identification of an adenylyl cyclase inhibitor for treating...

DOI: 10.1126/scitranslmed.3001269, 65ra3 (2011);3 Sci Transl Med

et al.Hansen Wangand Inflammatory PainIdentification of an Adenylyl Cyclase Inhibitor for Treating Neuropathic

Editor's Summary

humans, it may prove useful to eliminating seemingly purposeless pain from our lives.If the selective action of NB001 on neuropathic pain and its lack of serious side effects also holds true in

pain-related brain region.−hippocampus, a noneffect may underlie its analgesic ability, a conclusion consistent with the fact that it does not alter such plasticity in the

Thisdorsal horn of the spinal cord and the anterior cingulate cortex to ''learn,'' a process triggered in neuropathic pain. A clue to how NB001 works can be gleaned from the result that it extinguishes the ability of synapses in the

profile of this drug.safetymotor function, and fear all showed that NB001 had no effects on these endpoints, a good sign for the potential

affect neurotransmission of the critical hormone glutamate or the size of glutamate-induced currents. Tests of anxiety,not interfere with normal nociception, the sensation that allows the animal to escape dangerous heat. It does not

Just as important as these effects of NB001 on chronic pain are the effects that it does not have. NB001 does

also prevented allodynia, although to a lesser extent, suggesting that NB001 acts on multiple sites in the body.drug was injected directly into the anterior cingulate cortex (a brain region involved in neuropathic pain generation), it were ligated or in mice with chronic inflammatory pain, produced by an injection of an irritant into a paw. And when theNB001 prevented allodynia (a condition in which an innocuous stimulus causes pain) in mice in which certain nerves NB001, was most effective and also inhibited cyclic AMP production in mouse brain slices and human neurons.production and of the transcription factor CREB in human cells transfected with adenylyl cyclase 1. One of these, thought to underlie this kind of pain. The authors screened chemical compounds for inhibition of cyclic AMPactivity-dependent enzyme, expressed selectively in neurons, that is critical for the pain-related neural plasticity

Adenylyl cyclase 1 has many characteristics of a good drug target for neuropathic pain: It is an

identify one, NB001, which can block this type of pain in rodents without apparent side effects.for drugs that selectively block a type of calcium-activated adenylyl cyclase that participates in neuropathic pain. They

have now identified a promising new candidate by screeninget al.neuropathic pain are desirable but scarce. Wang seems to have no purpose. Analgesics that block only−−also called neuropathic pain−−burning, or aching pain

Pain from a hot stove or an injury can be a good thing. It can help to prevent more serious damage, but chronic,

No Gain from Pain

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R E S EARCH ART I C L E

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Identification of an Adenylyl Cyclase Inhibitor forTreating Neuropathic and Inflammatory PainHansen Wang,1* Hui Xu,1* Long-Jun Wu,1* Susan S. Kim,1* Tao Chen,1

Kohei Koga,1 Giannina Descalzi,1 Bo Gong,1 Kunjumon I. Vadakkan,1 Xuehan Zhang,1

Bong-Kiun Kaang,2 Min Zhuo1†

Neuropathic pain, often caused by nerve injury, is commonly observed among patients with different diseases.Because its basic mechanisms are poorly understood, effective medications are limited. Previous investigationsof basic pain mechanisms and drug discovery efforts have focused mainly on early sensory neurons such as dorsalroot ganglion and spinal dorsal horn neurons, and few synaptic-level studies or new drugs are designed to targetthe injury-related cortical plasticity that accompanies neuropathic pain. Our previous work has demonstrated thatcalcium-stimulated adenylyl cyclase 1 (AC1) is critical for nerve injury–induced synaptic changes in the anteriorcingulate cortex. Through rational drug design and chemical screening, we have identified a lead candidate AC1inhibitor, NB001, which is relatively selective for AC1 over other adenylate cyclase isoforms. Using a variety ofbehavioral tests and toxicity studies, we have found that NB001, when administered intraperitoneally or orally,has an analgesic effect in animal models of neuropathic pain, without any apparent side effects. Our study thusshows that AC1 could be a productive therapeutic target for neuropathic pain and describes a new agent for thepossible treatment of neuropathic pain.

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INTRODUCTION

Acute or physiological pain is necessary for animals and humans tosurvive in a changing environment. Persistent neuropathic pain char-acterized by chronic shooting pain and burning sensations, however,is usually caused by injury or diseases such as cancer or AIDS (or evenAIDS medications) and has no physiological benefit (1–5). Unfor-tunately, many conventional analgesics are not selective and affect bothphysiological and pathological pain. Treatment of pathological painrequires selective drug targets and compounds that preferentially in-hibit pathological pain without or with minimal effect on protectivephysiological pain (3, 4, 6, 7).

Recent molecular studies of different levels of the central nervoussystem (CNS) have underscored the fact that the mechanisms leadingto chronic neuropathic pain are far more complicated than previouslyassumed (2, 4, 8–11). At peripheral sites, injuries trigger sensitizationand induce prolonged abnormal neural activity along primary afferentfibers. In the spinal cord, long-term potentiation (LTP) of sensory syn-aptic transmission in the spinal dorsal horn projecting neurons oftenoccurs (10). Recent work demonstrates that rapid synaptic plasticityalso takes place in cortical areas that participate in pain perception,such as the anterior cingulate cortex (ACC) (4, 12, 13). Peripheral in-flammation or nerve injury produces LTP of glutamate release and glu-tamate receptor–mediated synaptic responses (14–16). Protein and ionchannels that are involved in the induction and expression of plasticchanges in somatosensory pathways are potential drug targets for treat-ing chronic pain (17–19). Targeting the signaling mechanisms involved

1Department of Physiology, Faculty of Medicine, University of Toronto Centre for theStudy of Pain, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario,Canada M5S1A8. 2National Creative Research Initiative Center for Memory,Department of Biological Sciences, College of Natural Sciences, Seoul NationalUniversity, Seoul 151-747, Korea.*These authors contributed equally to this work.†To whom correspondence should be addressed. E-mail: [email protected]

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in injury-related plasticity may be particularly helpful in discoveringmore effective drug treatments for neuropathic pain. Among severalcandidates, the N-methyl-D-aspartate (NMDA) receptor, a major gluta-mate receptor that is important for synaptic LTP, has been intensivelyinvestigated as an analgesic target by academic laboratories andpharmaceutical companies. However, the use of a general NMDA re-ceptor antagonist is limited by side effects such as ataxia and sedation(20). Still, signaling proteins downstream from the NMDA receptor re-main viable targets for neuropathic pain treatment. Our previous studieshave found that one of these targets, calcium-stimulated adenylyl cy-clase 1 (AC1), plays a critical role in pain-related LTP in both the spinalcord dorsal horn (21) and the ACC (22). Behaviorally, AC1 and AC8knockout mice show reduced inflammatory, deep muscle pain and neu-ropathic pain (15, 23, 24), whereas other physiological functions remainintact in AC1 knockout mice, including acute pain (24), hippocampalLTP, and related Morris water maze performance, as well as anxiety-like behaviors and motor functions (25). Considering that AC1 is mainlyexpressed in the CNS (26–28), we propose that AC1 may be a suitableneuron-specific drug target for treating neuropathic pain.

