975; Cheney et al., 1975), w h ilst several studies indicate

that longer treatm ent (2-4 weeks) appears to result in increase levels or

decreased acetylcholine u tiliza tio n (e.g. Domino and Wilson, 1973). A

possible c lin ica l application to the use o f a cholinergic drug in the trea tm ent

o f dependence and w ithdrawal has been proposed by Albin et al. (1975). They

reported that nalorphine-precipitated w ithdrawal signs fo llow ing constant

morphine plus an anticholinesterase (tetrahydroam inoacridine, THA) dosing in

rhesus monkeys were markedly less than that observed a fte r morphine alone

treatm ent. Indeed, an earlie r study indicates that THA adm in istra tion

reduces physical dependence in humans (Stone et al., 1961).

There is evidence fo r a cholinergic involvement in a ffec tive disorders (e.g.

depression) in humans, as fo llow ing adm inistration o f physostigmine in normal

subjects resulting symptoms include many o f those.associated w ith depressive

symptomatology (Risch et al., 1980). In addition, in patients w ith a h istory o f

a ffe c tive disorder, but w ith c lin ica l remission, infusion o f low doses o f

physostigmine produces transient depressive symptomatology (Janowsky et

al., 1974). More recent studies (Risch e t al., 1981) have shown tha t

adm inistration of physostigmine or ajrecoline to both normal and a ffe c tiv e -

disorder subjects produces altered mood and a ffe c tive changes, resulting in

increased depression. These observed e ffects were highly corre lated w ith

increased plasma 3-endorphin im m unoreactiv ity (but not co rtiso l or

pro lactin) and the authors suggest the existence o f a cholinerg ically mediated

3-endorphin pathway modulating a ffec tive and cognitive states in humans.

A classical response to opioid adm inistration in mice is to increase the ir

locomotor a c tiv ity . This stereotypic running is believed to be dependent on

dopamine (Kushinsky and Hornykiew icz, 1974; Eidelberg and Erspamer,

1975), but there is also evidence that a component o f morphine's action

involves the cholinergic system. Adm inistra tion of the muscarinic receptor

agonist oxotremonine (Seidel et a l., 1979), or the anticholinesterase agent,

DFP (Su and Loh, 1975), attenuate morphines e ffe c t on locomotion, w h ils t the

muscarinic antagonist, atropine enhances morphines action (Seidel e t al.,

1979). Cholinergic drugs appear then to influence opioid-induced running.

The mechanism is unclear, but may w e ll involve other neurochemicals, such

as catecholamines.

1.6.10. Conclusions

Several agents which in te rfe re w ith the cholinergic system have been shown

to be antinociceptive. These include reversible and irreversib le

anticholinesterases and agonists at muscarinic and n ico tin ic receptors. To

a ffirm that cholinergic drugs potentia te the analgesic action o f opioids is

perhaps an overgeneralisation. However many experimental studies have

been able to show that enhancement o f cholinergic a c tiv ity can lead to

enhancement o f opioid antinociception. In addition there is evidence to

suggest that endogenous analgesia, induced by stress may in part be

modulated by cholinergic inputs.

The in teractions between the cholinergic and opioid systems probably

prim arily occurs w ith in the centra l nervous system as studies w ith quaternary

compounds have been mostly negative whilst in teractions fo llow ing centra l

adm inistration are generally observed. The molecular mechanisms involved

are not understood but most o f the evidence points to a pharmacodynamic

rather than a pharmacokinetic in teraction.

Too few studies have used receptor selective antagonists and where

cholinergic antinociception has been observed the involvement o f both

cholinergic and opioid receptors is often the case. Our understanding o f

in teractions at the receptor level require more selective pharmacological

tools espeically w ith respect to opioid pharmacodynamics. I t is also probably

an overs im plifica tion to th ink o f an opio id/cholineric in te raction involving

only one mechanism and there may be in terrelationships w ith other neuronal

systems. In particu la r the evidence points to a close relationship w ith

monamines. Whether manipulation o f cholinergic and opioid system produces

more successful pharmacological agents are analgesics remains to be seen.

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CHAPTER 2

Antinociceptive Activity of Morphine, Fentanyl and Alfentanil

in Mice and Rats:

Effect of DFP and Drugs Used in the Treatment of DFP Poisoning

INTRODUCTION

2.1. An tinoc icep tive Models

There are many tests available to determ ine the antinociceptive a c tiv ity of

drugs. The m a jo rity o f these tests u tilize the response to experim ental pain

in animals (Linebery, 1981). Anim al testing can only serve as a model of

human c lin ica l pain as the pain response in man is a subjective param eter

w ith large va riab ility between individuals. Extrapolation from animal data to

humans must be viewed w ith caution as false negatives and positives may be

encountered.

Wood (1984) presented a lis t o f five c r ite r ia fo r consideration when

in te rpre ting data from animal screening tests:

1 Test V a lid ity : Is the test predictive o f the c lin ica l u t i l i ty o f known

analgesics?

In considering the most appropriate antinociceptive test in this

study, these points were taken in to consideration. I t was decided to

employ the hot-p la te test (Woolfe and MacDonald, 1944) fo r mice

and the paw pressure test (Randall and Se litto , 1957) fo r rats.

2 Test R e lia b ility : Is the test response reproducible throughout an

experimental day and from day to day?

3 Test S im p lic ity : Does the procedure use a standard defined noxious

stimulus, measure a well-defined animal response, and is the test

easily utilized?

The mouse hot-p la te is an extensively used procedure and unlike the

ta il f lic k test does not re ly on a re flex ive response. The paw lick

response in the hot-p la te test relies on integrated behaviour

involving processes occuring at a high level w ith in the centra l

nervous system. The ra t paw pressure test was chosen to determ ine

antinociceptive drug a c tiv itie s in an additional species using a

d iffe re n t noxious stimulus, and in this test the responses are not

re flex ive in nature.

An tinociceptive responses are subject to wide variations, so care

was taken to account fo r and contro l environmental variables.

Test S ensitiv ity : Can d iffe ren t analgesic doses be discrim inated and

can the procedure detect weak and agonist/antagonist analgesics?

The paw pressure test, but not the mouse hot-p la te test is sensitive

to agonist/antagonist analgesics. In th is study, this would not be so

im portant as only strong analgesics were investigated.

Data Analysis: Varies between laboratories. Is the method of

analysis appropriate? Methods o f analysis should be described.

The analysis o f the data was given fu ll consideration, and a model

was developed to accurately f i t the form of the observed data (see

Section 2.10.).

2.2. In it ia l Experiments

Ample evidence has been presented in Section 1.6. fo r the role of cholinergic

agents in opioid antinociception. The in it ia l studies, which are presented in

this section, were designed to determ ine the in teractions between the

anticholinesterase agents and opioid antinociception in the models described

above.

MATERIALS AND METHODS

2.3. Anim als and Experim ental Conditions

Male albino mice (CD-I strain; 25-30g) were purchased from Charles R iver.

Male albino Wistar rats (Surrey U nivers ity strain; 140-210g) were bred at the

University 's Anim al U n it. Upon delivery, animals were housed in the Anim al

U n it under contro lled temperature, hum id ity and lighting conditions (12h

ligh t/da rk cycle; lights on 0700h). Mice remained in the U n it fo r at least 5

days before use. Tw enty-four hours before experimentation, the animals

were equilibrated in a quiet laboratory where a ll procedures were carried out.

Animals had free access to food and water up to experim entation. A ll

experiments were carried out between 0900h and 1300h.

The quiet laboratory (3.3m x 3.5m x 2.75m high) was windowless and had the

same lighting conditions as the Anim al U n it. The room was ventila ted but

had no independent heating or hum id ity controls. Am bient tem perature was

monitored during experimentation, and stayed w ith in the range 20-25°C

throughout the year. So as to minimise disturbance there was res tric ted

access to the room before and during experimental procedures.

2.4. Drugs

The fo llow ing drugs were used:

DRUG NAME SOURCE

Di-isopropylfluorophosphate Sigma

A tropine sulphate Sigma

Morphine sulphate M acfarlan Smith

Fentanyl c itra te Janssen Pharm aceutical*

A lfe n ta n il hydrochloride Janssen Pharm aceutical*

Pralidoxim e m ethyl sulphonate CDE, Porton Down*

Diazepam Roche*

Neostigmine bromide Sigma

Pyridostigm ine bromide Roche*

Naloxone hydrochloride Endo*

* These compounds were generously donated by the manufacturers.

Drugs were dissolved in 0.9% saline (Travenol) im m ediate ly prior to use, w ith

the fo llow ing exceptions: in some experiments DFP was dissolved in peanut

o il (Sigma), and as diazepam is not soluble in saline i t was f irs t dissolved in

polyethylene glycol 200 (PEG) to which an equal volume of saline was added

to give the correct drug concentration. Great care was taken in handling

DFP due to its highly tox ic nature. The supplied Safety Data Sheet was used

as a guideline to handling procedures outlined below:

2.5. Safety Procedures fo r DFP

DFP was stored, re frige ra ted at about 4°C, and kept w ith in the lidded m eta l

container in which i t was supplied. The v ia l containing DFP was handled only

in a fume cupboard, w ith the user wearing gloves and an overall w ith tig h t

cuffs.

Appropriate quantities o f DFP were obtained by penetrating the seal w ith a

fixed needle Ham ilton syringe and w ithdrawing liquid. The DFP was

im m ediate ly transfered to a vo lum etric flask to which saline was added. The

flask was shaken thoroughly fo r at least 2 minutes to ensure tha t the DFP had

dissolved. The fume cupboard also contained a bucket containing 2% sodium

hydroxide. Any accidental spillages or contaminated equipment could be

neutralised in copious quantities o f the sodium hydroxide solution.

DFP degrades w ith tim e due to hydrolysis (Leopold and Krishna, 1963) and

therefore stock was replaced every six months. In addition, a lfen tan il

potentia ting a c tiv ity was confirm ed (see Section 2.13.).

2.6. Dosing Protocols in M ice

Mice were scruffed and drugs were in jected in 0.1ml volumes subcutaneously

(s.c.) in the back o f the neck, w ith the exception o f diazepam and

neostigmine. Diazepam was in jected in a 0.03ml volume in tram uscularly

(i.m .) into the hind leg, because o f a local in teraction w ith DFP (D. Green,

CDE, personal communication), and neostigmine was in jected

in traperitonea lly (i.p.) in 0.1ml volumes.

Drug dosing protocols are illus tra ted in Figure 2.1. When used, atropine

(17mg/kg) and pralidoxime (15mg/kg) were mixed and in jected together 1

minute a fte r DFP, according to Gordon et al (1978). Diazepam (6.25pm ol/kg)

was in jected im m ediate ly prior to the atropine/pralidoxim e dose (D. Green,

personal communication).

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For the neostigmine experiments, e ither saline or neostigmine were

administered (i.p.) at tim e 0, a lfen tan il 25 minutes la te r, and antinociceptive

testing carried out at 30 minutes.

Pyridostigm ine (lm g/kg) was administered 1 hour before antinociception. The

times fo r the in jection o f morphine, fentanyl and a lfen tan il were 30, 50 and

55 minutes a fte r pyridostigm ine, respectively.

In those experiments when naloxone was used, i t was administered 30 minutes

before testing.

2.7. Dosing Protocols in Rats

Rats were scruffed fo r s.c. in jection (0.1ml volumes) in the back o f the neck.

DFP was administered at tim e 0 and morphine, fentanyl or a lfen tan il

administered 30, 50 and 55 minutes la te r respectively. Each animal was

tested four times; once im m ediate ly before the f irs t in jection and again at

three times to include peak antinociceptive e ffe c t. The tim ings fo r\

in jections and fo r post-drug testings are given in Fig 2.2.

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2.8. Antinociceptive Testing in Mice

The hot-p la te test (Woolfe and MacDonald, 1944) was employed fo r

antinociceptive testing. The hot plate was made in the Biochem istry

workshop, University o f Surrey, and consisted o f an aluminium plate (30cm

xl5cm) surrounded by a transparent perspex w a ll (22cm high). This was

placed in a therm osta tica lly contro lled w ater bath w ith the water maintained

at 33°C. The same apparatus and temperature setting was used throughout

a ll experiments.

When tested mice were placed on the hot-p la te and a hand-held d ig ita l

stopwatch started as soon as the mouse touched the plate, the nociceptive

end-point was taken as paw-licking (in most cases this was the fore-paw) or

jumping on the few occasions when the lick ing response was absent. I f the

mice fa iled to respond a fte r 60 seconds they were removed from the plate

and scored as >60 seconds. As the walls o f the hot-p la te were perspex and

unheated, occasionally some mice spent long periods on the ir hind paws w ith

fore-paws resting on the walls; these mice were excluded.

2.9. Antinociceptive Testing in Rats

The non-inflamed paw pressure test was employed fo r nociceptive

determ inations (Randall and Se litto , 1957) using an Ugo Basile Analgesy m eter

(A .R .Horwell, London). This apparatus (Fig. 2.3. ) consists o f a p lastic pusher

(P) positioned about 2mm above a Teflon p lin th (B). Animals were held

f irm ly , but not tig h tly around the upper part o f the ir body, so they could be

held in the correct position w ithout the animal struggling. The animals paw,

was placed under the pusher and subjected to a force increasing linea rly w ith

tim e as a pivoted weight (W) moved horizonta lly. A foot-pedal contro lled the

M i mm 'M: s m v

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B a s i le A n a lg e s S M et

■ fig

weight's movement which v/as marked o ff on a graduated scale (SC). The

score on the graduated scale was converted to the force applied in grams by

m ultip ly ing by 30.

The nociceptive end-point was taken as a w ithdrawal or attem pted

w ithdrawal o f the animal's paw, or when the animal struggled. With practice

i t was possible^ distinguish between struggling in response to the noxious

stimulus and non-specific struggling. Both hind paws, in turn, were placed

under the pusher and a mean value taken as the score fo r tha t animal. To

avoid damage to the animal's paws, a maximum c u t-o ff weight o f 750g was

employed. Those animals which did not respond before the 750g c u t-o ff was

reached were scored as 750g.

2.10. S ta tis tica l Procedures

2.10.1. H ot-P late Data

Four doses fo r each opioid were used to obtain a dose-response curve. Each

point was the mean of 12 or 18 observations made on 2 or 3 separate days.

The dose-response relationship was represented by an AD^-g value, which was

defined as the dose required to increase the hot-p la te latency to 30 seconds

(ie., 30% of the maximal response tim e). Unpaired Student's t- te s t (see

Appendix A) was used to.compare AD<-g values.

A ll dose response curves included censored values, tha t is mice fa iling to

respond before the 60 seconds c u t-o ff, which were scored as >60 seconds.

Though employing a c u t-o ff tim e is essential to avoid inducing tissue damage

in the animal's paws, i t does d is to rt the dose-response relationship. However,

this d istortion may be compensated fo r by using appropriate m athem atical

models. Such a model was applied to the data to obtain AD^g values and is

described in Appendix B. The Fortran listings fo r the test procedures (run on

the Prime computer operating system) are given in Appendix B.

2.10.2. Paw Pressure Data

A m athematical model was applied to data giving the tim e course of drug

e ffe c t to obtain values fo r peak responses fo r each dose. Comparison

between groups was by unpaired *t’ test. Results were norm ally mean values

o f at least six animals. To obtain a value fo r peak response, a quadratic

equation was f it te d to the data: y = a - b (x-c) , where: a = peak response

score, b = curvature o f line and c = tim e o f peak response

RESULTS

2.11. Pilot Studies

Repeated exposure to antinociceptive testing procedures of naive mice (ie.,

non-injected) was assessed on the hot plate (Table 2.1.). Re-testing the same

mouse resulted in s ign ifican tly increased latency. A ll subsequent

antinociceptive data presented represent latencies from mice tested once

only.

Table 2.2. shows the e ffe c t of repeated saline in jection on nociceptive

responses o f mice. There were s ign ificant changes (eg., single in jection 11.7 -

0.73, tr ip le in jection 9.2 - 0.85, t- te s t p<0.05), but these were quan tita tive ly

small.

2.12. E ffect of DFP on Nociception in Mice

Table 2.3. shows tha t DFP at lm g/kg did not exh ib it antinociceptive a c tiv itie s

a t the times studied. However, a t a 2mg/kg dose a 2- to 3-fo ld increase in

hot-p la te response tim e was observed over th is period. A t th is higher dose,

mice exhibited symptoms of anticholinesterase poisoning, such as trem ors and

fasiculations. Mice showed hind-limb abduction and impaired and reduced

ambulation.

When peanut o il was used as the vehicle fo r DFP (Table 2.4.) no a lte ra tion in

hot-pla te response tim e was observed compared w ith saline.

The e ffe c t o f the opioid antagonist, naloxone, and the muscarinic cholinerg ic

antagonist, atropine, on the antinociceptive a c tiv ity o f DFP at 2mg/kg is

shown in Table 2.5.

Table 2.1. E ffect of Repeated Exposure to Antinociceptive Testing

Procedures on Hot-Plate Times of Naive Mice

One exposure

Second exposure (+ 30 mins)

Third exposure (+ 4 hours)

H ot-P la te Latency (Secs)

9.3 - 2.0

17.0 - 3.3

16.8 i 2.0

Each value represents the mean - SEM of 12 observations.

Table 2.2. E ffect of Multiple Saline Injections on Hot-Plate Response Time in Mice

H ot-P la te Latency (Secs)

Number o f Time from last in jection to test

Injections

30 mins 10 mins 3 mins

1 11.7 - 0.7 (56) 10.3 - 1.1 (12) 9.6 - 0.7 (18)

2 9.3 - 0.8 (12) 14.6 - 1.4 (6) 11.1 - 1.6 (18)

3 9.2 - 0.8 (17) 10.4 - 1.2 (11) 9.3 - 1.0 (12)

Each value represents mean - SEM. Number o f observations are given in

parentheses.

Mice receiving two in jections were f irs t in jected 60 minutes before testing,

and those receiving three in jections had the ir f irs t two in jections 60 and 59

minutes before testing.

Table 2.3. E ffect of DFP on Hot-Plate Reaction Times in the Mouse

Saline

DFP (lm g/kg)

DFP (2mg/kg)

H ot-P la te Latency (Secs)

Time a fte r In jection

0.3 hours

11.9 - 1.6

9.7 - 0.8

28.4 - 5.6*

1 hour

13.2 - 1.1

9.4 - 0.7

19.0 - 2.0*

2 hours

12.7 - 1.5

11.8 - 1.2

18.7 - 2.8

4 hours

11.7 - 1.0

12.0 - 0.9

33.0 - 8.7*

Each value represents mean - SEM o f at least 6 observations. Unpaired t -

test vs. saline *p<0.05.

Table 2.4. E ffect of Different Vehicles on the Antinociceptive Activity of DFP

Vehicle used

Peanut O il

Saline

H ot-P la te Latency (Secs)

lm g/kg

9.8 - 1.1 9.2 t 2.3

9.4 - 0.7

2mg/kg

18.6 +4.5

19.0 - 2.0

Each value represents the mean - SEM of 6 observations.

Table 2.5.

Treatm ent

Naloxone

Atropine

Effect of Atropine and Naloxone on 2mg/kg

DFP Induced Antinociception in Mice

H ot-P la te Latency (secs)

Saline 33.9 - 5.9 (13)

0.1 mg/kg 26.0' - 4.9 (12)

1.0 mg/kg 29.9 - 7.5 (6)

10 mg/kg 33.6 - 5.0 (12)

Saline 28.1 - 2.6 (6)

1 mg/kg 16.4 - 2.1 (6)*

10 mg/kg 16.2 - 3.8 (6)*

20 mg/kg 12.1 - 0.9 (6 )**

Each value represents mean - SEM. Number of observations are in

parentheses. Unpaired t-te s t vs saline *p<0.005 **p<0.001.

Doses o f naloxone between 0.1 and lOmg/kg had no e ffe c t on the hot-p la te

reaction times of mice receiving DFP (2mg/kg). However, when mice

received atropine (1 to 20mg/kg) hot-p la te reaction tim e was s ign ifican tly

reduced. A 20mg/kg, response times were not s ign ifican tly d iffe re n t from

mice receiving saline in jections.

2.13. E ffect of DFP and Drugs Reversing the Effects of DFP on Opioid

Antinociception in Mice

The antinociceptive ac tiv ities of morphine, fentanyl and a lfen tan il are shown

in Table 2.6. A c tiv itie s are represented in terms o f computed ADj-g values as

a measure o f antinociceptive potency.

In mice pre-treated w ith lm g/kg DFP the AD^g values of morphine and

fentanyl were unaltered. However, a tw o-fo ld increase in the antinociceptive

a c tiv ity o f a lfen tan il (as shown by a reduced AD^g), was observed fo llow ing

DFP pre-treatm ent.

The atropine-pralidoxim e mix was administered as a trea tm ent fo r DFP

poisoning. The e ffe c t o f DFP on the antinociceptive ac tiv ities o f fentanyl

and a lfen tan il was unaltered by the atropine-pralidoxim e mix trea tm ent,

however, an increased antinociceptive a c tiv ity o f morphine (denoted by

s ign ifican t decreases in AD^g) was observed by this treatm ent.

Diazepam was used as an additional component in the trea tm ent o f DFP

poisoning. No s ign ificant changes in AD^g values fo r the opioids were

observed fo llow ing additional adm inistration o f diazepam (Table 2.6.).

Table 2.6. E ffect of DFP (Img/kg) and of Drugs Reversing DFP

Poisoning on Opioid-Induced Antinociception in Mice

A D cn Values

A lfe n ta n il

(gg/kg) .

329.7 - 23.3

164.3 - 11.0**

175.0 - 1 5 .0 **

207.3 ± 3 3 .0 **

AD^g values are the mean - SEM (of at least 9 observations). AD^g values

are doses required to increase hot-p la te reaction tim e to 30 seconds and are

calculated from the fu ll dose response curves fo r each agonist. Normal

deviate fo r comparison of means vs. saline **p<0.01; ***p<0.001.

Treatm ent Morphine Fentanyl

(mg/kg) (gg/kg)

Saline 20.7 - 1.9 66.4 - 6.7

DFP 24.7 - 2.5 52.5 - 5.6

DFP + A tropine +

Pralidoxime

12.0-1.0* 54.3 - 5.9

DFP + Atropine +

Pralidoxime + Diazepam

10.7 ± 1.7* 53.3 ± 8.3

2.14. E ffect of Atropine and Pralidoxine and Diazepam on Nociception

As a control, atropine-pralidoxim e mix, and diazepam were administered

alone, to determ ine the ir e ffe c t on mouse hot-p la te latency.

When the atropine-pralidoxim e mix was administered at 1 m inute, w ith saline

given at 0 and 30 minutes, testing at 60 minutes gave a response tim e o f 10.5

- 0.7 seconds (n=12), which is not s ign ifican tly d iffe ren t from saline contro l

values.

The e ffe c t o f diazepam on nociceptive responses is shown in Table 2.7.

Diazepam did not a ffe c t hot-p la te response times, even at doses which

produced clear motor incapacitation.

Using a PEG/saline in jection vehicle o£ in jecting saline i.m . did not give hot­

plate reaction times s ign ifican tly d iffe re n t from saline s.c. (Table 2.7.)

2.15 E ffect of Neostigmine and Pyridostigmine on Opioid Antinociception

The e ffe c t o f the quaternary anticholinesterase agents neostigmine and

pyridostigm ine opioid antinociception is shown in Tables 2.8 and 2.9.

Neostigmine did not produce any s ign ifican t potentia tion o f a lfen tan il over

the dose range studied. Pyridostigm ine pre-treatm ent did not s ign ifican tly

a lte r AD^g values fo r the opioids studies, though there was a small

enhancement o f a lfen tan il antinociception (p=0.105).

Table 2.7. E ffect of Intramuscularly-Administered Diazepam

and Injection Vehicle on Hot-Plate Reaction Times in Mice

Treatm ent

Saline

PEG/Saline

Diazepam 6.25 pm ol/kg

Diazepam 12.5 pmol/kg

H ot-P la te Response

Time (Seconds)

8.7 - 1.3 (12)

8.2 - 0.5 (6)

8.7 - 1.6 (6)

10.2 - 2.1 (6)

Values show means - SEM o f hot-p la te response times. Number

observations is shown in parentheses. PEG = polyethylene glycol.

Table 2.8. E ffect of Various Doses of Neostigmine on the

Antinociceptive Activity of Alfentanil

Saline

Neostigmine 50gg/kg

Saline

Neostigmine lOOgg/kg

Saline

Neostigmine 200gg/kg

A lfe n ta n il AD,-g (gg/kg)

143 - 18

146 1 13

178 - 13

170 - 11

155 - 14

128 - 14

Values are mean + SEM o f 6 observations on hot-p la te reactions times.

Contro l (saline) AD^g values were obtained on the same day fo r each dose o f

neostigmine.

Table 2.9. E ffect of Pyridostimine (Img/kg) On Antinociceptive

Potencies of Morphine,Fentany 1 and Alfentanil

Treatm ent

Saline

Pyridostigm ine

Morphine

(mg/kg)

16.8 - 1.3

lm g/kg 16.1 - 1.4

AD^g Values

Fentanyl

(pg/kg)

37.6 - 4.3

36.7 - 2.8

A lfe n ta n il

(pg/kg)

220 - 17.9

172 - 18.9

Values are mean - SEM of 6 observations on hot-p la te reaction tim es.

Control (saline) AD ^q values were obtained on the same day fo r each opioid.

Table 2.10.

Treatm ent

A lfe n ta n il

DFP +

A lfen tan il

Naloxone Antagonism of Antinociception Induced by

Alfentanil With and Without DFP

Naloxone a d 50+s e m Dose Ratio Slope of Apparent pA2 values

(pg/kg) pg/kg pA2 plot (95% confidence in terva

0 0.22 - 0.02

10 0.26 - 0.03 1.18

100 0.57 - 0.07 2.59

1000 3.20 - 0.30 14.55 -1.00 8.30 (8.26 - 8.35)

0 0.13 - 0.07 -

10 0.25 - 0.06 1.92

100 0.56 - 0.06 4.31

1000 3.30 - 0.09 25.39 -0.99 8.95 (6.30 - 11.60)

AD^g values are the mean doses - SEM required to increase hot-p la te reaction tim e to 30 seconds.

2.16 Antagonism of Alfentanil, Alone and With DFP, on Naloxone

The e ffe c t o f naloxone antagonism on the antinociceptive a c tiv ity o f

a lfen tan il and the e ffe c t o f naloxone on DFP plus a lfen tan il antinociception

is shown in Table 2.10.

Naloxone antagonism was measured as 'apparent pA£' according to Hayashi

and Takemori (1971). The pA2 value represent the a ff in ity fo r the antagonist

naloxone fo r its receptor. The values obtained fo r 'apparent PA21 were very

s im ilar indicating tha t the compounds were acting through the same

receptors. The slope o f the pA2 p lo t were very s im ila r to the theore tica l

value of -1 fo r com petitive antagonism.

2.17. E ffect of DFP upon Nociceptive Responses in the Rat

Figure 2.3. shows the tim e course fo r the e ffe c t o f DFP adm in istration on the

nociceptive responses o f the ra t in the paw pressure test.

Saline base-line responses remained constant over the tim e studied and w ith

repeated testing. A t lm g/kg, DFP did not a ffe c t the nociceptive response.

However, there were s ign ificant (p<0.001) increases in response thresholds fo r

a ll the tim e points studied fo llow ing DFP at 2mg/kg (Fig 2.3.) A t the

2mg/kg dose DFP produced clear signs o f anticholinesterase poisoning, such

as marked tremors. However, the leg w ithdrawal and struggling responses

were not impaired.

2.18 E ffe c t o f DFP P re-Treatm ent on Opioid Antinociception in Rats

The e ffe c t o f the sub-antinociceptive dose o f DFP (Img/kg) on the

antinociceptive ac tiv ities o f morphine, fentanyl and a lfen tan il are shown in

(Figs. 2.4., 2.5. and 2.6.). DFP pre-treatm ent did not a ffe c t the

antinociceptive a c tiv ity o f morphine, but both fentanyl and a lfen tan il were

s ign ifican tly potentiated at low doses o f these opioids, (p < 0.01, unpaired t -

test).

RESSURE(g)00

0 0 -

00 -

0 0 -

0 0 -

00 -

yy 90 TIME(mins)Pre ao 45 60

Fig. 2.3. Time Course for the Effect of DFP on the Nociceptive Responses in the Rat Paw

Pressure Tests

Closed circles: saline treated animals. Open triangles: DFP (lmg/kg) treated animals. Closed triangles: DFP(2mg/kg) treated animals. Points represent means and SEM of 10 observations.

PRESSURE(g)8OO1

700-

1 0 0 -

2.5 20 DOSE(mg/kg)

Fig. 2.4. Effect of DFP on Morphine Antinociception in the Rat Paw Pressure Test

The abscissa shows dose for morphine (s.c.) in mg/kg Closed circles: control treated. Open circles: DFP (lmg/kg) treated. Each histogram represents mean and SEM of 10 observations.

Means represent response at peak.antinociceptive times

PRESSURE(g)800 - i

700

600

500-

400-

300-

2 0 0 -

10 0 -

5h T5o 260 400 DOSE(jjg/kg)

Fig. 2.5. Effect of DFP on Fentanyl Antinociception in the Rat Paw Pressure Test

The abscissa shows dose for fentanyl (s.c.) in yg/kg.Closed circles: control treated. Open circles: DFP (lmg/kg) treated. Each histogram represents mean and SEM of 10 observations.

I Means represent response at peak antinociceptive times

PRESSURE(g)

700-

600-

500

400-

300

200

100

80 DOSE(|jg/kg)4020

Fig 2.6. Effect of DFP on Alfentanil Antinocoception in the Rat Paw Pressure Test

The abscissa shows dose for alfentanil (s.c.) in yg/kg. Closed circles: control treated. Open circles: DFP (lmg/kg) treated. Each histogram represents mean and SEM of 10 observations.

Means represent response at peak antinociceptive times

DISCUSSION

2.19. Drug Injection Times

The choice o f in jection times and in it ia l doses o f drugs were determined from

p ilo t experiments or were based on available lite ra tu re .

Neostigmine adm in istration (30 minutes before antinociceptive testing) and

in it ia l dose were based on studies o f Lawrence and Livingstone (1979).

Pyridostigm ine has a longer duration of action than neostigmine (B ritish National

Form ulary, 1983) and so fo r these studies pyridostigm ine was in jected one hour

before testing. A high dose of lm g/kg O-D^g = 1.3mg/kg mice; Mestinon data

sheet) was used so tha t any e ffe c t would more like ly be revealed.

Naloxone was administered 30 minutes before testing, a tim e generally given in

the lite ra tu re (eg, Koehn and Karczm ar, 1978).