Here, we carried out integrative chemical screening and biologicalexperiments to identify selective inhibitors for AC1 and examined thepotential analgesic effects of the AC1 inhibitor in neuropathic and in-flammatory pain models. By chemical screening, we have successfullyidentified a lead AC1 inhibitor, NB001, and explored its effects in vitroand in vivo.

RESULTS

General consideration of AC1 as a therapeutic pain targetOne major challenge to developing a clinically useful analgesic drug isto avoid side effects in non-neuronal tissues. Many clinically proveneffective drugs have been removed from the market as a result of their

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side effects in non-neuronal organs such as the cardiovascular systemand liver. Ways to avoid side effects include (i) identifying drug targetsthat are mainly expressed in neurons; (ii) choosing target proteins orchannels that are activity-dependent, with an almost “silent” status innormal physiological status; and (iii) choosing target proteins that arecritical for chronic pain–related neuronal plasticity, but not for othercognitive functions (4). Few target proteins meet all three criteria. Onthe basis of previous studies and our recent studies with geneknockout mice, we proposed that AC1 would be a good target fortreating chronic pain. First, AC1 is primarily expressed in neurons,and no AC1 gene expression was found in heart, liver, or kidney cells(Table 1). Second, AC1 is activated in a calcium-calmodulin (CaM)–dependent manner (24) (Fig. 1A). Third, it acts downstream from theglutamate NMDA receptors and contributes to chronic pain–relatedneuronal plasticity in the cortex and spinal cord (3, 4, 15). Finally,gene knockout mice lacking AC1 showed reduced or blocked behav-ioral sensitization to non-noxious mechanical stimuli in animalmodels of inflammatory and neuropathic pain.

Biochemical screening and identification of an AC1 inhibitorTo our knowledge, there is no selective inhibitor for AC1. Thus, wedecided to first perform chemical screening experiments to identifyAC1 inhibitors, using a heterologous expression system, in which AC1was stably expressed in human embryonic kidney (HEK) 293 cells,which do not express AC1 (29, 30). HEK293 cells were stably trans-fected with the AC1 expression vectors, and AC1 expression was con-firmed by reverse transcription polymerase chain reaction (RT-PCR).AC1 expression was not observed in the control cells (Fig. 1B). Wethen tested the stimulatory effects of forskolin and the calcium ion-ophore A23187 in these cells. We found that co-application of forskolinand A23187 induced an increase in cyclic AMP (adenosine 3′,5′-monophosphate) in AC1-expressing cells, but caused a significantlysmaller effect in control HEK293 cells, indicating little or no basal

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AC activity (Fig. 1B). On the basis of the chemical structures of forsko-lin and adenosine 5′-triphosphate (ATP), we screened a family of com-pounds (NB001 to NB016) for potential AC1 inhibition. We tested theireffects on forskolin- and A23187-stimulated cyclic AMP concentrationsin HEK293 cells stably expressing AC1. At a concentration of 100 mM,we found that NB001 to NB007 significantly reduced cyclic AMP con-centrations compared to other compounds, with inhibition of 69.5 ± 0.2to 96.9 ± 0.1% (n = 4 wells) (table S1). Among them, NB001(C12H20N6O) (Fig. 1E) was the most effective inhibitor, with inhibitionof 96.9 ± 0.1% at 100 mM (n = 4 wells) (table S1).

Additionally, we measured the transcription factor cyclic AMP re-sponse element–binding (CREB) protein activity, using a dual luciferasereporter system. In this assay, we measured changes in the amount offirefly luciferase, the transcription of which is regulated by CREB bindingto an upstream cyclic AMP response element (CRE) (31). We measuredthe effect of NB001 to NB007 on CREB activity in AC1-expressingHEK293 cells stimulated by forskolin and A23187. Consistent with thecyclic AMP assay data, NB001 to NB007 inhibited CREB activity at aconcentration of 100 mM (table S1). NB001 was again the strongest in-hibitor among the compounds (n = 4 wells) (table S1).

Selectivity of NB001Like AC1, AC8 is also stimulated by CaM, although it is less sensitiveto calcium (32, 33). We have performed experiments similar to thosewith AC1 by expressing AC8 in HEK293 cells. The inhibitory effects ofNB001 were tested at different concentrations (between 0.2 and 200 mM)in AC1- or AC8-expressing HEK293 cells after the application of forskolinand A23187. By cyclic AMP assay, we found that NB001 inhibitedcyclic AMP production in a concentration-dependent manner, with anestimated median inhibitory concentration (IC50) of 10 mM in AC1-expressing HEK293 cells (Fig. 1C). However, in AC8-expressingHEK293 cells, NB001 produced significantly less inhibition, with anestimated IC50 of 139 mM (Fig. 1D). We also tested the inhibitory


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Table 1. Distribution patterns of target proteins for treatment of neu-ropathic pain. Blank, no data found; +++, strongly expressed or veryeffluent; +, less effluent; 0, no expression. The distribution patterns ofsome proteins are based on in situ hybridization data. DH, dorsal horn;

DRG, dorsal root ganglion; GABAR, GABA receptor; NMDAR, NMDAreceptor; COX-2, cyclooxygenase 2; NOS, nitric oxide synthase; 5-HT5-hydroxytryptamine (serotonin); NA, noradrenaline; VR1, vanilloidreceptor 1.