2.20. E ffect of DFP on Nociceptive Responses in Mice and Rats

Though at lm g/kg DFP did not appear to show any appreciable antinociceptive

a c tiv ity in the mouse hot-p la te test, at 2mg/kg the increase in hot-p la te reaction

tim e was marked. This apparent antinociceptive a c tiv ity o f DFP was long-

lasting. However, at this higher dose mice showed pronounced signs o f

anticholinesterase poisoning, including tremors and fasiculations, decreased

ambulation and apparent sedative ataxia. The nociceptive response in the hot­

plate test involves the animal lif t in g and lick ing one or more of its paws. As this

response requires a complex set of co-ordinated non-reflexive movements, i t is

possible tha t the apparent antinociceptive e ffe c t observed fo r DFP a t 2mg/kg

was due to impaired muscular function. This im pairm ent could be o f centra l

origin, eg, disruption of contro l of movement or peripheral orig in, eg, muscular

weakness, or indeed, both.

I t has been reported that in rats at 2mg/kg s.c. DFP caused hind-lim b abduction

(Fernando et al, 1984) and at 2.2mg/kg i.m . the animals appear behaviourally

depressed and le tharg ic (Samson, 1984). Even at doses below lm g/kg, DFP is

reported to produce physical incapacita tion (Harris and S titcher, 1984). I t is not

unreasonable to presume from observations in this study and the available

lite ra tu re , tha t DFP adm in istration could im pair the nociceptive response

w ithout necessarily possessing antinociceptive a c tiv ity . The e ffe c t o f DFP on

the locomotor a c tiv ity of mice and the im plications in nociceptive testing is

given fu ll consideration in Section 3.13.

The data presented in this thesis are consistent w ith the findings o f Cox and Tha

(1972) who did not observe any antinociceptive fo r DFP up to 2mg/kg in the

mouse hot-p late test.

In contrast, studies in the ra t indicate tha t DFP is antinociceptive in this

species. Doses as low as 0.lm g/kg s.c. have been shown to induce antinociception

in the ra t hot-p la te test (Koehn et al, 1980) and 0.5mg/kg s.c. in the ra t ta il f lic k

test (Koehn and Karczm ar, 1978). The results presented in this thesis show tha t

DFP at 2mg/kg produced marked antinociception in the paw pressure test.

Though this antinociceptive dose is much higher than tha t reported by Koehn et

al (1980), this may be a re flec tion on the use o f pressure as the nociceptive

stimulus. The nociceptive response to pressure is believed to be predom inantly

mediated through k -opioid receptors, whilst heat noxia are believed to act

through p-opioid receptors (Tyers, 1980; Upton et al, 1983).

The observation tha t naloxone up to lOmg/kg did not a lter the reaction tim e

fo llow ing adm in istration o f DFP (Table 2.3.) suggests tha t the apparent

antinociceptive a c tiv ity o f DFP. is not mediated through an opioid system.

A tropine, however, did antagonize the increased latencies, and at 20mg/kg

to ta lly reversed DFP's e ffe c t. The apparent antinociceptive a c tiv ity is therefore

only mediated via muscarinic receptors and this contrasts w ith e ffec ts in the ra t

where both opioid and muscarinic receptors mediates its action (Koehn and

Karczm ar, 1978).

Clear species differences emerge, w ith mice being insensitive to an

antinociceptive action o f DFP w h ils t in rats, DFP may product potent

antinociception.

2.21. E ffect of DFP on Morphine Antinociception

DFP had no e ffe c t on morphine antinociception (Table 2.6.) and this contrasts

w ith the findings o f Bhargava and Way (1972). They reported po tentia ting

a c tiv ity o f DFP upon morphine antinociception, though i t should be stressed tha t

these effects, while consistent, fa iled to reach s ta tis tica l significance. The

possibility o f vehicle-re la ted e ffects must be entertained since Bhargava and

Way (1972) used peanut o il fo r DFP adm in istration and 0.9% saline was used in

the experiments described in this thesis. However, though peanut o il has been

shown to induce sedation (Pinsky et al, 1982) no antinociceptive e ffec ts fo r DFP

in th is vehicle could be shown (Table 2.4.) and this possibility can be re a lis tica lly

excluded.

A t a dose of 0.2mg/kg, Saxena (1938) found that DFP did not potentia te morphine

in the ra t ta il c lip test. Though there are many reports o f cholinerg ic drugs

potentia ting opioid antinociception (see Table 1.2.) the available data appears to

m ilita te against a potentia ting e ffe c t o f DFP in morphine-induced

antinociception.

2.22. Effect of DFP on Fentanyl and Alfentanil Antinociception

Though the a c tiv ity of the synthetic opioid analgesic fentanyl was not found to

be potentia ting by DFP in the mouse hot-p late test, the s truc tu ra lly -re la ted

compound a lfen tan il was potentia ted tw o-fo ld (Table 2.6.).

In addition, a lfen tan il antinociception in the ra t paw pressure test appeared to be

potentiated by DFP pre-trea tm ent. Though this potentia ting e ffe c t was only

observed at low doses o f a lfen tan il, i t seems like ly tha t employment o f a c u t-o ff

weight masks potentia tion at higher doses. This problem was also encountered in

the hot-p la te data (see Section 2.11.1).

Fentanyl also appeared to be potentiated in rats by DFP in the same way as

a lfen tan il. So a lfen tan il is potentiated by DFP in both mouse and ra t

antinociceptive test, though fentanyl was only potentiated in the ra t. This could

be a re flec tion o f d iffe ren t nociceptive mechanisms in the ra t compared to those

in mice.

2.23. Proposed Mechanisms fo r DFP Potentia tion o f A lfe n ta n il

Though morphine, fentanyl and a lfen tan il are a ll p rim arily u-opioid agonists,

DFP has been shown in this study to have a d iffe re n tia l potentia ting e ffe c t on

these opioids. There are several ways by which DFP could potentia te the

a c tiv ity o f opioids; these are a lterations in absorption, d is tribu tion, receptor

action, metabolism or excretion.

2.23.1. Absorption

There are a number o f factors which a ffe c t absorption of drugs from s.c.

in jection sites. A major fac to r is cutaneous blood flow and DFP trea tm ent is

believed to decrease c ircu la to ry rate (Ramachandran, 1967). In addition, DFP

dose dependently induces hypothermia (Kenley, e t al, 1982) which would decrease

peripheral blood flow . These factors would tend to reduce drug absorption from

a s.c. site and furtherm ore would not d iscrim inate between the opioids studied.

I t is also probable that drug absorption is not a lim itin g fac to r fo r fen tanyl and

a lfen tan il. Both these drugs are lipid-soluble w ith octanokw ater p a rtition

coeffic ien ts o f 860 and 130 fo r fentanyl and a lfen tan il respective ly (Meuldemans

et al, 1982). Morphine has low lip id so lub ility w ith an octanokw ater p a rtition

coe ffic ien t o f 1.42 (Kaufman et al, 1975). A d iffe re n tia l in te raction between

DFP and a lfen tan il at the site o f in jection is therefore unlike ly.

2.23.2 D is tribution

Many drugs are bound to plasma proteins and may compete fo r the same binding

sites. Such com petition could result in displacement and altered

pharmacokinetic behaviour o f one drug. This type of drug in te raction would be

pa rticu la rly im portant fo r drugs which have high frac tiona l binding (see

MacKichan, 1984 fo r review).

Both fentanyl and a lfen tan il have high frac tiona l binding to blood proteins

(Meuldemans, 1982) as does DFP (M artin, 1985). I t is possible tha t DFP could

displace a lfen tan il from binding sites, allowing more a lfen tan il to enter the

brain. This possib ility was studied and is described in Chapter 5.

2.23.3 Receptor Action

DFP may act at the receptor level to increase the potency o f a lfen tan il. For

example, DFP may be producing opioid receptor super-sensitivity to a lfen tan il.

P ilo t binding studies using a lfen tan il were performed, but were inconsistent.

This is most probably due to the very rapid dissociation from the receptor site

tha t has been reported fo r this ligand (Leysen et al, 1983).

In fac t, an in te raction between DFP at the receptor level would not explain the

d iffe re n tia l action o f DFP on a lfen tan il, since a ll three opioids studied exert

pharmacological e ffec ts through p-opioid receptors (Upton et al, 1983; Leysen et

al, 1983).

2.23.4. Metabolism

In addition to being a potent inh ib ito r o f cholinesterase, DFP may w e ll in h ib it

other enzymes including those involved in drug metabolism (see O’N e ill, 1981).

For example, a s ign ificant inh ib ition o f m itochondrial aldehyde dehydrogenase

occurs fo r at least 85 days fo llow ing adm inistration o f 0.5mg/kg DFP (Messiha,

et al, 1983).

DFP potently inhib its the enzymes hydrolysing substance P (Kato et al, 1980) and

metabolising enkephalins (Snead et al, 1980). A lfen tan il is rapid ly metabolised

fo llow ing i.v. adm inistration, w ith 50-60% metabolised a fte r 4 minutes (M ichie ls

et al, 1981) by N -dealkylation and O-demethylation (Meuldemans, 1980). However

the term ination of a lfentan il's action is predominantly dependent on a

red istribu tion mechanism (Stanski and Hug, 1982). I t is conceivable tha t DFP

could inh ib it the enzymes responsible fo r the metabolism of a lfen tan il, thereby

increasing its antinociceptive a c tiv ity . However, i t must be borne in mind tha t

antinociceptive testing was carried out five minutes a fte r a lfen tan il

adm inistration and over such a short tim e period, DFP would have to be a very

potent inh ib ito r to produce a tw o-fo ld potentia tion o f a lfen tan il.

2.23.5.Excretion

If, as mentioned above, red istribu tion is the main means o f te rm ination o f

action, an in te raction between DFP and a lfen tan il in excretion processes would

probably not a ffe c t a lfen tan il action. Moreover in rats only 0.2% o f the parent

drug is excreted in the urine (Meuldemans, 1980b), and so i t is un like ly tha t

impaired renal excretion would have any e ffe c t on the antinociceptive a c t iv ity

o f a lfen tan il.

2.24. Effect of Atropine and Pralidoxime on DFP Potentiation of Alfentanil

Antinociception

The potentia ting e ffe c t o f DFP on a lfen tan il antinociception may be independent

o f an action on the cholinergic system as the adm inistration o f the m uscarinic

receptor antagonist atropine and pralidoxime to regenerate cholinesterase

enzymes did not a lte r the AD ^q value fo r DFP plus a lfen tan il. However,

pralidoxime is unable to cross the blood-brain barrier, so tha t brain

cholinesterase would remain largely inhib ited by DFP even a fte r pralidoxim e

adm inistration. Though atropine would block muscarinic receptors, i t is possible

tha t an in teraction between DFP and a lfen tan il could involve unprotected

centra l n ico tin ic receptors. Other possibilities regarding the nature o f DFP and

a lfen tan il in teraction are given in Section 4.11.

2.25. E ffe c t o f A trop ine and Pralidoxim e on the A n tinociceptive A c t iv ity o f

Morphine

DFP alone did not a ffe c t the AD^g fo r morphine, but when atropine and

pralidoxime was also administered morphine's antinociceptive a c tiv ity was

enhanced. As mentioned in Section 2.24., the atropine-pralidoxim e mix does not

pro tect centra l n ico tin ic receptors and so i t may be tha t by blocking the

muscarinic receptors w ith atropine, more o f the non-metabolised acetylcholine

would be able to in te rac t w ith n ico tin ic receptors. In other words, the presence

o f a muscarinic antagonist in a cholinesterase inhib ited system could lead to

increased n ico tin ic response. In consideration o f this, i t is worth noting tha t

there have been several studies in mice, ra t and dog showing potent

antinociceptive a c tiv ity o f n ico tin ic agonists (see Table 1.3.).

This in teraction, however, would have to be specific fo r morphine and the

n ico tin ic response since neither fentanyl or a lfen tan il had altered AD^g

fo llow ing atropine and pralidoxime.

An a lte rna tive explanation fo r these findings is tha t morphine may be in te rac ting

w ith atropine. Indeed, i t has been reported tha t in the ra t hot-p la te test,

atropine at 2mg/kg s ign ifican tly potentiated the antinociceptive a c tiv ity o f

morphine (Malec and Langwinski, 1982). This report also notes tha t atropine did

not a lte r the antinociceptive a c tiv ity o f fentanyl or o f pentazocine. The results

of this study showing d iffe re n tia l potentia tion o f morphine antinociception by

atropine are supportive o f the results presented in this thesis.

An additional mechanism for the potentia tion of morphine by atropine which

should be considered is that respiratory alkalosis enhances the percentage of

unionised morphine in the c ircu la tion and thereby increasing the amount entering

the brain (Schulman et al., 1984). I t is possible tha t the additional adm in istra tion

of atropine and pralidoxime produces a degree o f respiratory alkalosis, which

through pharmacokinetic mechanisms potentiates the antinociceptive a c tiv ity o f

morphine plus DFP.

2.26. Effect of Diazepam on the Antinociceptive Activity of Opioids in Mice

Receiving DFP, Atropine and Pralidoxime

Diazepam administered alone did not produce any increase in ho t-p la te la tency

in the mouse. Diazepam has, in fac t, been reported to possess antinociceptive

a c tiv ity in humans (Haas et al, 1979) though at doses much higher than those

described in this thesis.

Diazepam has been shown to both reduce (Mantegazza, 1982) and to potentia te

(M atla and Langwinski, 1982) the antinociceptive a c tiv ity o f opioids including

morphine and fentanyl. In this study, co-adm in istration of diazepam did not

a lte r the AD^g values o f the three opioids studied. I t should be realised,

however, tha t the animals were receiving four d iffe ren t drugs in addition to the

opioid, so tha t any e ffects o f diazepam on antinociceptive ac tiv itie s could be

obscured by non-specific drug in teractions.

2.27. Variation in Antinociceptive Potency of Alfentanil in Mice

The values given fo r AD^g scores fo r a lfen tan il in Tables 2.6. and 2.8. are

d iffe ren t. The orig in of this disparity is not obvious as the animal stra in, test

conditions and experimentor were the same fo r both sets of experiments. Two

differences do exist however, and these are, f irs tly , tha t d iffe re n t a lfen tan il

batches were used and, secondly, these experiments were carried out at d iffe re n t

tim es of the year. Considering the second proposition, i t has been reported tha t

stim ulation-induced antinociception is subject to circadian varia tion (Buckett,

1981) and this has been confirm ed w ith morphine using the same species and

apparatus described in this thesis (P.G.Green, unpublished observations). In

addition, there are reports of circannual varia tion in opioid response as Buckett

(1981) reports tha t stim ulation-induced antinociception as measured by latencies

in mice vary throughout the year. In v itro response of vasa deferentia to

2 5morphine and D -A la , Met enkephalinamide have also been shown to exh ib it

circannual varia tion, w ith a five -fo ld varia tion in potency (De Ceballos and De

Felipe, 1984). The varia tion o f a lfen tan il potency is also considered in Section

5.18. This in form ation described above highlights the importance o f carrying out

appropriate controls in para lle l w ith the experiment.