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n

Target protein
CNS distribution
Heart
Lung Liver Kidney Testis Skeletal muscle Adrenal Uterus Brain DH DRG
AC1
+++ +++ + 0 0 0 0 0 0 + 0
GABAR
+++ +++ + + + + + + +
Gabapentin-binding sites

NMDAR
++ ++ +? + + + + +
Opioid receptors
+++ +++ ++ ++ ++ ++ ++ ++ + + +
COX-2
+ + + ++ ++ ++ ++ ++ + + +
NOS
++ ++ ++ + + + + + + + +
Cannabinoid
++ + + + + + + +
Calcium channels
++ + + + + + + +
5-HT/NA
++ ++ ++ + + + + + +
Adrenergic receptors
++ ++ + ++ + + + + + + +
VR1
++ ++ ++ ++ + + + + + +


effect of NB001 on other ACs including AC5, AC6, and AC7, which aremostly coupled to G protein (heterotrimeric guanosine 5′-triphosphate–binding protein)–coupled receptors. The effects of NB001 at variousconcentrations between 0.2 and 200 mM were evaluated in AC5-, AC6-,or AC7-expressing HEK293 cells. The estimated IC50s of NB001 forAC5, AC6, and AC7 were 210, 167, and 191 mM, respectively (Fig.

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1D). These results indicate that NB001is relatively selective for AC1.

Effects of NB001 on cyclicAMP production in adultmouse brain slicesTo investigate the effect of the AC1 inhib-itor NB001 in brain tissues, we studied theeffects of NB001 using brain slices of theACC from adult mice. We first charac-terized the stimulatory effects of forskolinand calcium ionophore A23187 in ACCslices. We found that co-application offorskolin (10 mM) and A23187 (10 mM)induced significant increases in the cyclicAMP concentration, whereas applicationof either forskolin (10 mM) or A23187(10 mM) alone caused less cyclic AMPproduction (Fig. 2A). We then tested theeffect of NB001 on cyclic AMP produc-tion in ACC slices stimulated by forskolinand A23187. Application of NB001 (0.1 to200 mM) 15 min before and during thetreatment with forskolin and A23187(10 mM for each) for 30 min inhibited cy-clic AMP production in a dose-dependentmanner (Fig. 2B). However, the inhibitoryeffect of NB001 was limited, even at a highconcentration (100 or 200 mM). One pos-sible explanation is that other isoforms ofACs were also activated in ACC slices,and NB001 is ineffective for other iso-forms of ACs in ACC neurons.

The activation of NMDA receptors byglutamate can elicit a calcium-dependentincrease in cyclic AMP in the hippocam-pal CA1 region (34, 35). Our previous studyhas shown that AC1, but not AC8, con-tributes to the elevation of cyclic AMPelicited by NMDA receptor activation incortical neurons (32). To investigate the ef-fect of NB001 at a more physiological con-dition, we tested the stimulatory effectsof glutamate in mouse ACC slices. Wefound that application of glutamate (0.25to 1 mM) could induce significant cyclicAMP increases in mouse ACC slices (Fig.2C). Application of NB001 (0.1 to 200 mM)15 min before and during the treatmentwith glutamate (0.5 mM) for 30 min signif-icantly inhibited the cyclic AMP productionstimulated by glutamate in a dose-dependent

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manner; the effect of glutamate was completely blocked by NB001 atthe concentration of 100 or 200 mM, and the estimated IC50 is 5.1 mM(Fig. 2D). These results indicate that NB001 can inhibit calcium-stimulated AC1 during glutamate treatment in adult mouse ACCneurons, which suggests that it might be effective at physiological orpathological conditions in adults.

Fig. 1. AC1 stably expressed in HEK293 cells. (A)A model shows that AC1 acts downstream fromthe glutamate NMDA receptors (NMDAR) and isactivated in a calcium-dependent manner. (B)Upper panel: AC1 expression was verified by RT-PCR. 1, 100–base pair (bp) DNA ladder marker; 2,HEK293 cells transfected with AC1 plasmid; 3,HEK293 cell control; 4, mouse brain cortex sample.The size of PCR products is 384 bp. Lower panel:stimulatory effect of forskolin and calcium ion-

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ophore A23187 in HEK293 cells stably transfected with AC1. HEK293 cells were stimulated by forskolin(10 mM), A23187 (10 mM), or forskolin (10 mM) plus A23187 (10 mM) for 30 min before cyclic AMP wasmeasured. n = 4 wells. (C) Inhibitory effects of NB001 on AC1-expressing HEK293 cells. n = 4 wells. (D)Effect of NB001 on AC5, 6, 7, or 8 activities. HEK293 cells were pretreated with NB001 for 15 min andthen stimulated by 10 mM forskolin (AC5, AC6, and AC7) or 10 mM forskolin plus 10 mM A23187 (AC1 andAC8) for 30 min in the presence of NB001. n = 4 wells. AC activity was detected by cyclic AMP assay. (E)Chemical structure of NB001.

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Effect of NB001 in human neural cellsTo investigate whether NB001 is effective in mammalian neurons, wetested the effect of NB001 in the human neuroblastoma SH-SY5Ycells, which have been shown to express AC1 (36). In addition, SH-SY5Y cells express glutamate receptors and respond to glutamate orNMDA stimulation (37, 38). Indeed, we found that bath application ofglutamate (0.25 to 1 mM) could induce significant increases at cyclicAMP levels in SH-SY5Y cells (Fig. 2E). Application of NB001 (0.1 to200 mM) 15 min before and during the treatment with glutamate(0.5 mM) for 30 min inhibited the cyclic AMP production in a dose-dependent manner; the effect of glutamate was completely blocked byNB001 at a concentration of 100 or 200 mM, with an estimated IC50

of 8.3 mM (Fig. 2F). These results indicate that NB001 can inhibitcalcium-stimulated AC during glutamate treatment in SH-SY5Y cells,suggesting that it might be effective in human neurons at physiologicalconditions.

Analgesic effects of NB001 in animal models of neuropathicand inflammatory painPrevious studies with genetic knockout mice lacking AC1 showed thatbehavioral allodynia (pain experienced to a usually innocuous stimulus)in animal models of neuropathic pain and inflammatory pain was sig-nificantly reduced (24). However, it is difficult to rule out the possiblecontribution of developmental defects in AC1 knockout mice that maycontribute to behavioral results. With the identification of NB001 as aninhibitor for AC1, we examined the effects of NB001 on behavioral al-lodynia in animal models of neuropathic pain induced by nerve ligation(39). Similar to our previous report (15, 39), we found that behavioralallodynia reached its peak at day 7 after the nerve injury. Intraperitonealadministration of NB001 (0.1 mg/kg) given 30 min before behavioralallodynia testing produced a significant analgesic effect. NB001 at ahigher dose of 1 mg/kg produced a greater inhibition of behavioralallodynia (Fig. 3A).