CHAPTER 3

Locomotor Activity of Mice

Effect of DFP and Drugs Used in the Treatment of DFP Poisoning

INTRODUCTION

3.1 Assessment of Drug Effect on Spontaneous Locomotor Activity

As a behavioural measure, locom otor a c tiv ity is probably one o f the most

simple. Its s im p lic ity enables many experiments to be carried out

simultaneously and data to be collected autom atica lly. On the other hand, its

s im p lic ity means tha t only general ambulation is measured and in form ation

on stereotypic resonses (eg, sn iffing and grooming), co-ordination and

muscular r ig id ity cannot be recorded. I t is well known tha t po ten tia lly useful

in form ation goes unrecorded (Krsiak et al, 1970). The in form ation gained

from these locomotor studies should therefore be viewed as only a basic

measure o f a lterations in behaviour and too detailed an in te rp re ta tion o f the

results should be avoided. Nevertheless, w h ils t bearing in mind the

lim ita tions o f these techniques, drug-inducing a ltera tion o f locom otor

function does give an indication of certa in drug-receptor in teractions w ith in

the centra l nervous system. Indeed, i t has been suggested tha t in order to

assess the behavioural e ffects o f a drug, data must be obtained on its

influence on spontaneous m otor a c tiv ity (Robbins, 1977).

Data presented in this thesis shows tha t DFP at 2mg/kg produced apparent

antinociception (Table 2.3.). Though the hot-p la te reaction tim e was

s ign ifican tly increased compared to saline controls, mice exhibited reduced

ambulation and muscular weakness (as evidenced by hind limb abduction). I t is

possible tha t impaired muscular function and co-ordination could delay the

nociceptive end-point (the licking response). Experiments were therefore

devised to assess the e ffects of DFP on spontaneous motor a c tiv ity , and

a c tiv ity fo llow ing opioid adm inistration. The e ffects o f drugs used in

trea ting anticholinesterase poisoning were also assessed fo r e ffec ts on

locomotor a c tiv ity .

MATERIALS AND METHODS

3.2. Animals and Experimental Conditions

Male albino mice (CD-I strain; 25-30g) were used as described in Section

2.2.1. Procedures fo r equ ilibration were also as those previously described.

3.3. Drugs

The drugs used were obtained from sources listed in Section 2.6.

3.4. Dosing Protocols

Mice were in jected subcutaneously using the same tim ings to para lle l the

antinociceptive procedures described in Section 2.3.3. Doses o f opioids used

were 20mg/kg morphine, 80ug/kg fentanyl and 400pg/kg a lfen tan il.

3.3. Locomotor Activity Testing

Locomotor a c tiv ity in mice was measured using a system identica l to tha t

described by Francis et al (1983) in which a photobeam m onitoring system was

linked to a m icrocomputer. The apparatus consisted of pairs o f industria l

grade in fra -red source and detector pairs. Three in fra -red ligh t beams cross

each test box 0.3cm above the box base, and are detected by appropriate

sensors. When the beam path is blocked or unblocked, a score is registered.

Data is collected and displayed on a m icrocom puter (BBC B) via an in te rface .

The software employs an in te rrup t routine to assess the state o f the

photocells (on or o ff) every 10ms, and a change in state is recorded as a score.

Locomotor hardware was b u ilt in the B iochem istry Workshops, U nivers ity of

Surrey. O riginal software w ritte n fo r this apparatus was adapted to display

the mean and standard deviation of counts (see Appendix C fo r listing).

Individual mice were placed in each testing box (18.3 x 11cm), allowing five

mice to be tested simultaneously. Locom otor a c tiv ity was measured

im m ediate ly a fte r the last in jection, w ith the exception o f some experiments

where DFP or saline were in jected 1 hour before testing. In jection tim ings

were the same as those described in Section 2.2.3.(a). Testing was m onitored

fo r 15 5-m inute epochs, so tha t changes in a c tiv ity over tim e could be

assessed.

3.6. Statistical Procedures

Locomotor scores were analysed fo llow ing data reduction. Mean locom otor

scores fo r each 5-m inute period were summed over the f irs t 15 and to ta l 75

minute periods. Means fo r the 15 and 75 m inute periods were obtained from

10 or 15 animals recorded on one, two or three separate days.

E ffe c t o f drug trea tm ent was analysed using m ultip le analysis of variance

followed by a posteriori examination using Duncan's M ultip le Range Test

(Duncan, 1955). These s ta tis tica l analyses were carried out using the SPSS

software on the University 's Prime computer (S ta tis tica l Packages fo r the

Social Sciences, Pennsylvania State University, Version M, Release 9.1.).

RESULTS

3.7. E ffect of DFP on Locomotor Activity

DFP at lm g/kg produced a s ign ificant decrease in locomotor a c tiv ity only

over 75 minutes compared to saline (Figure 3.1. and Figure 3.2.). However,

when DFP was in jected 1 hour before testing, DFP s ign ifican tly decreased

exploratory a c tiv ity over the f irs t 15 minutes. This depression o f locom otor

a c tiv ity was fu rthe r decreased fo llow ing DFP 2mg/kg .

3.8. E ffect of Atropine and Pralidoxime on Locomotor Activity

The atropine-pralidoxim e m ix produced hyperlocomotion which was not

decreased in animals pre-treated w ith DFP. The e ffe c t o f DFP alone was

reversed by the atropine-pralidoxim e m ix adm inistration.

3.9. E ffect of Diazepam on Locomotor Activity

Diazepam alone greatly depressed locomotor a c tiv ity and fu rthe r decreased

the hyperlocomotory e ffe c t o f DFP.

3.10. Effect of Various Treatments on Alfentanil Induced Hyperlocomotion

The hyperlocomotion fo llow ing a lfen tan il adm inistration was s ign ifican tly

decreased by DFP (Fig. 3.3.).

Over the f irs t 15 minutes, atropine-pralidoxim e mix adm in istra tion did not

s ign ifican tly a lte r the DFP e ffe c t, but over 75 minutes there was a reversal,

w ith increased locomotor a c tiv ity . The additional in jection o f diazepam

produced a marked decrease in a c tiv ity .

SCORE800 i

700-

600-

500-

400-

300-

2 0 0 -

1 0 0 -

10 15 20 25 30 35 40 45 50 55 6 0 65 70 755

TIME(mins)

Fig 3.1. Graph Showing the Effect of Various Drug Treatments on Locomotor Activity in Mice

Close circles: saline controls. Closed triangles: DFP (lmg/kg) treated. Open triangles: DFP (2mg/kg) treated. Open squares: morphine (20mg/kg) treated. Each graph represents the mean of at least 10 observations.

— p- I

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breviations: ALF

=

Alfentanil,

FENT

= Fentanyl,

MORPH

= Morphine,

SAL

= Sa

line

DFP

=

Di-i

sopr

opyl

fluo

roph

osph

ate,

ATR/PRAL

= Atropine

and

Pralidoxime, DIAZ =

Diaz

epam

Arrows

in this

table

indicate

significant

changes

in locomotor

activity

in mi

ce

10000-J

frJRi FF jfiL+flLFENT DFP+ftLFENT DFP+RTR/PR OFP+DIflZ. SRLINE+RTR

+RLFENTRNIL +R/P+RLF /F ’RhL+hLF

Fig 3.3. Effect of DFP on Alfentanil-inducedhyperlocomotion over 15 and 75 minutes

Black histograms: Grey histograms: SAL + ALFENT:DFP + ALFENT:DFP + ATR/PR + ALFENTANIL:

locomotor score over 15 minutes locomotor score over 75 minutes Saline and alfentanil treated. DFP and alfentanil treated.DFP, atropine and pralidoxime and alfentanil treated.

DFP + DIAZ+ A/P + ALF: DFP, diazepam, atropine and

pralidoxime and alfentanil treated.

Values represent mean - S.E.M. o f at least 10 observations.

Saline contro l scores: 15 mins 1442 - 108

(n=5) 75 mins 5630 ^479

3.11. E ffect of Various Treatments on Fentanyl Induced Hyperlocomotion

The hyperlocomotory a c tiv ity o f fentanyl was not s ign ifican tly altered by

adm in istration o f DFP or DFP + atropine/pra lidoxim e over 15 or 75 m inute

periods (Fig. 3.4.). The additional in jection o f diazepam, however,

s ign ifican tly depressed locom otor a c tiv ity scores.

3.12. E ffect of Various Treatments on Morphine Induced Hyperlocomotion

Morphine-induced hyperlocomotion was s ign ifican tly decreased fo llow ing DFP

pretreatm ent, but only over the 75 m inute period (Fig.3.5.). The atropine-

pralidoxime m ix had no e ffe c t on morphine hypolocomotion. Diazepam

produced marked decreases in locom otor scores, and though this was

s ign ificant over the f irs t 15 minutes s ta tis tica l s ign ificant differences were

not observed over the 75 minutes, despite there being nearly a 34-fo ld

d ifference; very high pooled standard deviations existed in these groups.

LOCOMOTOR SCORE 10000-

5000-1

DFP+FEHT DFP+ftTR/PR QFP+DIflZ, +FENT fi/P+FENT

SflLINE+flTR/P+FENT

FENTRNIL SflL+FENT

Fig. 3.4. Effect of DFP on fentany1-inducedhyperlocomotion over 15 and 75 minutes

Black histograms Grey histograms: SAL + FENT:DFP + FENT:DFP + ATR/PR + FENTANYL:

DFP + DIAZ + A/P + FENT:

locomotor score over 15 minutes locomotor score over 75 minutes Saline and fentanyl treated.DFP and fentanyl treated.DFP, atropine and pralidoxime and fentanyl treated.

DFP, diazepam, atropine and pralidoxime and fentanil treated.

Values represent mean t S.E.M. o f at least 10 observations.

Saline contro l scores: 15 mins 1384 - 141

75 mins 5957 - ~531

B$^^/+++////B

LOCOMOTOR SCORE 1000th

5000-

PH

' ••••

SfiL+MORPH DFP+hORPH OFP+HTR/PRtilORPH I HE

DFP+DI.RZ,+ii/P+nuRPH

SRLINE+flTR/P+MmPPm

Fig 3.5. Effect of DFP on morphine-inducedhyperlocomotion over 15 and 75 minutes

Black histograms Grey histograms: SAL + MORPH:DFP + MORPH:DFP + ATR/PR + MORPHINE:

locomotor score over 15 minutes locomotor score over 75 minutes Saline and morphine treated.DFP and morphine treated.DFP, atropine and pralidoxime and morphine treated.

DFP + DIAZ + A/P + MORPH: DFP, diazepam, atropine and

pralidoxime and morphine treated.

Values represent mean - S.E.M. of a t least 10 observations.

Saline contro l scores: 15 mins 1541 - 122

(n=15) 75 mins 6095 - 404

^147901691^8944691576436^1079456^14795

DISCUSSION

3.13. E ffect of DFP on Locomotor Activity

There are several reports in the lite ra tu re describing the e ffec ts o f

anticholinesterase agents in im pairing or reducing ambulatory behaviour in

rodents. This e ffe c t has been shown fo r DFP (Koehn and Karczm ar, 1977,

1978; Koehn et al, 1980; Raslear and Kaufman, 1983; Samson, 1984; M artin ,

1985), physostigmine (Chaturvedi, 1984; Gonzales, 1984; Harris e t al, 1984;

Wolthuis and Vanworsch, 1984), pyridostigm ine (Harris et al, 1984; Wolthuis

and Vanworsch, 1984), sarin (Landauer and Romano, 1984b), soman (Wolthuis

and Vanworsch, 1984) and tabun (Landauer and Romano, 1984a; Wolthuis and

Vanworsch, 1984). The w e ll documented e ffe c t o f cholinergic agents on

locom otor a c tiv ity has been described as a le rt non-m otile behaviour (ANMB)

(Karczmar, 1977) and is believed to involve a cholinergic-dopam inergic dipole

in n ig rostria ta l and extrapyram idal pathways.

In this study, s ign ificant and marked decreases in locomotor a c tiv ity were

observed fo r DFP at lm g/kg in mice. When administered 1 hour before testing

to paralle l the antinociceptive testing procedures, DFP reduced exploratory

motor a c tiv ity . In in terpre ting the consequences of this data, i t is not

unreasonable to suggest tha t DFP induced 'antinociception' was observed as a

result of impaired motor co-ordination and muscular function ra ther than an

inh ib ito ry action on nociceptive systems. This suggestion gains credence

when the antagonist data is taken into consideration (Table 2.5). The opioid

antagonist naloxone did not a lte r the DFP antinociception but atropine which

reversed the hypolocomotor e ffec ts of DFP (see below), also reversed

antinociceptive a c tiv ity .

3 . 1 4 . Effect of Atropine and Pralidoxime Alone and With DFP on Locomotor Activity

DFP induced reduction o f locom otor a c tiv ity appeared to involve the

cholinergic system as DFP's e ffec ts were com pletely reversed fo llow ing

atropine-pralidoxim e mix adm in istration. When administered alone, atropine

and pralidoxime produced hyperlocomotion. Hyperlocomotion has been

previously reported fo r atropine (Chatervedi, 1984; Seidel et al, 1979) and fo r

another muscarinic antagonist, hyoscine (Sansone, 1983; Kuribara and

Tadokar, 1983).

3.15. Effect of Diazepam Alone and With DFP on Locomotor Activity

Inh ib ition o f locomotor a c tiv ity fo llow ing diazepam adm inistration was

marked and this is in contrast w ith some reports in the lite ra tu re . For

example, i t has a c tiv ity been shown tha t in mice, diazepam up to Im g/kg did

not a ffe c t locomotion (Sansone, 1982). In rats at 2.5mg/kg diazepam enhanced

ambulatory a c tiv ity w hilst at 20mg/kg i t was inhib ited (Matsubara and

Matshushita, 1982). In this study the dose of diazepam used was 1.78mg/kg,

which suggests tha t a marked motor depression was observed, m ice may be

more sensitive than rats to the locomotor depressant action o f diazepam.

When administered a fte r DFP the locomotor a c tiv ity o f diazepam was

decreased s t ill fu rthe r, so tha t the hypolocomotory e ffects o f these drugs

were addictive.

3.16. Effects of DFP on Opioid-Induced Hyperlocomotion

The action o f opioids in mice is to produce a stereotypic running response (eg,

C arro ll and Sharp, 1972) and in this study morphine, fentanyl and a lfen tan il a ll

produced this running response. This hyperlocomotion was reduced fo r a ll

opioids by DFP pretreatm ent. This is consistent w ith the lite ra tu re as DFP

at 2mg/kg has been reported to decrease morphine-induced running in mice

(Su and Loh, 1975). and oxotremorine has also been shown to decrease

morphine hyperlocomotion (Seidel, 1979). I t has been suggested tha t the

augmentation of the opioid-induced running response by cholinergic agents

may be due to an increase in dopamine u tiliza tio n produced by muscarinic

agonists (Seidel, 1979).

The data indicates tha t the e ffe c t o f DFP on opioid induced running is most

pronounced w ith a lfen tan il. However, i f DFP was enhancing the e ffe c t of

a lfen tan il in the same way as tha t observed fo r the antinociceptive a c tiv ity ,

one m ight have expected increased locomotor scores. A possible explanation

o f this apparent anomaly highlights a drawback encountered in this form of

measurement o f locomotor a c tiv ity . Observation o f mice receiving opioids,

especially a lfen tan il, revealed tha t during peak opioid a c tiv ity mice had

running f its ie, periods of intense running a c tiv ity interspersed w ith periods

o f im m ob ility (c f. dopamine catatonia). These periods o f catatonia appeared

w ith greater frequency and duration w ith increasing dose, at 800pg/kg, ha lf

or sometimes all o f the 60 seconds antinociceptive testing period m ice were

catatonic. I f DFP potentiated a lfen tan il, the measured locomotor a c tiv ity

score could have been reduced fo llow ing DFP i f the mice spent a greater

proportion of the ir tim e in a catatonic im m obile state. A c t iv ity is not a

unitary measure and photocell measured locomotor a c tiv ity cannot detect

such stereotyped ac tiv ities such as c irc ling , excessive grooming, lick ing or

b iting or d iffe ren tia te between walking and running ac tiv ities . More

sophisticated a c tiv ity monitors or scored human observation would be

required to iden tify these specific changes in a c tiv ity . So i f DFP potentia ted

a lfentanil's a c tiv ity and produced increased catatonia, this would be re flec ted

in a decreased locomotor a c tiv ity score.

3.17. The Effect of Atropine and Pralidoxime on the Locomotor Activity of

DFP and Opioid-Treated M ice

In DFP and opio id-treated mice the e ffe c t o f the atropine-pralidoxim e mix

trea tm ent on locom otor a c tiv ity was only s ign ificant fo r a lfen tan il over 75

minutes. I t is quite like ly tha t no s ign ificant e ffe c t was observed fo r the

other opioids due to the in te raction o f the four drugs administered and the ir

e ffects on locomotor a c tiv ity . For example, the atropine-pralidoxim e m ix in

addition to possessing hyperlocom otory a c tiv ity (see Section 3.18.), would

tend to antagonize the hypolocomotory e ffects o f DFP. This antagonism of

DFP could therefore decrease the incidence o f a lfen tan il catatonia (see

Section 3.20.). Furtherm ore, atropine is reported to potentia te morphine-

induced c irc ling (Seidel, 1979).

In summary, the combination o f opioid, DFP, and the atropine-pralidoxim e

mix produce a complex e ffe c t on locomotor behaviour. The observed

enhancement of a lfen tan il locomotion w ith atropine-pralidoxim e m ix is like ly

to have been a result o f both a reduced degree o f catatonia and an add itive

cholinergic hyperlocomotion.

3.18. E ffe c t o f Diazepam on Opioid-Induced Locomotion

The large decrease in locomotor score fo llow ing diazepam was common to a ll

three opioids studied. This e ffe c t has been reported previously fo r morphine-

and fentanyl-induced hyperlocomotion (M atla and Langwinski, 1982).

Benzodiazepines are known to have a sedative action and i t could w e ll be this

action on benzodiazepine GABA receptors which produced the decrease in

locom otor score. A lte rna tive ly , diazepam could be acting through another

receptor system; the role of the dopaminergic system has already been

mentioned in its involvement in locomotor a c tiv ity (Section 3.17.) and i t is

possible tha t the observed e ffe c t o f diazepam on locomotion was due to its

action inh ib iting dopamine function (Reubi et al, 1977). I t may also be

possible tha t opioid catalepsy (and hence im m obility ) was enhanced fo llow ing

diazepam, as catalepsy has been shown to involve inh ib ition of the centra l

dopaminergic pathway (Kushinsky and Hornykiew icz, 1972).

3.19. Interpretation of Locomotor Data

Locomotor measurement is a s im plis tic measure o f a ltera tion o f behaviour.

I t is subject to many errors and i t is well known tha t large quantita tive

differences between individual animal a c tiv ity scores are observed (Izazole-

Conde et al, 1983).

Observation of animals during locom otor testing procedures suggested tha t at

least part of the large varia tion o f scores between animals were probably due

to behaviours such as grooming. Mice were occassionally observed to be

grooming d irec tly in the path o f the in fra -red lig h t beam so tha t th e ir rapid

head movements increased the locom otor score.

As mentioned above, production o f catalepsy is produced w ith high doses o f

opioids and would tend to increase varia tion between animals. This form of

error has been reported in the lite ra tu re where alcohol at certa in doses

produces unchanged or increased locomotor a c tiv ity and simultaneously large

increases in im m ob ility (Smoothy and Berry, 1984). Observation revealed tha t

this was due to alcohol's e ffe c t of decreasing non-locomotor a c tiv ity such as

rearing which is not recorded by simple locomotor a c tiv ity monitors. These

lim ita tions notwithstanding, the locomotor a c tiv ity m onitor used in this study

gave a general indication o f the effects o f drugs on locomotor a c tiv ity o f

mice.

CHAPTER 4

Distribution of H Morphine, Fentanyl and Alfentanil

In Mouse Brain: E ffect of DFP Administration

INTRODUCTION

4.1 Distribution Studies

The d iffe re n tia l potentia tion o f a lfen tan il by DFP led to the conclusion tha t

the mechanism probably involved an e ffe c t on pharmacokinetics (Section

2.28) ra ther than altered pharmacodynamic parameters. A simple measure of

d is tribu tion u tilizes radiolabel tracing methodologies, wherein plasma and

brain levels o f a radiolabelled drug are determined. Drug concentration in

contro l and treated animals can then be compared.

A drawback to the method used in this thesis is tha t only to ta l ra d io ac tiv ity

was determined. The presence o f radioactive m etabolites, which may or may

not possess pharmacological a c tiv ity are not distinguished. However, fo r

fentanyl and a lfen tan il the short tim e period between adm in istration o f the

drug and k illing would mean tha t i t is like ly tha t the m a jo rity o f the

rad ioac tiv ity in the brain is the parent drug. For morphine the glucuronide

metabolites do not contribute s ign ifican tly to uptake in to the brain of

labelled morphine (H artv ig et al, 1984). However, morphine-3-glucuronide

does possess some antinociceptive a c tiv ity (P.G.Green, unpublished

observations).

MATERIALS AND METHODS

4.2. Animals and Experimental Conditions

Male albino m ice (CD-I strain; 25-30g) were used as described in Section

2.2.1. Procedures fo r equ ilibration were also as those previously described.

Radiolabelled morphine and fentanyl were supplied in ethanol, and

radiolabelled a lfen tan il was suplied in w a te r:ace ton itrile 30/70, ethanol 100.

The solvent was removed under a gentle stream o f dry nitrogen at room

temperature.

4.3. Drugs

The drugs used were obtained from sources described in Section 2.4. In

addition, the fo llow ing radiolabelled compounds were used:-

DRUGS

^ H Morphine sulphate

Specific A c t iv ity 20-24 C i/m m ol

Amersham International

3 H Fentanyl c itra te

Specific A c t iv ity 15-18 C i/m m ol

IRE, Fleurus (Belgium)

3 H A lfe n ta n il hydrochloride

Specific A c t iv ity 23.1 C i/m m ol

Janssen Pharmaceuticals

3 H Hexadecane tr it iu m standard Amersham International

Specific A c t iv ity 1.69 qC i/m l

* 3 H A lfen tan il was generously donated by Janssen Pharmaceutical

4.4. Dosing Protocols

Drug adm in istration procedures were carried out by identica l to tha t

described in Section 2.6. fo r DFP and opioid adm in istration.

Mice received either 0.9% w /v saline or DFP (Img/kg) s.c. This was followed

by s.c. in jections o f radiolabelled morphine, fentanyl or a lfen tan il

administered 30, 30 or 33 minutes la te r respectively. Two doses o f each

opioid were studied and each in jection contained 7pCi o f ^H-labelled opioid.

4.5. Distribution Studies Protocol

A t times o f peak opioid antinociceptive a c tiv ity (see Figure 2.1.) (also 1 hour

a fte r DFP or saline adm inistration) animals were k illed by decapitation and

trunk blood collected in to heparinised tubes. The brains were rapid ly

removed and cooled over ice on a moistened f i l te r paper covered pe tri dish.

Brains were then dissected in to eight regions, fo llow ing a protocol s im ila r to

tha t described by Glowinski and Iversen (1966). Each region was weighed and

transferred to glass sc in tilla tion vials to which 1ml o f Soluene-100 (Packard)

was added. Plasma was obtained by centrifugation a t 2500g fo r 10 minutes

and a 20pl o f plasma was solubilized w ith 0.5ml Soluene-100. Brains were le f t

overnight to solubilize. With the larger brain regions, cerebellum and

cortices, solubilization was promoted by warming the vials to 50°C fo r 10 to

30 minutes, and allowing to cool.

4.6. Scintillation Counting

Follow ing solubilization, sc in tilla tion flu id consisting o f 1.5 litre s o f sulphur-

free toluene, 750ml o f synperonic NX PO POP 225mg and PPO 12g (Wood et3

al, 1975) was added to samples. H a c tiv ity was determined in an LKB 1210

U ltrobe ta sc in tilla tion counter. Chemiluminescence was avoided by

acid ify ing the sc in tillan t w ith 10ml glacia l acetic acid per 2.5 litre s of

sc in tillan t.

Presence of solubilised brain tissue has a quenching e ffe c t on detection of

sc in tilla tions. As varying amounts o f brain tissue were present in each v ia l,

i t was im portant to take in to account and compensate fo r variable quenching.

A quench correction curve was therefore set up. Brain tissue weighing 10, 20,

40, 60, 80, 100 and 120pg were solubilized in 1ml o f Soluene-100. To each via l,

10 pi o f hexadecane standard was added, and the vials counted on the 1210

U ltrobeta . Decay tables fo r allowed to ta l a c tiv ity per v ia l to be

calculated. With c.p.m. and R values from these vials, the coe ffic ien ts o f the

data were calculated by applying a Basic regression programme (see Appendix

D (i). This program yielded values fo r AO, A l, A2 and A3 which were

programmed in to the sc in tilla tion counter, enabling rad ioac tiv ity to be given

in d.p.m. The quench curve was also p lotted using the d.p.m. package on a

RackBeta 1215 (see Appendix D (ii)).

A s im ilar procedure was carried out fo r plasma as occasionally samples were

haemolysed. Plasma samples o f varying degree o f haemolysis were counted

w ith 10pi hexadecane standard. The coeffic ien ts were determined as fo r

the brain samples.

RESULTS

4.7. Effect of DFP Pretreatment on Alfentanil Brain and Plasma Levels

Figure 4.1. represents the e ffe c t of DFP on levels in various brain regions o f

a lfen tan il, expressed in d.p.m ./g wet weight o f tissue.

P retreatm ent w ith DFP at Img/kg caused a s ign ificant increase in the levels

in a ll brain regions o f a lfen tan il at 400pg/kg (Figure 4.1.). This increase was

most marked in the hypothalamus w ith a 3.2-fo ld increase in levels compared

to saline controls. There was a marked reduction in plasma a lfen tan il

rad ioac tiv ity (Table 4.1.). A t the 200pg/kg dose o f a lfen tan il, DFP appeared

to l i t t le a ffe c t brain levels. I f brainrplasma ratios are considered (Table

4.2.), DFP pre-trea tm ent produced a s ign ificant e ffe c t in a ll brain regions

and at both doses.

4.8. E ffect of DFP Pretreatment on Morphine and Fentanyl Brain and Plasma

Levels

DFP appeared to have l i t t le e ffe c t on brain levels o f fentanyl or morphine

(Figure 4.2. and 4.3.). There was a slight decrease in morphine levels

fo llow ing DFP but this only reached significance in the hypothalamus at the

higher dose. S im ilarly, DFP did not s ign ifican tly a lte r plasma ra d io a c tiv ity

o f e ither morphine or fentanyl, (Table 4.1.) and so brain:plasma ra tios fo r

these two opioids were very s im ilar in contro l- and DFP-treated animals

(Table 4.2.).

DPM g'"1'{ x10-3)

4 0 0

3 8 0 -

360

340

320

200

180

160

120

100

80 ■

6 0 •

4 0 -

20 -

CB PM HYP STR MB HIP HCX FCX CB PM HYP STR MB HIP HCX FC>

Fig. 4.1. Effect of DFP on brain levels (dpm g -*-) ofalfentanil (A = 200yg/kg“l, B = 400yg/kg_^)

Open histograms: control treated. Dotted histograms: DFP (ling/kg--1-) treated. Each histogram represents the mean *s.e. mean of at least six observations. CB = cerebellum,PM = pons and medulla, HYP = hypothalamus, STR = striatum, MB = mid brain, HIP = hippocampus, HCX = hind cortex,FCX = frontal cortex.

DPM g '1

(x10“3) B2 4 0 i

220 1

200

160

80 i

60 i

40 -j

20 i

CB PM HYP STR MB HIP HCX FCX CB PM HYP STR MB HIP HCX FCX

Fig. 4.2. Effect of DFP on brain levels (dpm g_l) of fentanyl ( A = 40yg/kg“l, B = 80yg/kg--*-)

Open histograms: control treated. Dotted histograms: DFP (lmg/kg-l) treated. Each histogram represents the mean 1 s.e. mean of at least six observations. CB = cerebellum, PM = pons and medulla, HYP = hypothalamus, STR = striatum, MB = mid brain, HIP = hippocampus, HCX = hind cortex,FCX = frontal cortex.

3 D M g " 1

1 x 1 0 ~ 3 )

-5 iA B

4 0 -

3 0

25

20

15 -

10 -

X

jL

JL

I1

1I I

I

I 4-X J L

1 X

LL

[I

I

1

JL

X

i l

X

CB PM HYP STR MB HIP HCX FCX CB PM HYP STR MB HIP HCX FCX

Fig. 4.3. Effect of DFP on brain levels (dpm g- -) of morphine (A = lOmg/kg-1, B = 20mg/kg-1)

Open histograms: control treated. Dotted histograms: DFP (lmg/kg-1) treated. Each histogram represents the mean t s.e. mean of at least six observations. CB = cerebellum, PM = pons and medulla, HYP = hypothalamus, STR = striatum, MB = mid brain, HIP = hippocampus, HCX = hind cortex,FCX = frontal cortex.

Plasma R ad ioactiv ity dpm m l" (x 10” )

Opioid

M orph ine, (10mg/kg~ )

Morphine (20mg/kg" )

Fentanyl , (40pg/kg" )

Contro l

197 - 7

118 - 10

7 3 - 7

DFP-Treated

174 - 6 *

97 - 8

90 -13

Fentanyl , (80pg/kg” )

67 - 4 5 7 - 3

A lfe n ta n il . (200pg/kg )

166 - 10 110 ± 12*

A lfe n ta n il (400pg/kg" )

147 +34 9 1 - 6

Each value is the mean o f six observations, t test, DFP vs. contro l * p < 0.05

Table 4.1. E ffe c t of DFP (lm g/kg"^) on Plasma Levels o f H Morphine, Fentanyl and A lfe n ta n il

i—i CL * * * * * * * *, 1cn

c5 ■*

Ll.Q 1

.3

CM

<—1 3.3

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DISCUSSION

4.9 Opioid Lipophilicity and Access to the CNS

Opioids exert the ir antinociceptive e ffe c t in the centra l nervous system. To

gain access to the ir site o f action, opioids must cross the blood-brain barrier.

Generally speaking, the greater the lip o p h ilic ity o f an opioid (or any other

drug) the greater the ease w ith which i t is able to cross this barrier and enter

the brain (Jacobson, e t al, 1977).

A measure o f lip o p h ilic ity is the octanohwater pa rtition coe ffic ie n t at pH7.4

and the lite ra tu re values fo r the opioids studied are 1.42 fo r morphine

(Kaufman et al, 1975), 130 and 860 fo r a lfen tan il and fentanyl respective ly

(Meuldermans e t al, 1982). These values para lle l the order o f brain:plasma

ratios obtained in this study. Levels o f rad io ac tiv ity in the brain compared

w ith the plasma are highest fo r fentanyl, followed by a lfen tan il and then

morphine. The times fo r measurement o f morphine, fentanyl and a lfen tan il

levels were chosen to correspond to peak antinociception (see Section 2.6.).

These times agree w ith lite ra tu re values fo r these opioids (Gardocki and

Yelnoski, 1963; Johannesson and Becker, 1973). There is also a good

corre lation between levels o f these opioids in the brain and the ir

antinociceptive e ffects (Johannesson and Becker, 1973; Hug and Murphy, 1981).