To address the possible central, spinal, or peripheral action ofNB001, we performed site-directed local administration of NB001 inmice with nerve injury. We found that multiple microinjections ofNB001 into the dorsal and ventral ACC on both sides of the brain(a total of 75.6 nmol, 0.5 ml for each site) produced a significant in-hibitory effect on behavioral allodynia (n = 5 mice, P < 0.05) (fig. S1A),whereas NB001 (189.0 nmol, 5.0 ml) intrathecally applied to the spinalcord or subcutaneous injections of NB001 (378.0 nmol, 10 ml) did notproduce any significant inhibitory effect on nerve injury–induced me-chanical allodynia. These results suggest that NB001 is likely actingcentrally at supraspinal sites to produce analgesic effects (fig. S1, Band C). The inhibitory effects on mechanical allodynia produced byNB001 applied locally in the ACC (even with multiple injection sites)were smaller than that applied systemically, suggesting that NB001may also act at other cortical and subcortical sites.

In general, application of NB001 at different dosages did notcause any abnormal behaviors in animals. Animals injected withNB001 were calm and less responsive to behavioral allodynic mea-surement than were control animals. To compare our results withNB001 with the analgesic effect of currently available drugs for neu-ropathic pain, we also performed experiments with gabapentin. In-traperitoneal injection of gabapentin (30 mg/kg, n = 4 mice) producedsignificant analgesic effects in the animals with neuropathic pain. Theinhibitory effects were comparable to those produced by NB001 at0.1 mg/kg (Fig. 3A).

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Behavioral allodynia induced by the hindpaw injection of completeFreund’s adjuvant (CFA) (50%) has been commonly used for evaluat-ing a drug’s analgesic effects in chronic inflammatory pain. In previ-ous studies, we reported that AC1 knockout mice showed significantreduction in behavioral allodynic responses induced by hindpaw CFAinjection (24). Furthermore, peripheral neurogenic plasma extravasa-tion induced by capsaicin injection was the same in AC1 knockout

Fig. 2. Effect of NB001 in mouse ACC slices and human neural cell line.(A) Cyclic AMP production in ACC slices stimulated by forskolin (10 mM),
A23187 (10 mM), or forskolin plus A23187. Cyclic AMP was measured at30 min after treatments. n = 4 mice. (B) Effect of AC1 inhibitor NB001(0.1 to 200 mM) on cyclic AMP production stimulated by forskolin (10 mM)and A23187 (10 mM) in ACC slices. NB001 was applied 15 min before andduring the treatment with forskolin and A23187 for 30 min. n = 4 mice.(C) Cyclic AMP production in ACC slices stimulated by glutamate (0.25 to1 mM) for 30 min. n = 4 mice. (D) Effect of AC1 inhibitor NB001 (0.1 to200 mM) on cyclic AMP production stimulated by glutamate (0.5 mM).NB001 was applied 15 min before and during the treatment with gluta-mate for 30 min. n = 4 mice. (E) Cyclic AMP production in SH-SY5Y cellsstimulated by glutamate (0.25 to 1 mM) for 30 min. n = 4 wells. (F) Ap-plication of AC1 inhibitor NB001 (1 to 200 mM) 15 min before and duringthe treatment with glutamate (0.5 mM) for 30 min could inhibit cyclicAMP production stimulated by glutamate in a dose-dependent manner.n = 4 wells.



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mice and in littermate wild-type mice (24).Therefore, we expect that NB001 wouldbe analgesic in the animal model of in-flammatory pain induced by CFA injec-tion. Application of a 0.4-mN von Freyfiber to the dorsum of a hindpaw elicitedno response in control mice, but at 1 and3 days after CFA injection, mice with-drew their hindpaw in response to stim-ulation of the ipsilateral or, to a lesserextent, the contralateral hindpaw. Oneday after the CFA injection, NB001 [1 and3 mg/kg intraperitoneally (ip)] producedsignificant analgesic effects. The inhibitoryeffect was dose-related: Greater inhibitionwas found with a higher dose of NB001(n = 5 mice for each group) (Fig. 3B). Sim-ilar analgesic effects were also obtained 3days after CFA injection (Fig. 3B).

Effect of NB001 onnociceptive responsesNext, we carried out behavioral tests todetermine whether NB001 affects physi-ological nociceptive responses in normalmice. We tested the effect of intraperi-toneally injected NB001 in different noci-ceptive tests. For noxious thermal pain,NB001 affected behavior in neither thespinal nociceptive tail-flick reflex nor thehot-plate test (at 55°C) (Fig. 3, D to F).To determine potential effects at a lowertemperature, we also tested the animalson a hot plate set at 50°C and found thatbehavioral response latencies were notaffected (Fig. 3E). For mechanical thresh-old, we measured hindpaw withdrawal re-sponses to von Frey filaments in normalmice. Again, NB001 did not produce anysignificant effect (Fig. 3C). These resultssuggest that NB001 did not affect acutenociception, in good accord with previ-ous findings of unchanged physiologicalpain in AC1 knockout mice (24).

Effect of NB001 administered orallyin a rat model of neuropathic painWe next examined the effects of NB001in a rat model of neuropathic pain (seeMaterials and Methods) to see whetherorally delivered NB001 can produce anal-gesic effects, a common method of drugdelivery in patients. As shown in Fig. 3G,we found that NB001 at 1 mg/kg (orally)produced a significant reduction in be-havioral allodynia 45 min after oral inges-tion, and that the analgesic effect lasted forat least 2 hours (n = 6 rats). The analgesic

Fig. 3. Effect of NB001 on acute pain and neuropathic pain. (A) Effect of NB001 on allodynia after nerveligation. Intraperitoneal administration of NB001 (0.1 mg/kg body weight) given 30 min before behavioralallodynia testing produced a significant analgesic effect. NB001 at a high dose of 1 mg/kg produced agreater inhibition of behavioral allodynia. Intraperitoneal injection of gabapentin (30 mg/kg; n = 4 mice)produced significant analgesic effects in the animals with neuropathic pain. The inhibitory effects werecomparable to those produced by NB001 at 0.1 mg/kg. (B) Effect of NB001 on inflammatory pain. At1 day after the CFA injection, NB001 (1 and 3 mg/kg ip) produced significant analgesic effects. The inhib-itory effect is dose-related; greater inhibition was found with a higher dose of NB001 (n = 5 mice). Similaranalgesic effects were also obtained at 3 days after CFA injection. (C) Comparison of behavioral responsesto non-noxious mechanical stimulus. There was no significant difference in hindpaw withdrawal to von Freyfilaments before and after intraperitoneal injection of saline (n = 5) and NB001 (n = 6). (D) Effect of NB001 intail-flick (TF) test. NB001 (10 mg/kg ip) did not affect spinal nociceptive TF reflex. (E) Effect of NB001 in hot platetest at 50°C. Therewasno significantdifference in response latencybefore andafter intraperitoneal injection ofsaline (n= 5) and NB001 (10mg/kg) (n = 6) in hot plate test set at 50°C. (F) Effect of NB001 in hot plate testat 55°C. There was no significant difference in response latency before and after intraperitoneal injection ofsaline (n = 5) and NB001 (10 mg/kg) (n = 6) in hot-plate test set at 55°C. (G) Oral application of NB001(1mg/kg, 3ml per injection) produced significant analgesic effects in adult ratswith neuropathic pain (n=5).Allodynia was measured at 45 min, 2 hours, and 4 hours after oral application. *P < 0.05.

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effect of NB001 is reversible, because be-havioral allodynia returned 4 hours afterthe oral application, suggesting that theeffects of NB001 are unlikely to be toxicor to induce nonselective side effects.

Effect of NB001 on anxiety, motorfunction, and fear memoryTo test the potential side effects of NB001,we carried out a battery of behavioralanxiety and motor function tests withthe same analgesic dose of NB001. First,in two tests of anxiety-like behavior, ele-vated plus maze (EPM) and open-fieldtest (40, 41), we found that NB001 didnot produce any significant effects (Fig.4, A to D). The NB001-injected mice didnot make a significantly different numberof entrances into the arms of the EPM, nordid they spend more time in the openarmswhencompared to the saline-injectedmice (Fig. 4, A and B). Also, the distancetraveled in the center of the open-field testdid not differ between NB001- and saline-injectedmice (Fig. 4C), againdemonstratingthat NB001 had no effect on anxiety-likebehavior. We also tested the motor func-tion of the mice injected with NB001. Inthe open-field test, we found that NB001only produced a significant change duringthe first 5min after the injection, and therewere no differences in the total distancetraveledbetweenNB001- and saline-injectedmice (Fig. 4, E and F). In the RotaRodmo-tor test, NB001 did not cause any signifi-cant changes (Fig. 4G), illustrating thatNB001 had no effect on the motor func-tion of the mice.

Cyclic AMP is a key second mes-senger in the CNS, and calcium-stimulatedcyclic AMP signaling pathways have beensuggested to contribute to learning andmemory (42, 43). However, in previousstudies of AC1 knockout mice, severalforms of learning and memory were notaffected in AC1 knockout mice, includingthose measured by classic fear memorytests and the Morris water maze (25). Oneexplanation for this lack of effect may bethat the function of AC1 may be compen-sated for by other AC isoforms such as AC8and other calcium/calmodulin-dependentprotein kinases, including CaMKII andCaMKIV.We testedwhetherNB001affectsfearmemory inducedbynoxious foot shock.Consistent with previous genetic studies(25), we found that pretreatment withNB001 (1 mg/kg ip) did not significantly

Fig. 4. Effect of NB001 on anxiety-like behavior, locomotor activity, and motor function. (A) There was
no significant difference in the number of arm entries (open + closed) between saline-injected (n = 5)and NB001-injected (n = 7) mice. (B) There was no significant difference in the percentage of timespent in the open arms between saline- and NB001-injected mice. (C) From left to right: diagram ofthe EPM, where filled boxes represent closed arms and open boxes represent open arms; represen-tative traces showing the movement of saline- and NB001-injected mice in the EPM for 5 min. (D) Rep-resentative traces showing the movement of saline-injected (n = 6) and NB001-injected (n = 6) mice inthe open field for 30 min. (E) NB001-injected mice traveled significantly more within the first 5 minduring the 30-min session than did saline-injected mice. *P < 0.001. (F) There was no significant differ-ence in the total distance traveled between saline- and NB001-injected mice. (G) There was no signif-icant difference in latency to fall between saline-injected (n = 5) and NB001-injected (n = 6) mice. Ineach experiment, mice were given an intraperitoneal injection of NB001 (10 mg/kg) or saline 30 minbefore the test.
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affect fear memory (n = 4 mice). Furthermore, we also injected NB0011 day after the fear conditioning and found that fear memory was notsignificantly affected (n=4mice), indicating thatNB001 at the analgesicdosage does not affect the formation and expression of fear memory.

Effect of NB001 on glutamate receptor–mediated currentsGlutamate AMPA receptors mediate most of the basal synaptic trans-mission in the spinal dorsal horn and the ACC (44, 45), whereasNMDA receptors are important for producing synaptic potentiation(4, 46). It is thus important to determine whether NB001 producesanalgesic effects by affecting these glutamate receptor–mediated re-sponses in sensory neurons. We performed whole-cell patch-clamprecordings from ACC pyramidal cells, a key cortical area for process-ing pain information in animals and humans (4). We first measured

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the effects of NB001 on AMPA receptor–mediated responses. AMPAreceptor–mediated excitatory postsynaptic currents (EPSCs) wererecorded from cingulate pyramidal cells before and after the ap-plication of NB001. We did not see any significant change in AMPAreceptor–mediated EPSCs after the perfusion with NB001 (50 mM)(n = 8 neurons, 6 mice) (Fig. 5A). Paired-pulse facilitation (PPF), asimple form of synaptic plasticity, was also measured by applicationof paired stimuli at different intervals. We found that PPF tested atdifferent interpulse intervals also did not show any significant change[PPF in the presence of NB001, n = 7 neurons, 2 mice; control PPF inthe absence of NB001, n = 17 neurons, 7 mice; two-way analysis ofvariance (ANOVA), P > 0.05] (Fig. 5B).