4.10. E ffect of DFP on Opioid Brain Levels

DFP pretreatm ent produced a disparate e ffe c t on the brain levels and

distribution of radiolabelled morphine, fentanyl and a lfen tan il. The most

marked e ffe c t o f DFP is on the d istribution o f a lfen tan il where plasma levels

are lower and at 400pg/kg a lfen tan il there is a concom itant rise in a lfen tan il

in the brain. This enhanced entry could account fo r the observed increase in

the antinociceptive a c tiv ity o f a lfen tan il fo llow ing DFP (see Section 2.24.).

The absence of an apparent increase in brain levels o f a lfen tan il at 200pg/kg

by. DFP may be partly due to the decrease in plasma levels, since the blood

present in the brain was not accounted fo r and contributes to the to ta l

rad ioac tiv ity . A lte rna tive ly , DFP may be a ltering pharm acokinetic

parameters so tha t peak brain a lfen tan il levels could occur earlie r. Peak

opioid receptor occupancy or antinociceptive e ffe c t could persist fo r a short

tim e a fte r peak drug level (see Figure 4.4.)

4.11. Mechanism of Increased Levels of Alfentanil Produced by DFP

There is no lite ra tu re at present describing a potentia tion o f a lfen tan il by

other drugs. However, there are some reports in the lite ra tu re describing the

action of some drugs in increasing the potency o f morphine by a mechanism

o f increasing the plasma and brain levels o f this opioid. For example, a

synergism has been demonstrated between diazepam and morphine (Sanchez

et al, 1982). This appears to be dependent on diazepam com petitive ly

inh ib iting morphine glucuronization and dem ethylation. This results in higher

plasma and hence higher brain levels o f morphine. Brain uptake o f morphine

was shown to be increased fo llow ing adm in istration of the antih istam ine

tripelennam ine (Vadlamani, 1984) possibly by increasing perm eability o f the

blood-brain barrier. By contrast, the potentia tion o f morphine by

physostigmine did not appear to be dependent on a ltera tion in morphine

plasma or brain levels (Bhargava and Way, 1972).

I t is possible tha t DFP could increase a lfentan il's entry into the brain also by

a ffecting the blood-brain barrier. Indeed, DFP has been shown to increase

BRAINALFENTANIL

CONC.

DFP+400pg/kgALFENTANIL

\400pg/kg''

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Fig. 4.4. Scheme for Explanation of Observed Increased Brain Alfentanil Levels for DFP (lmg/kg) Administration

This figure illustrates that by assessing brain levels at one time point (5 minutes), misleading information may be obtained (see text).

the perm eability of capillaries in the anterior segment o f the eye (von

Sallman and D illon, 1947). Furtherm ore, in considering the mechanism of

potentia tion o f morphine antinociception by the two anticholinesterases,

physostigmine and neostigmine, Rypka and N a vra til (1982) showed tha t the

blood-brain barrier perm eability to acridine orange was markedly increased

fo llow ing adm in istration o f physostigmine and neostigmine. I f however, DFP

exerted this same e ffe c t on blood-brain barrier perm eability, this would be

non-selective and i t m ight be expected tha t a ll three opioids would a ll have

enhanced entry in to the brain. Moreover, as a lfen tan il and fen tanyl are

highly lip id soluble, the blood-brain barrie r would not norm ally l im it the entry

of these drugs into the brain. On the other hand, the low lip id so lub ility o f

morphine accounts fo r the low penetration o f morphine into the brain

(H artv ig et al, 1984). This means tha t an increase in the perm eability o f the

blood-brain barrier would tend to increase the entry o f a low perm eability

agent such as morphine, ra ther than an agent which is highly lip id permeable.

The observation tha t morphine levels are unchanged by DFP tends to m ilita te

against an action on blood-brain barrier perm eability by DFP.

The evidence suggests tha t DFP enhancement o f a lfen tan il entry in to the

brain is probably more like ly to be dependent on pre-blood-brain barrie r

mechanisms. One such candidate fo r this mechanism is a lte ra tion o f

a lfen tan il binding to plasma proteins.

CHAPTER 5

3In Vivo and In Vitro Plasma Protein Binding of H Alfentanil

3and H Fentanyl: Effect of DFP Administration

INTRODUCTION

I A35.1 Plasma Protein Bind[of Drugs

The a b ility of plasma proteins to bind and transport chemicals and drugs has

been known fo r many years (Rosenthal, 1926). The potentia l binding capacity

is enormous w ith an estimated to ta l surface area o f plasma proteins per

person being between 130,000 and 735,000m (Bennhoid, 1967). In practice,

only a frac tion of this area is available as compounds tend to bind to specific

sites (B ickel, 1978).

The binding o f drugs to plasma proteins has been comprehensively reviewed

over the years (Goldstein, 1949; Meyer and Guttman, 1968; Jusko and Gretch,

1976). I t has become apparent tha t the protein binding o f a drug can

influence its d is tribu tion which d irec tly and ind irec tly influences the action

o f the drug by determ ining its ava ilab ility at its sites o f action.

5.2 Plasma Protein Binding of AlfentaniljFentanyl and DFP

Studies in ra t, dog and man have shown tha t fentanyl and a lfen tan il have high

frac tiona l binding to blood proteins (Meuldemans et al, 1982). As only free

unbound drugs may cross the blood-brain barrier increased entry o f drug could

occur if , fo r example, the proportion o f opioid bound to plasma proteins is

altered fo llow ing DFP adm inistration. I t is possible tha t DFP may a lte r

binding of opioids to plasma proteins, as its adm inistration may produce

e ither acidosis or alkalosis by e ither stim ulating respiration at high doses or

depressing i t at low doses (Karczmar, 1967) since fentanyl binding is markedly

affected by a lterations in blood pH (Meuldermans et al, 1982). In addition,

a lte ra tion in pH would a lte r the per cent ionization (ionized drugs cannot pass

through the blood-brain barrier) and this could be im portant fo r fentanyl

where only 9% is unionized at normal pH. Though a lfen tan il would be l i t t le

a ffected as 89% is unionized, change in per cent ionization has been shown to

markedly potentia te the brain uptake o f morphine as shown in a lka lo tic rats

(Schulman et al, 1984). However i t appears tha t altered blood pH is not an

im portant fac to r as o f the three opioids studied a lfen tan il pharmacokinetics

are the most pH-independent.

DFP has also shown to bind, irrevers ib ly , to plasma proteins (Truhaut, 1966)

and so i t is worth enterta in ing the notion tha t DFP competes fo r or inhib its

a lfen tan il binding, thereby increasing the proportion o f free a lfen tan il which

is able to enter the brain. This form of in te raction is well known, and has

also been shown fo r irreversib le anticholinesterase agents; i t has been

suggested tha t the potentia tion o f sodium pentobarbita l anaesthesia by soman

is dependent on com petition fo r plasma protein binding (Clement, 1984). This

mechanism of in teraction is considered in th is section.

5.3 Assessment of Drug Binding to Plasma Proteins

There are two main methods fo r determ ining free drug levels: equilibrium

dialysis and u ltracen trifugation . Equilibrium dialysis is the most used o f

these methods but suffers from a number of methodological drawbacks.

These include Donnan effects, changes in ion concentration which may

compete w ith drugs fo r binding or a lte r protein conform ation (Bowers e t al,

1984). U ltra filtra t io n has the advantage o f being a more rapid procedure

though one drawback is tha t there may be non-specific adsorption o f drug to

the u ltra filtra tio n membrane (Zhirkov and P iotrovski, 1984), however th is

adsorption may be determined and compensated fo r. I t has been concluded

tha t ultracentrifuga'cion is a more sensitive method fo r determ ining small

changes in protein binding (Coyle and Denson, 1984) and be tte r re flec ts the

real values than those obtained by equilibrium dialysis (Tuila, 1984).

I N V I T R O I N V I V O

Mice in jec tedwith DFP or saline

Mice in jec ted with -o p io id

Trunk blood co l lec ted ^

Plasma o b t a i n e d . by centr i fugat ion

Plasma aliquot incubated with

-op io idU ltracentr i fugat ion to obtain unbound

drug f ract ion

^ Liquid scint i l la t ion , counting

Fig. 5.1. Protocol for Plasma Protein Binding Experiments Iri Vivo and Iri Vitro. (See text for details)

MATERIALS AND METHODS

5 .4 Animal and Experimental Conditions

Male albino mice (CD-I strain; 25-30g) were used as described in Section

2.2.1. Procedures fo r equ ilibration were also as those previously described.

5.5 Drugs

The drugs used were obtained from sources described in Section 2.6. and 4.6.

5.6. In Vivo Protein Binding Protocol

Timings o f drug adm inistrations were the same as the previous experiments

(Section 4.4) where DFP or 0.9% w /v saline was given s.c. at tim e 0, fen tanyl

(80pg/kg) at 50 minutes, and a lfen tan il (400pg/kg) was administered at 55

minutes. Each in jection contained 7pCi o f opioid. Trunk blood was

collected fo llow ing decapitation at 60 minutes, and plasma obtained by

centrifugation, (Fig 5.1.).

5.7 In Vitro Protein Binding Protocol

Mice received s.c. DFP or 0.9% w /v saline and were sacrificed by

decapitation 60 minutes la te r. A 250pl a liquot of plasma obtained fo llow ing

centrifugation was incubated fo r 30 minutes at 37°C w ith 25pl ^ H a lfen tan il

or ^ H fentanyl. The incubation medium contained additional cold opioid so

tha t drug concentrations paralleled those fo llow ing a s.c. antinociceptive

dose (Fig 5.1.).

5.8. Ultracentrifugation Protocol

Free drug concentrations were determined by u ltracen trifuga tion o f plasma

samples in an Amicon m icropartition MPS-1 k it (Figure 5.2.). A 200pl aliquot

was pipetted in to the sample reservoir o f the assembled unit. The un it was

centrifuged in a bench top centrifuge at 1500g fo r 10 minutes. A 20pi a liquot

o f the u ltra f ilt ra te (i.e. unbound drug) and 20p i o f the plasma (i.e. to ta l drug)

were added to polythene tubes in duplicate. 3-5mls o f Unisolve-1 sc in tilla tio n

flu id (Koch L ight) was added and tubes were counted in a RackBeta 1215 or

1216.

5.9. Calculation of Free and Bound Drug Concentration

Calculation o f the free and bound drug concentrations were made from data

o f u ltra f ilt ra te and plasma rad ioactiv ities using the equation:

Total drug concentration = bound concentration + Free concentration Plasma Concentration)

So, % Bound = Total - Free 100

or % Bound = 100 - % Free

5.10. Determination of Drug Adsorption in Ultracentrifugation Apparatus

Recovery experiments were performed to determine whether any loss o f

a lfen tan il or fentanyl occurred during the u ltracen trifuga tion process through

adsorption on the membrane or plastic reservoir. A stock of mouse plasma

3 3contaimrKj e ither H a lfen tan il or H fentanyl was aliquoted and

ultracentrifuged as described in Section 5.6. R ad ioactiv ity was determ ined

fo r the stock and fo r u ltra f iltra te s in duplicates. Recovery could then be

determined by comparing rad ioactiv ities in the original stock w ith the

u ltra f ilt ra te .

RESERVOIR ' IC A P ___________________ i

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RESULTS

5.11. Recovery Experiments

Recovery experiments fo r a lfen tan il and fentanyl showed tha t the a lfen tan il

u ltra f ilt ra te contained 103 - 11% (n=4) o f the orig inal stock and the fentanyl

u ltra f ilt ra te contained 80.7 - 1.3% (n=6).

5.12. Percentage of Plasma Protein Binding of Fentanyl and Alfentanil

Contro l binding o f a lfen tan il were fa ir ly s im ilar fo llow ing in vivo and in v itro

binding protocols (Table 5.1). The figures fo r fentanyl binding in contro l mice

were quite d iffe ren t, dependent on whether _in vivo or in v itro protocols were

used.

5.13. Effect of DFP on Binding Fentanyl and Alfentanil to Plasma Proteins

Table 5.1. shows the e ffe c t o f DFP (lmg/kg) on the levels o f plasma to ta l and

plasma free a lfen tan il and fentanyl fo llow ing incubation in v itro . The values

presented have been adjusted fo r losses during u ltracen trifuga tion in the in

v itro experiments. There was no a ltera tion in free or bound fentanyl in the

presence o f DFP (Table 5.2.), however, there was a greater than 40% increase

in free a lfen tan il in DFP treated plasma.

The e ffe c t o f DFP on m vivo adm inistration o f a lfen tan il (Table 5.2.) is more

marked w ith nearly a 60% increase in free a lfen tan il. For in vivo binding of

fentanyl, DFP has very l i t t le e ffe c t on free levels (Table 5.2.)

Table 5.1.

Treatment

Saline

DFP

Img/kg

Effect of DFP Treatment on In Vivo and In Vitro

Plasma Levels of Alfentanil

and fentanyl

Alfentanil

ng/ml

In vivo

Fentanil

ng/ml

In v itro In v itroin vivo

Tota l 485 - 25.2 519 - 5 16.87^0.36 13.25 - 0.76

Free 118 - 6.1 140 ± 10 2.01 - 0.14 7.09 - 0.54

Total 286 - 40.2 517 - 9 16.30 - 0.59 12.25 - 1.51

Free 110 - 13.0 195 - 12 1.83 - 0.06 5.63 - 0.68

Values are means ^ SEM of 6 observations.

Table 5.2. E ffect of DFP Treatment on Levels of In Vivo and in Vitro

Free Plasma Levels of Alfentanil and Fentanyl

% Free levels

Treatment Alfentanil Fentanyl

In V itro In Vivo In V itro In Vivo

Saline 24.3 26.9 53.5 11.9

DFP lm g/kg 38.5 37.8 46.0 11.2

DISCUSSION

5.14 DFP Binding to Plasma Proteins

DFP is known to bind plasma proteins in several species including man

(Jandoff and McNamara, 1950; Cohen and Warringa, 1954; Murachi, 1963;

Truhaut, 1966; Ramachandran, 1967; Hansen et al, 1968; Schuh, 1970; M artin ,

1985). DFP is capable o f combining covalently w ith most proteins (M artin ,

1985) in a general type o f a lky l phosphorylation reaction (Murachi, 1963; see

Figure 5.3.). The percentage of DFP bound to plasma proteins is high

fo llow ing in jection; a dose o f lm g/kg DFP i.v . in mice results in over 95% o f

plasma DFP being bound.

There has been l i t t le work on a ttem pting to iden tify to which protein DFP is

bound. Schuh (1970) however, has indicated that albumin is the main prote in

as the same values fo r percentage DFP binding can be obtained by

substituting a 3.2% albumin solution, equivalent to normal plasma

concentrations.

5.15. Plasma Protein Binding of Alfentanil and Fentanyl

Both a lfen tan il and fentanyl have been shown to have high frac tiona l prote in

binding (Meuldemans et al, 1982). The results in this thesis showing around

75% binding fo r a lfen tan il are consistent w ith those obtained in the

lite ra tu re : fo r ra t, dog and man and the fraction bound is 84, 73 and 92%

respectively (Meuldermans, et al. 1982). Values obtained in this thesis fo r

fentanyl binding vary depending on the method used. Follow ing in v itro

methodology, fentanyl binding was found to be sim ilar to that obtained in ra t,

Fig. 5.3.

DFP Phosphorylation

of Protein

by an

Alkyl Phosphoyrlation

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dog and man (Meuldermans, et al, 1982). In vivo binding yields a lower

percentage level which is sim ilar to tha t obtained by Murphy et al (1979) in

the dog. The discrepancy observed in the study presented here between jn

vivo and jn v itro binding could depend on several factors; fentanyl binding is

temperature and pH dependent (H o llt and Teschemacher, 1975) so tha t change

from pH 7.4 to pH 7.55 increases fentanyl binding from 67% to 76%. The

37°C incubation step in the in v itro binding protocol could produce the

observed increase in fentanyl binding compared to the in vivo protocol.