NMDA receptors are important for pain-related plasticity in thespinal cord and cortex (2, 4, 10), and NMDA receptor antagonists areanalgesic in different animal models of chronic pain (47). To rule outthe possibility that NB001 may produce analgesic effects through in-hibition of NMDA receptor–mediated responses, we performed two dif-ferent types of experiments. We first evaluated the effect of NB001 onthe input-output curves of NMDA receptor–mediated EPSCs. NB001(50 mM) application did not produce any reduction of NMDA receptor–

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Fig. 5. Effect of NB001 on AMPA receptor–mediated synaptic responses.(A) Bath application of NB001 (50 mM) did not affect AMPA receptor–
mediated basal synaptic responses in the ACC neurons (n = 8). Inset: rep-resentative traces and pool data of AMPA receptor–mediated EPSCs inACC neurons with the perfusion of NB001 (50 mM) for 10 min. (B) PPFwas recorded at intervals of 35, 50, 75, 100, and 150 ms. Open circles:neurons in the absence of NB001 (n = 17 neurons); filled circles: neuronsin the presence of NB001 (100 mM) applied in the pipette (n = 7 neurons).Representative traces of PPF with an interval of 50 ms recorded in theACC are shown in the insets.
Fig. 6. Effect of NB001 on NMDA receptor–mediated responses. (A) NB001application did not affect NMDA receptor–mediated EPSCs recorded from
ACC pyramidal cells. Representative traces and pooled data of synapticNMDA receptor–mediated input-output curve in ACC neurons in the ab-sence (open circles; n = 6 neurons, 3 mice) and presence (filled circles;n = 6 neurons, 2 mice) of NB001 (100 mM) in the pipette. (B) NB001 ap-plication did not affect the I-V response curves of NMDA receptor–mediatedresponses. Representative traces and pooled data of synaptic NMDAreceptor–mediated I-V curve in ACC neurons in the absence (open circles;n = 6 neurons, 3 mice) and presence (filled circles; n = 6 neurons, 2 mice) ofNB001 (100 mM) in the pipette.



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mediated input-output curves (Fig. 6A). Next, we also tested whetherNB001 affected the I-V (current-voltage) curve of NMDA receptor–mediated EPSCs. Bath application of NB001 did not significantly af-fect the functions of NMDA receptor–mediated I-V curves (Fig. 6B).Voltage dependence of NMDA receptor–mediated EPSCs evoked byfocal electrical stimulation was not affected by the treatment withNB001 (50 mM). These findings indicate that NB001 does not producebehavioral analgesic effects by inhibiting NMDA receptors. Finally, wealso examined whether NB001 affects AMPA and NMDA receptor–mediated responses. We tested the effects of NB001 on the AMPA andNMDA receptor–mediated EPSC ratio. We found that NB001 (50 mM)did not affect the AMPA/NMDA receptor–mediated EPSC ratio inACC neurons (ratio in the absence of NB001: 1.8 ± 0.3, n = 7 neurons,5 mice; ratio in the presence of NB001: 2.5 ± 0.6, n = 7 neurons, 5 mice;P > 0.05).

Effect of NB001 on sensory LTP in the spinaldorsal horn and ACCLTP of excitatory synaptic transmission in the spinal dorsal horn andACC are proposed to be cellular mechanisms for chronic pain (4, 10).Previous studies with AC1 knockout mice found that AC1 is requiredfor synaptic LTP in the dorsal horn neurons and ACC (21, 22). AlthoughNMDA receptor–mediated responses are intact in AC1 knockoutmice, it is difficult to completely exclude potential developmental de-fects in AC1 knockout mice that may contribute to diminished LTP.Thus, we tested whether NB001 inhibits sensory-related LTP in thesetwo areas. In mouse ACC slices, LTP in ACC pyramidal neurons wasnot significantly different in the presence of NB001 (10 mM) in thepipette (control LTP: 164.6 ± 11.2%, n = 15 neurons, 8 mice; LTP inthe presence of NB001, 154.8 ± 16.8, n = 6 neurons, 4 mice; P > 0.05).The application of NB001 (50 and 100 mM) in the pipette completelyprevented the induction of LTP in ACC pyramidal neurons (controlLTP: 164.6 ± 11.2%, n = 15 neurons, 8 mice; LTP in the presence of50 mMNB001: 117.4 ± 9.1, n = 8 neurons, 3 mice, P < 0.05; LTP in thepresence of 100 mM NB001, 111.8 ± 8.8%, n = 7 neurons, 3 mice, P <0.01) (Fig. 7A). The spinal dorsal horn is also critical for pain transmis-sion. Previous studies reported that activation of NMDA and substanceP receptors are critical for spinal LTP (48). AC1 is also expressed inspinal dorsal horn neurons; LTP induced by a pairing protocol is abol-ished in AC1 knockout mice (21). To confirm the requirement of AC1activity for spinal LTP, we tested the effects of NB001 on spinal LTPinduced by the pairing protocol. We found that synaptic potentiationwas significantly reduced (control: 157.8 ± 10.0%, n = 8 neurons, 3 mice;LTP in the presence of 100 mM NB001: 95.3 ± 10.1%, n = 6 neurons,5 mice; P < 0.05) (Fig. 7B). These results provide strong evidence forcritical roles of AC1 in both spinal and ACC LTP, one possible cel-lular mechanism contributing to neuropathic pain.

Effect of NB001 on memory-related hippocampal LTPOne potential side effect of NB001 is alteration of hippocampal LTP,which is important for the formation of new spatial memories. To ex-amine the effects of NB001 on LTP in the CA1 region of the hippo-campus, we tested LTP with a bath perfusion of 50 mM NB001. LTPwas induced in the CA1 region of mouse hippocampal slices by strongtetanic stimulation (two trains, 100 Hz for 1 s per train, delivered at a20-s interval) (49, 50). We found that the amount of potentiation 45 minafter tetanic stimulation with NB001 perfusion was similar to the poten-tiation in slices of control animals (control: n = 5 slices, 169.5 ± 12.7%;

Fig. 7. Effect of blockade of ACC LTP and spinal LTP by postsynapticapplication of NB001. (A) Representative traces and pooled data of LTP
induced by pairing protocol in ACC neurons in the absence (open squares;n = 15 neurons, 8 mice) and presence (filled squares; n = 7 neurons, 3 mice)of NB001 (100 mM) in the pipette. (B) Pooled data of LTP induced bypairing protocol in spinal lamina II neurons in the absence (open squares;n = 8 neurons, 3 mice) and presence (filled squares; n = 6 neurons, 5 mice)of NB001 (100 mM) in the pipette. (C) Pooled data of two-train tetanicstimulation–induced LTP in the CA1 region of the hippocampus in controlslices (open squares; n = 5 slices) and in slices with the perfusion of 50 mMNB001 (filled squares; n = 5 slices).



NB001: n = 5 slices, 163.9 ± 20.7%; comparing 40 to 45 min after tetanicstimulation to before the stimulation; P = 0.83) (Fig. 7C), indicating thatNB001 did not affect hippocampal LTP.

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DISCUSSION

The present study is built on two previous discoveries. First, peripheralinjuries trigger plastic changes in cortical areas of the brain (includingthe ACC), induce an array of activity-dependent immediate-earlygenes, and enhance synaptic responses (3, 5, 12, 24, 51). In a recentstudy, we elucidated the synaptic mechanism for this neuronal plasticityafter nerve injury in the ACC, namely, presynaptic enhancement of glu-tamate release and postsynaptic enhancement of AMPA receptor–mediated responses (15). Second, AC1 and AC8, two major formsof calcium-stimulated ACs, are critical for chronic pain, includingneuropathic pain (4, 23, 24). Here, we have identified a candidate mol-ecule that may be useful as a selective AC1 inhibitor. Biochemical andbehavioral studies indicate that NB001 could prove to be a useful an-algesic drug for neuropathic pain.

A previous study has shown that AC1 messenger RNA is highlyexpressed in pain-related cortical areas such as the ACC and insularcortex, and that AC1 activity contributes to behavioral nociceptive re-sponses to nerve injury and inflammation (24). In mice lacking AC1,chronic pain, including neuropathic pain, is significantly reduced,whereas acute pain to noxious thermal or mechanism stimuli remainsintact (24). These results suggested that AC1 is selectively involved inpersistent, chronic pain. In neurons, it has been proposed that AC1couples NMDA receptor–induced cytosolic Ca2+ elevation to cyclicAMP signaling pathways (25, 32, 35). Because activation of NMDAreceptors contributes to persistent, chronic pain (3–5, 7, 51), it is likelythat AC1 acts downstream from the NMDA receptors and thuscontributes to chronic pain.

Systemic administration of NB001 (0.1 to 1 mg/kg), whetheradministered intraperitoneally or orally, inhibited mechanical allo-dynia. Application of gabapentin produced a similar magnitude ofanalgesia at a much larger dose (higher by a factor of >50 to 100).One possible reason for this difference is that NB001 targets pain-related synaptic plasticity, whereas gabapentin affects neurotransmitter re-lease in a nonselective manner. When we tested whether microinjectionof NB001 directly into the ACC could reproduce the effects of systemicNB001 administration, we found that the AC1 inhibitor did not producecomplete blockade of behavioral allodynia. Furthermore, the analgesiceffects were smaller than that produced by NB001 administered system-ically. Several possible factors may explain this difference. First, micro-injection of NB001 into the ACC may not have completely blockedAC1 activity within the ACC, although we performed multiple injectionsof NB001 to inhibit AC1 activity in both the dorsal and the ventral partsof the ACC. Second, AC1 activity in other cortical structures, such as theprefrontal cortex, insular cortex, and somatosensory cortices, may also beimportant for chronic pain (4). Finally, we also cannot rule out the pos-sible contribution of subcortical structures to the effects produced by AC1on neuropathic pain. Thus, it is likely that the strong analgesic effectproduced by NB001 applied systemically is due to inhibition of chronicpain–related plasticity within cortical and subcortical neural networks.

Recently, we found that both presynaptic enhancement and post-synaptic changes in AMPA receptor–mediated responses were blockedin AC1 knockout mice, indicating that AC1 may contribute both pre-

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synaptically and postsynaptically to injury-induced potentiation inthe ACC. In fact, stimulation of AC1 is critical for mossy fiber LTP(52, 53). Because basic properties of synapses, including paired-pulsefacilitation, AMPA receptor–mediated responses, and NMDA receptor–mediated responses, are not affected in AC1 knockout mice, we believethat the involvement of AC1 is an activity-dependent rather than a non-selective side effect. In addition to cortical plasticity, AC1 is also likelyinvolved in spinal cord plasticity. In previous studies, we have shownthat genetic deletion of AC1 inhibits spinal cord LTP induced by thepairing training protocol and that synaptic facilitation is induced byco-application of forskolin and serotonin (21, 54). Considering theincreasing evidence that spinal LTP and descending facilitation con-tribute to chronic pain, it is conceivable that AC1 in spinal cells mayalso contribute to chronic pain.

In addition to its neuron-specific distribution, AC1 has been pro-posed to be an intracellular coincidence detector acting downstreamfromNMDA receptors. Inmany neurons, AC1 is activated by increasesin postsynaptic calcium levels. This provides an ideal target for treat-ment of chronic pain (23, 54). Unlike a single learning event, injury islikely to trigger a series of plastic changes in the CNS. The selectiveeffects of NB001 on neuropathic, but not acute, pain support the hy-pothesis that LTP can be used as a useful cellular model for persistentpain in sensory synapses. Unlike other drug targets that have widedistribution in non-neuronal tissue, AC1 is mainly expressed in neu-rons (26–28). Although one potential side effect of NB001 is a deficitin cognitive functions, AC1 gene knockout mice did not show anymarked memory defects (25).AC1 knockout mice showed normal hip-pocampal late-phase LTP and long-term memory (25). Here, we haveshown that AC1 inhibitor NB001 did not affect anxiety-like behavior ormotor functions. It is possible that cognitive functions, including learningandmemory,may be compensated for by other isoforms ofACs, such asAC8, and other key signaling proteins, including CaMKII and CaMKIV.Further experiments are needed to clarify the potential side effects ofNB001 before it can be tested in patients with neuropathic pain.

We have identified NB001 as a relatively selective inhibitor of AC1,as demonstrated by in vitro biochemical assays. Furthermore, NB001showed inhibitory effects similar to those seen in AC1 knockout mice.Neither genetic deletion of AC1 nor NB001 affected acute pain re-sponses to noxious thermal andmechanical stimuli, but both selectivelyreduced neuropathic pain. Genetic deletion of AC1 and NB001 pro-duced similar inhibitory effects on ACC LTP, and AC1 may also con-tribute to pain-related synaptic plasticity in other cortical areas. NB001is effective in reducing neuropathic pain even after nerve injury. Onepossible explanationmay be that the centralmechanismof chronic painis a multiple-stage learning process. After the initial injury, only somesynapses may undergo potentiation, and many of these potentiatedevents may be reversible as a result of long-term depression (LTD). Forchronic pain to develop, continuous ongoing LTP in other synapses is re-quired; therefore, preventing LTP (through inhibition of AC1 activity) byapplication of NB001 can reduce allodynia seen in neuropathic painmodels even after nerve injury. Allodynia is therefore likely a conse-quence of a long-lasting neuronal circuit abnormality, and it is unlikelyto be explained by a simple form of LTP in an individual synapse. Thisongoing plasticity may also explain why AC1 is preferentially involvedin chronic pain. The lack of an effect of the AC1 inhibitor on memorymay be due to compensation by other key cognitive protein kinasessuch as PKC (protein kinase C), CaMKII, and CaMKIV. Searching fortarget proteins with a cellular model such as ACC LTP may be useful




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for identifying drugs that interfere with or inhibit such network activ-ity. Future studies to design safe compounds with greater efficacy andselectivity on AC1 will improve prospects for clinical treatment ofchronic pain in patients.