Though s truc tu ra lly s im ilar, these drugs do not apparently bind to the. same

sites. Fentanyl has been shown to bind red blood cells, glycoproteins and

lipoproteins, w h ilst a lfen tan il is predominantly bound to the acute phase

reactant protein a^-acid glycoprotein (a^-AGP) (Meuldemans et al, 1982). In

fac t, Meuldemans et al (1982) have calculated tha t i f a^-AGP were the only

binding protein, i t could account fo r a ll a lfen tan il binding in human

plasma but only about ha lf of fentanyl binding. Volunteer plasma samples

showed a linear relationship between a^-AGP concentration and free drug

frac tion only fo r a lfen tan il (Meuldermans et al, 1982).

5.16. E ffect of DFP on Plasma Protein Binding of Alfentanil

The presence o f DFP decreased the frac tiona l binding o f a lfen tan il to plasma

proteins. As a lfen tan il is highly lipoph ilic , the blood-brain barrier does not

l im it the entry o f free a lfen tan il into the brain. I t is reasonable to suppose,

therefore, tha t by increasing free a lfen tan il increased brain levels and

increased antinociceptive e ffe c t would fo llow . DFP did not a lte r fentanyl

plasma protein binding and so an explanation fo r the observation o f

d iffe re n tia l potentia tion by DFP o f a lfen tan il antinociception could be made

on the basis of DFP specifica lly inh ib iting the binding o f a lfen tan il to plasma

proteins.

Though Schuh (1970) suggested that DFP binds to albumin (see Section 5.12.),

this does not rule out the possib ility that DFP also binds a^-AGP because as

already mentioned, DFP reacts w ith most proteins and w ill phosphorylate

several classes o f proteins when incubated w ith plasma in v itro (Murachi,

1963).

Moreover, i t has been recently shown that albumin potentiates the binding o f

propanolol to a^-AGP (Chauvelot-Moachon e t al, 1985) so tha t i f this held

true fo r a lfen tan il, binding to a^-AGP could be a ffected by DFP

phosphorylation o f albumin.

a-^-AGP is a high a ff in ity low capacity binding protein (Piafsky, 1981;

Mackichan and Zola, 1982), so that i f DFP were to compete fo r and saturate

binding sites on the a-^-AGP molecule this would lead to raised levels o f free

a lfen tan il. Fentanyl, on the other hand, i f displaced from these sites, would

not necessarily result in increased free levels as i t could be sequestered in

other high capacity binding depots such as red blood cells.

Several drugs have been shown to bind a-^-AGP and there is often a m utual

displacement o f drugs as there is only a single binding site (MUller e t al, 1983).

Furthermore, the binding site m ight represent a remote hydrophobic area

w ith in the protein part of the glycoprotein molecule (Brunnmer and MUller,

1985). So not only is the binding site probably a protein to which DFP could

covalently bind, but i t is also a hydrophobic region where the DFP molecule

would tend to go due to its lipoph ilic nature (Karczmar, 1967). There is a

good corre lation between displacing potency against ^ H -im ipram ine from

a^-AGP and lipoph ilic parameters for a varie ty o f compounds (MUller et a l,

1983).

5.17. Consequences of Alfentanil Displacement from Plasma Protein

Binding Sites

As the e ffe c t o f a drug appears to be more closely related to the free

unbound levels rather than the to ta l concentration (Lima et al, 1981) the

question arises as to what the consequences o f the displacement o f bound

a lfen tan il by DFP.

The degree of prote in binding o f a lfen tan il lim its its entry in to the brain.

This is re flected in the brainrplasma ratios which average about 0.6 at

400ug/kg (see Table 4.2.). L ite ra tu re values measuring unchanged a lfen tan il

ra ther than to ta l rad io ac tiv ity show braintplasma ratios averaging around 0.13

in the ra t fo r 10 minutes fo llow ing i.v. adm in istration (Michaels et a l, 1981).

I f binding to plasma proteins lim its a lfen tan il entry in to the brain,

displacement by DFP would tend to cause a proportional increase in entry o f

a lfen tan il. In this situation, displacement o f a lfen tan il would not a lte r the

average free drug concentration, though there would be a transient rise in

free drug concentration im m ediate ly fo llow ing displacement (Mackichan,

1984). This is borne out in the data presented in Table 5.1. fo llow ing in vivo

binding as free drug concentrations were l i t t le changed. The jn v itro binding

data shows tha t DFP increases free a lfen tan il concentrations. In vivo this

free a lfen tan il is able to red istribu te in to other tissues including the brain, so

tha t the actual steady state concentration is not altered jn vivo fo llow ing

DFP whilst the average to ta l drug concentration decreases.

5.18. Protein Binding and Relationship to Variation in Alfentanil Potency

As described in Section 2.27. in it ia l studies w ith a lfen tan il showed AD^g

values d iffe ren t from tha t obtained in experiments some months la te r. One

explanation fo r this anomaly was that there is a varia tion in circannual

sens itiv ity to a lfen tan il, however, though this may be true, the data from the

binding experiments which show the relevance o f a lfen tan il binding to its

potency, introduces the possibility tha t varia tion in binding could markedly

a ffe c t potency. If, fo r example, the batch of mice in the in it ia l experiments

were carried out had a low level v ira l in fection, this could greatly increase

a^-AGP levels and hence binding capacity. This could e ffe c tive ly increase

the true AD^-g value.

CHAPTER 6

GENERAL DISCUSSION

6 GENERAL DISCUSSION

In the foregoing chapters an account has been given o f the potentia tion of

a lfen tan il, but not morphine or fentanyl antinociception by DFP pre-trea tm ent in

mice. This e ffe c t was not reversed by drugs used to tre a t DFP poisoning. In

addition, DFP has d iffe re n tia l e ffec ts on opioid-induced running behaviour, w ith

the most marked e ffe c t observed in a lfen tan il-trea ted mice. Further

experiments identified tha t DFP enhanced the entry o f a lfen tan il in to the brain,

w h ils t the d istribu tion o f the other opioids was unaffected. This appeared to be

due to a displacement o f a lfen tan il from plasma protein binding sites, thereby

allowing more drug to enter the centra l nervous system.

The d iffe re n tia l potentia tion o f a lfen tan il may w e ll be dependent on the

observation tha t this opioid p rim arily binds to a^-AGP w hils t fentanyl binds

more than one protein (Meuldermans et al, 1982). Morphine's low frac tio na l

binding means tha t displacement from these sites would not greatly increase free

drugs.

There are im portant im plications to the findings presented here, demonstrating

functiona l increase in antinociceptive potency o f a lfen tan il fo llow ing

displacement from its plasma protein binding sites. In these studies, the data

shows that in mice, 75% of a lfen tan il is bound to plasma protein, w h ils t in

humans the figure is 92% bound (Meuldemans et al, 1982). The higher the

frac tiona l binding the greater the percentage increase in free drug fo llow ing

displacement, thus in humans, potentia tion o f a lfen tan il action could be even

greater than that observed in mice.

A lfe n ta n il binding may vary in patients i f they are not receiving any drug

therapy, as the normal levels o f a^-AGP varies in normal healthy volunteers

from 55 to 140mg/100ml w ith a mean of 66mg/100ml (Piafsky et al, 1978).

Increased levels are probably due to subclin ical or m inor in fections. a^-AGP

levels are also increased as a result o f the physiological stress associated w ith

in flam m ation, cancer (Piafsky, 1980) and surgery (Fremsted e t al, 1978).

This in form ation suggests tha t a lfen tan il potency may be sensitive to

pharmacological and physiological variables, which may be im portan t c lin ica lly .

I t should be borne in mind, however, tha t i t is an assumption to extrapolate the

demonstrated in teraction in an experimental animal to an in te raction in humans.

An assessment o f the potentia l in teractions of a lfen tan il w ith other drugs should

therefore be carried out w ith other drugs and using human plasma.

The property o f the potentia ting a c tiv ity o f DFP does not appear to be

dependent on its anticholinesterase a c tiv ity per se, as the carbamate

anticholinesterases neostigmine and pyridostigm ine, did not a lte r a lfen tan il

antinociception. A plausible in te rpre ta tion o f th is is tha t DFP may produce a

stable phosphorylated plasma protein (which competes fo r a lfen tan il binding) and

this may hold true fo r other organophosphorous compounds including

organophosphates. Given tha t in DFP treated mice, morphine antinociception is

altered fo llow ing atropine and pralidoxime adm in istration, fentanyl appears to be

the opioid of choice and the least like ly to cause in te raction problems w ith

organophosphates or drugs used in the trea tm ent o f anticholinesterase poisoning.

This study has shown the existance and probable mechanisms of DFP potentia tion

o f a lfen tan il antinociception. This is of importance not only as an example o f

the relevance of plasma protein binding on drug e ffe c t, but also in term s of the

possible im plications fo r the c lin ica l use o f a lfen tan il fo llow ing adm in istra tion o f

other drugs. There is clearly scope fo r fu rthe r research in this area.

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aj-PENDIX a

Equation for Unpaired Student’s t-test

t = x

(SEM^2 + (SEM2)2

APPENDIX B

C om puter P rogram fo r A n tin o c ice p tive D ata in Mouse H o t-P la te Test

A set o f data has th ree components, the mean response tim e , the va ria tio n o f

response tim e about the mean, and the e rro r d is tr ib u tio n . For th is data, i t

was found th a t a W eibu ll d is tr ib u tio n could successfu lly be applied to th is la s t

com ponent. The data may also be lin e a rlize d , such th a t log (mean response

tim e ) is linea r in dose. S tra ig h t- lin e regression is ca lcu la ted by a m ethod

s im ila r to the usual least-squares method, using a general procedure ca lled

m axim um like lihood es tim a tion . The sum o f squares is rep laced by the

like lihood fun c tio n , which represents the p ro b a b ility o f obta in ing data under

the model. This model enables p red ic tions to be made on the percentage o f

response tim es th a t exceed 60 seconds. This model also a llows fo r the

inclusion o f zero-dose (ie, saline) data. A fu lle r descrip tion o f th is m odel is

given by K itchen and C row der (1985).

A N A lG fM SCREEN i;A T A FEB S 3 ) : CENSORED RESPONSE H U E S UNDER DRUGS

ANA < O LS U . • >: REFN DATA <FF.R 6 3 M CENSORED RESPONSE T IM E S ..»NOEP DRUGS REAL XS ( .0 >. 1 0 ) . WX C c»X ; .WY< 6 0 0 > . R ( 1 5 ) . B L U ( 2 . 1 5 /DOUBLE PRF.C I S IO N WI ( I 5 / . W 2( 1 5 ) , W 3(1 5 . 1 5 ) . P I V ,T G L .L L K D COMMON -'R ID A T / Y < 6 0 0 > . N . YMS . VCNSR. X ( 6 0 0 . 10 > . NDRG EXTERNAL C R T0 12DATA IX / 6 0 0 . . I W / T O C / I . D - 1 0 /READ C.AfA E T C .READ; 1 . * > I RF>. Iw R , I CHECK

10 C ALL I k R T O l( IR D . IC H E C K )Z F ( N . lE . O ) STOP

20 UR IT E M i PC-1)R E A D H . * ) NOI E <N O .L E . 0 ) GOTO tOMLE F I T BY T R IA L AND ERRORN P -N P R O +2I F ( N P . 0 T . I 5 > STOPno so j«i , isRl.U< 1 , J ) = 0 .

3 0 D L U (2 .* J ) = 1 0 0 .B L U M . N P > * . 01 -C A L L F M IN !C R T 0 1 2 .N P .W 1 ,L L K D » W 2 .W 3 .W 3 ,IW .B L U )

r PARAMETER C O VARIAN C E M ATR IXA - W K 1 )DO 5 0 J s l .N D R G

5 0 B < J ) * W 1 ( J * l >G AM =W 1(NP)W R IT E (1 .9 0 © )R EAD ( I . * > IC O VIF < IC O V . N E .1 ) GOTO 7 0W R IT E ( IW R .9 0 2 ) (W 1 ! J ) , J = 1 »N P ) , LL K DC A L L C R T 0 0 2 ( A .B » G A M .L L K D .W 1 . W 3 » IW .2 . 1 )DO 5 5 J = 1 ,N PC A LL M X S W P 0 (W 3 .IW ■NP * N P »U »P I V » T G L )

5 5 W RITE< 1 ,9 0 .3 ) J . P I V DO 6 0 J - l . N P U 1 ( J ) * - W 3 ! J , J )IF ( W 1 ( J ) . G T . O . ) W 1 ( J )= D S Q R T ( W 1 (J ) )

6 0 C O N TIN UEW R I T E M . 9 0 7 ) (W1 ( J ) . J « I , N P )W R IT E ! IW R .9 0 8 )DO 6 5 J * l . N P

6 5 W R IT E !IW R ,S 0 2 T <U 3 < J . K ) . K = l . U )W R IT E M W R .9 0 6 )DO 6 7 J = 1 • NDRG E D 1 - . 6 9 3 1 5 / B ! J )E D 2 - ( 3 . 4 0 1 2 0 - A ) / B ( J )S D I» E D 1 * W 1 ( J + l ) / B ( J )S 0 2 — < W 3( 1 . 1 ) * 2 . «E D 2« W 3( 1 . J + l ) + E 0 2 *E D 2 *W 3 ( J + l . J + 1 ) ) t F ( S D 2 . G T . O . ) S D 2 -S Q R T ( S D 2 ) /B ( J >

6 7 W R IT E !IW R ,8 0 2 ) E D I* S D 1 . E D 2 .S D 2 C R E S ID U A LS

7 0 W R IT E !1 . 9 0 9 )R E A D M .* ) IR E SI F ! I R E S . E Q . 1 ) C A L L C R T 0 2 2 !A .B .G A M .W Y ,IW R )GOTO 2 0

8 0 1 FOR M AT( 2 0 1 4 )8 0 2 FO R M A T (1 P 8 G 10 . 3 )9 0 ? F O R M A T !/IO H P A R A M E T E R S /!1 P 6 G 1 2 .5 ) )9 0 3 F O R M A T (5 H P IV 0 T . 1 2 . IP G 1 0 . 3 )9 0 4 FORM AT( /4 5 H F I T : ORDER OF REGN ( l- L IN E A R .2 -Q U A D R A T IC .E T C ) )9 0 6 F O R M A T (/4 H E D 5 0 )9 0 7 F O R M A T !/1 0 H S T D E R R O R S / !1 P 8 0 I0 .3 ) >9 0 3 F O R M A T !/27H P A R A M E TE R C O VA RIAN C E M A T R IX )9 0 9 F O R M A T (/9 H R E S ID U A L S )

ENDSU BR O U TIN E IK R T 0 1 ( IR D .IW R )

C A N A L G E S IC SCREEN CENSORED RESPONSE T IM E DATA (F E B 8 3 )C X HAS COLS (DRUG NO. . D O SE. RESPONSE T IM E )

IN TEG E R N O ( 2 0 )R EAL D S ( 2 0 )C O M M O N /R TD A T /Y ( 6 0 0 ) > N .Y M S . YCNSR, X ( 6 0 0 . 1 0 ) . NDRG YUS— 9 9 .Y C N S R -6 0 .D 0 S D IV - 1 0 0 .N * 0R E A D !IR D ,8 0 3 ) NDRGIF ( N D R G .L E .0 ) RETURNDO 3 0 J D R G - l . NDRGR EAD ( IR D .8 0 4 ) NDSREAD < IR D . * ) ( D S ( J ) . J - 1 . N D S)R E A D M R O .* ) ( N D ( .J ) , J * 1 .N D S )IF ! I W R .G T .O ) W R IT E !IW R ,8 0 2 ) ( D S ! J ) , J « 1 . N D S )1 1-N-* 1DO 2 0 J - l . N D S N J - N D ( J )DO 2 0 I - l . N J N -N MX ! N . 1 >*UDRG

20 X ( N ,? ) - n S ! . J ) /D O S D IVR E A D !IR D .# ) ! X ( I * 3 ) . I - I 1 * N >

30 C i-in T IN IIFVC N SR -ALO G ( YCNSR>DO 4 0 1 = 1 ,N Y I = X < 1 .3 )I F ! V I . G T . O. ) Y ( I ) = 01 .00 ( Y I 5

4 0 CONTINUEI F ! I W R . L E . 0 ) RETURN W R IT E !IW R ,9 0 1 >DO 6 0 1 - 1 . N

6 0 W R IT F !IW R .8 0 ? > r X ( I ,..»> . J = 1 ..? ) , Y ! I >RETURN

8 0 2 FORM AT( 1 P 8 G 1 0 . 3■8 0 3 F O R M A T !5 X .1 3 )8 0 4 F O R M A T (J5 X . 13.)9 0 ) F O R M A T !/1 0 H 0 H F C K D ATA)

ENDSU BR O U TIN E C R T0 12 < N P .P A R ,F N , G R ,H . IH , I G R . IW R )

T P A R !. ) *A » B ( D . . . . , B (N D R G ). GAMR EAL B ( 1 5 )DOUBLE P R E C IS IO N F N ,P A R !N P ) . G R (N P ) . H ( I H . I H )C O M M O N /R TD A T /Y ! 6 0 0 ) .N .Y M S ,Y C N S R ,X ( 6 0 0 ,1 0 ) .NDRG A = P A R (1)DO 2 0 J * I ,N D R G

20 B ! J ) * P A R ( J + l )0A M =PAR !N P1CAL t. C R TO O ruA . B .G A M .F N .G R .H . IH , I OR, IW R)RETJIRN END

t IN S E R T IK 'CRTB * IN S E R T FMTNP

SUBROUTINE CRT002 <A,B.GAM.LLKD, Gl.»HL , IH, IGR, IWR)

SUBROUT IN E C R T 0 0 2 !A .6 , G A M .L L K D ,G L »H L , IH , IG R , IW R )M IN U S L O G -L IK D AND D E R IV S TOR L O G -W E IB U LL CENSORED RESPONSE T IM E S

f. M U /S IG MODEL S U P P L IE D BY S /R T N E C R T0 03REAL MU, B ! 1 5 )» GP ! 2 ) . GC ! 2 ) » H P ! 2» 2 ) » H C ! 2» 2 ) . GM! J 3 ) • O S ! 1 5 )»

1 H M <1 5 . 1 5 ) , H S ( 1 5 .1 5 )DOUBLE P R E C IS IO N L L K D ,G L ( I H ) , H L ( I H , I H )C 0 M M O N /R T D A T /Y I6 O 0 ) ,N .Y M S .Y C N S R ,X I6 0 0 ,1 0 ) .NDRG L L K D * 0 .N P -N D R G + 2 I F ( N P , G T . 1 5 ) STOP IF { IG R . I .E . 0 > GOTO 2 0 DO 15 J * 1 , NP G I „ ! J > * 0 .I F ( I G R . L E . l ) GOTO 15 DO 1 0 K = 1 ,N P

1 0 H L ( J . K ) = 0 .15 C O N TIN UE 2 0 DO 4 0 1 *1 ,N

Y I * Y ! I )IF ! Y I , F J 3 . YMS) GOTO 4 0 I D * I F I X ! X ! T » l ) + . 5 >D S * X ( 1 , 2 )C A L L C R T 0 0 3 ( A , B . NDRG. GAM, ID .D S .M U .S IG .G M .G S .H M .H S ,1 5 . IG R )IF IY I . L T . Y C N S R ) C A L L W B L L 0 1 < Y I, M U ,S IG ,P D .C O ,G P .G C ,H P .H C .IG R )IF IY I .G E .Y C N S R ) C A L L W B L L 0 1 < Y I, M U ,S IG .C D .P D ,G C ,G P .H C .H P ,IG R )I.L K D *L L K D --P DI F ( I G R . L E . O ) GOTO 4 0DO 3 5 J * 1 .N PI F ! I G R . L E . 1 ) GOTO 3 5DO 3 0 K * I , JH t L t , K ) * H L ! J . K ) - ! H P ! l . l ) # G M ! J ) + H P < t » 2 ) # G S ! J ) >*G M !K )-G P < 1 ) * H H < J ,K ) -

1 ( H P ( 1 , 2 ) « G M ! J ) + H P ! 2 . 2 ) * G S ! J ) > * G S ! K ) - G P ! 2 ) # H S ! J . K >3 0 H L ( K . J ) * H L ( J . K )3 5 G L ! J ) *G L .! J ) - G P ! t ) « G M !U ) - G P !2 ) * G S < J )4 0 C O N TIN U E

IF ( IW R .L E . O ) RETURN W R ITE < IWR * 9 0 1 )W R IT E ! IW R .8 0 3 ) LLK DW R IT E ! IW R .8 0 2 ) ! G L ! J ) » J * 1 . N P )DO 5 0 J * 1 * NP

5 0 W R IT E !T W R .8 0 2 ) ! H L ! J . K ) . K « 1 . J>RETURN

8 0 1 F O R M A T !2 0 I4 )8 0 2 F O R M A T !1P 8G 10 . 3 )8 0 3 F O R M A T !IP G 1 2 . 5 )9 0 1 F O R M A T !/2 5 H M IN U S L O G -L IK D AND D E R IV S )9 0 6 F O R M A T !2 I 3 .1 P 6 G 1 2 .5 / ! 6 X ,1 P 6 G 1 2 .5 ) )

ENDS U BR O U TIN E C R T 0 0 3 ! A , B »N B • GAM, ID ,D S ,M U .S IG ,G M ,O S .H M .H S ,IH . IG R )

C CENSORED RESPONSE T IM E SC REGN MODEL TYP E M U - A + B ! ID ) * D S !FO R DRUG ID ,D O S E D S ) , SIG *O AM »M UC RETURN M U ,S IG AND D E R IV S WRT < A ,B ( . )» G A M )

R EAL 8 ! N B ) . M U . G M ! I H ) , G S ( I H ) , H M ! I H , I H ) , H S ( I H , I H )B D * B ! ID ) « D SM U *A+BDS IG -G A M *M UI F ! I G R . L E . 0 ) RETURNN P »N B -f2DO 2 0 J - 1 , NPC M ! J ) * 0 .

2 0 G S ! . J ) * 0 .G M (1 ) * 1 .G M !ID + 1 ) * D S O S ( 1 ) *GAM G S (ID + 1 ) *G A M * D S O S ! N P ) *M UI F ! I G R . L E . 1> RETURN HS i 1 , N P ) * 1 .H S 1 N P ,1 ) * I .H S ! ID + 1 »NP)«=DS H S 1 N P ,ID + 1 ) * D S RETURN ENDS U D R O U TIN E C R T 0 2 2 !A ,B ,G A M .O R » IW R )

C 1 0 G -W E IB U L L CENSORED RESPONSE T IM E S ! E X P O N E N TIA L R E S ID U A LSC O R ! 1 ) , , . . . O R !N ) CORRESP TO NON-CENSORED Y 'S

IN TEG E R NW! 6 0 0 )R E A L M U , B ( 1 5 ) » O R !6 0 0 ) ,G M !1 > . O S !1 ) ,H M !1 , 1 ) , H S !1 » 1 )C O M M O N /R TD A T /Y ! 6 0 0 ) . N ,Y M S , YCNSR. X ! 6 0 0 , 1 0 ) , NDRG D ATA E G /O .5 7 7 2 1 5 6 6 4 ? / . P 6 / 1 .2 8 2 5 4 9 8 /I F ! IW R .G T .0 ) W R IT E !IW R ,9 0 1 ) N ,N D R G .Y M S .Y C N S RDO 2 0 1 * 1 . NI D * I F I X ( X ! t . t ) + . 5 >D S - X ! 1 , 2 )C A L L C R T 0 0 3 !A .B .N D R G ,G A M . ID ,D S .M U ,S IG ,C M .O S .H M ,H S ,1 . 0 )Y D * P 6 « ! Y ! I ) - M U ) /S I G O R !I> * E X P !Y O - E G )I F ! I W R . G T . 0 ) W R IT E !IW R .8 0 3 ) I D , D S . M U . Y ! I ) . Y D ,O R ! I )

7.0 C O N TIN UE RETURN

8 0 3 FORM AT! I 3 . 9 F 8 . 4 )9 0 1 FO R M A T !/ ? ! H E X P O N EN TIAL R E S ID U A L S .2 1 4 , F 6 . 1 . F 7 . 4 )

ENDSU BR O U TIN E W Bt.LO l ( Y ,M U . S IG ,P P . CD, G P ,G C , H P . HC, IG R )

C LO G -W E ID U L L D IS T N : P D -LO G D E N S IT Y F N , C D *l O G !1 -D IS T N F N ) . ANDr D E R IV S WRT M U ,S IG

R FAL M U ,G P !2 ) , G C ! 2 ) . H P !2 , 2 ) . H C ( 2 . 2 )DATA E G /O .5 7 7 2 1 5 6 6 4 9 / . P 6 / 1 . 2 8 2 5 4 9 8 /Y D = P 6 * ! Y - M U ) /S IG E Y *E X P (Y P -E G )P D *A LO G ! P 6 /S I G ) + Y D -E G -E Y C D *-E YI F ! IG R . L E . 0 ) RETltRNG P !1 ) * - P 6 * ! 1 . - E Y ) / S I G0 P ! 2 > * - ! ! . + Y 0 * ! 1 . - E Y > ) /S IGG C !1 > *P 6 « E Y /S IGG C !2 > * Y D « E Y /S IGI F ! I G R . L E . t ) RETURNS 0 2 * S IG * S IGH P ! 1 , I> * - P 6 * P 6 * E Y / S 0 2H P ! I , 2 ) * P 6 + ! 1 . - E Y -Y P # E Y ) / $G 2H P !2 » 1 ) « H P !1 , 2 )H P ! 2 , 2 ) * ! 1 . + ! Y D + Y D ) * ! 1 . —E Y ) -Y D * Y D * E Y ) /S G 2H C ( 1 . 1 )» - P 6 * P 6 * E Y /S G 2H C !1 . 2 ) = - P 6 * ( 1 . + Y D )+ E Y /S G 2M C ! 2 , 1 ) * H C ! J , 2 )H C !2 » 2 ) * - ! 2 . + Y D > » F Y *Y D /S G 2RETURNEND

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APPENDIX D ( i )

Program to obtain values for AO, Al, A2 and A3, as parameters for quench curve for 1210 Ultrobeta

10 REN NAME STANDARD PARAMETERS 20 REN PROGRAHHER A/SCOTT 30 REH DATE 2 4 . 0 7 . 8 040 REH T H I S PR0GRAHHE CALCULATES THE C O E F F I C IE N T S OF THE GIVEN DATA5 0 REM B E S T - F I T T I N G THIRD DEGREE P0LYN0HIAL60 REH E=A0+A1*R+A2*R*2+A3*R*3 BY THE HETH0D OF LEAST SQUARES70 DEFINE FILE #1= 'SCOTT",ASC,12071 REM AT FIRST N, THE TOTAL NUHBER OF POINTS.72 REH THEN A, THE ACTIVITY.73 REH THEN THE VALUES FOR R AND CPH IN TURNS.75 REH INSERT DATA IN FILE t i l . AT FIRST N, THE TOTAL NUMBER OF POINTS 80 REH AND A, THE ACTIVITY, THEN THE VALUES 90 REH FOR R AND CPH IN TURNS.95 DIM R<28)100 READ ti l ,N,A 110 FOR H=1 TO 10 120 S(H)=0 130 NEXT H 140 FOR 1=1 TO N 150 READ t i 1 , R( I ) , C 160 FOR J=1 TO 6 170 S ( J ) = S ( J ) + R ( I ) * J 180 NEXT J 190 FOR K=7 TO 10’200 S(K)=S(K) + 10"0*C*R( I) A ( K - 7 ) / A 210 NEXT K215 PRINT 'R = / , R ( I ) / E = ' , IN T<10A4 * C / A + . 5 ) / 1 0 0220 NEXT I230 D = S ( 2 ) - S ( 1 ) *2 /N240 E = S ( 3 ) - S ( 1 ) * S ( 2 ) / N250 F = S ( 4 ) - S ( 1) * S ( 3 ) / N260 G = S ( 8 ) - S ( 1) * S ( 7 ) / N270 A=EA2 - ( S ( 4 ) - S ( 2 ) " 2 / N ) * D280 B = ( S ( 5 ) - S < 2 ) * S ( 3 ) / N ) * D - E * F290 C = (S < ? ) -S (2 ) *S < 7 ) /N ) *D -E *G295 0 = ( A * ( ( S ( 1 0 ) - S ( 3 > * S ( 7 ) / N ) * D - F * G ) + B * C )300 A 3 = Q / ( A * ( ( S ( 6 ) - S ( 3 ) A2 / N ) * D - F * 2 ) + B "2 )310 A2=(B*A3-C) /A320 A 1 = (G -A 2 * t -A 3 * F ) /D330 A 0 = ( S ( 7 ) - A 1 * S ( 1 ) - A 2 * S ( 2 ) - A 3 * S < 3 ) ) / N335 PRINT340 PRINT 'AO = ' , INT<10 * A 0 + . 5 ) / 1 0 350 PRINT ' A l = ' , I N T ( 1 0 * A 1 + . 5)710 360 PRINT 'A2 = ' , I N T ( 1 0 * A 2 + . 5 ) / 1 0370 PRINT 'A3 = ' , IN T (1 0 * A 3 + . 5 ) / 1 0371 PRINT3.72 FOR i= 1 TO Ni 7 4 Y = A 3 * R ( I ) A3 + A 2 * R ( I ) ‘ 2+A1*R( I ) +A0 376 PRINT 'Y = ' , I N T ( 1 0 " 2 * Y + . 5 ) / 1 0 0 378 NEXT I 400 END

APPENDIX D ( i i )

Quench curve obtained from d.p.m. package on RackBeta 1215 * ’’

Ou6+JL+\ C lEFF1Z RATIO

8947.0 25.06 1.3128019.0 22.46 1.1877492.0 20.99 1.1464523.0 18.27 1.0505985.0 16.76 1.0094275.0 11.97 .8482531.0 7.09 .6721774.0 4.97 .5251743.0 4.88 .5911070.0 3.00 .460

PLOT QUENCH CURVES <R> ->R

IUSY CALCULATING

ISOTOPE 1. UINDOU 1

EFFZ25.0*4 *|

I **»I »*I *

22.50+I *I 0*I «*

2 0 . 00+ *I « +I «*I *0

17.50+ «

I II •I *«

15. 00+ «I ♦«I *I **

12.50+ **

II *I *»

10.00+ *•I •I *•I *

7.50+ ♦*I *0I ♦I +*

3.00+ •#«*«•*

I ••I *10II------♦--------------♦-----------♦-------------♦-------------♦................♦................♦-------------♦............................0

.5 0 0 .A 00 .7 0 0 .0 0 0 .9 0 0 1.000 ' 1.100 1.200 1.300