We also found that NB001 is effective in inhibiting behavioral al-lodynia in chronic inflammatory pain. The inhibitory effects of NB001in inflammatory pain are weaker than that in neuropathic pain, sug-gesting that different mechanisms may be involved. Among possibledifferences is a greater component of AC1-independent peripheralsensitization that contributes to behavioral allodynia in inflammatorypain than in neuropathic pain. This notion is also supported by theineffectiveness of NB001 when applied locally to the skin (fig. S1). Insummary, our study demonstrates that calcium-stimulated AC1 is crit-ical for nerve injury–triggered synaptic changes in the brain and that theAC1 inhibitor NB001 produced substantial analgesic effects in animalswith neuropathic pain. Because of the selective effect of NB001 on spi-nal and cortical LTP, we argue that NB001 produces its analgesic effectby affecting, at least in part, synaptic LTP in pain-related areas.

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MATERIALS AND METHODS

Cell cultureHEK293 cells [American Type Culture Collection (ATCC)] weregrown in Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% fetal bovine serum. Human neuroblastoma SH-SY5Y cells(ATCC) were grown in DMEM supplemented with 10% fetal bovineserum. The cell cultures were maintained at 37°C in a humidified (95%air/5% CO2) incubator.

Expression of ACs in HEK293 cellsFor AC1 expression vector pcDNA3-AC1 transfection, HEK293 cellswere plated onto 60-mm-diameter dishes at a density of 1 × 106 perplate, grown overnight, and transfected with pcDNA3-AC1 (0.8 mg ofDNA per plate) by Lipofectamine 2000 (Invitrogen). Stable transfectedclones were selected in culture medium containing G418 (0.8 mg/ml;Invitrogen) and maintained in this medium. For transient expressionof other AC isoforms in HEK293 cells, HEK293 cells were plated in96-well tissue culture dishes and transfected with plasmids for AC5, 6,7, and 8, respectively; experiments were carried out 48 hours aftertransfection.

RT-PCRTotal RNA was isolated from HEK293 cells with RNeasy Mini Kit(Qiagen Inc.). RT-PCR was performed in a 50-ml reaction volume withQiagen OneStep RT-PCR Kit, with the following amplificationconditions: initial denaturation at 94°C for 5 min, followed by 35cycles of 94°C for 45 s, 58°C for 30 s, and 72°C for 45 s, and a final7-min extension at 72°C. The PCR primers for AC1 are as follows: forward,5′-TGCCTTATTTGGCCTTGTCTACC-3′; reverse, 5′-GACACCCG-GAAAAATATGGCTAG-3′. PCR products were electrophoresed on a1.5% agarose gel and stained by ethidium bromide.

Brain slice preparations for cyclic AMP assayMouse brain slices were prepared as reported (55, 56). Briefly, micewere anesthetized with 2% halothane, and brain slices (300 mm) con-taining the ACC were cut at 4°C with a Vibratome, in oxygenated arti-ficial cerebrospinal fluid (ACSF) containing the following: 124 mM

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NaCl, 4 mM KCl, 26 mM NaHCO3, 2.0 mM CaCl2, 1.0 mM MgSO4,1.0 mM NaH2PO4, and 10 mM D-glucose (pH 7.4). The slices wereslowly brought to a final temperature of 30°C in ACSF gassed with95% O2/5% CO2 and incubated for at least 1 hour before the ex-periments. Slices were then exposed to different compounds of interestfor the indicated times. For cyclic AMP assay, the ACC regions weremicrodissected and lysed in 0.1 M HCl.

Cyclic AMP assayCyclic AMP assay was carried out as reported (40, 55). Briefly, HEK293cells expressing ACs were harvested and lysed in 0.1 M HCl after dif-ferent treatments. Direct cyclic AMP measurements were performedwith the Direct Cyclic AMP Enzyme Immunoassay kit (Assay Designs),and optical density values were measured at 405 nm by a microplatereader. Phosphodiesterase activity was inhibited by the addition of1 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma) to cultures.

CRE luciferase reporter assayThe HEK293 cells were subcultured into 96-well plates in the absenceof antibiotics and grown overnight and transfected with the pGL3–CRE–firefly luciferase and pGL3–CMV (cytomegalovirus)–Renilla lu-ciferase constructs (0.25 mg of DNA per well) with Lipofectamine 2000reagent. The transfected cells were incubated overnight, and media werechanged to DMEM containing 10% fetal bovine serum. After 48 hours,the cells were treated with 10 mM forskolin, 10 mM A23187, or 2 mMCaCl2, or a combination of 10 mM forskolin, 10 mMA23187, and 2 mMCaCl2, in the presence or absence of each chemical tested at the con-centration of 100 mM. At the end of 6-hour incubation, cells were har-vested and luciferase activity was assayed by Dual-Luciferase ReporterAssay System (Promega). Relative light units were measured by aSIRIUS luminometer.

Animal models for neuropathic and inflammatory painAdult (6 to 8 weeks old) male mice were used. C57BL/6 mice werepurchased from Charles River. Mice were maintained on a 12-hourlight-dark cycle. Food and water were provided ad libitum. AC1 knock-out mice were bred for several generations on a C57BL/6 background.Experiments were performed under protocols approved by the Univer-sity of Toronto Animal Care Committee. A model of neuropathic painwas induced by the ligation of the common peroneal nerve (CPN) asdescribed previously (39). Briefly, mice were anesthetized by intraperi-toneal injection of a mixture of saline, ketamine (0.16 mg/kg; Bimeda-MTC), and xylazine (0.01 mg/kg; Bayer). The left CPN was slowly ligatedwith chromic gut suture 5-0 (Ethicon) until contraction of the dorsi-flexors of the foot was visible as twitching of the digits. Allodynia wastested on postsurgical day 7, and the mice were used for electrophysio-logical studies on postsurgical days 7 to 14. Although initial behavioralexperiments with NB001 were performed not in a blind manner, mostof the subsequent experiments were performed blind, or the behavioralobservations were collected by an automatic computer program (forexample, the open-field test, freezing measurement) where personalfactors were avoided.

Acute nociceptionThe spinal nociceptive tail-flick reflex was evoked by focused, radiantheat (Columbus Instruments) provided by a 50-W projector lampfocused on a 1.5-mm by 10-mm area on the underside of the tail.The latency to reflexive removal of the tail from the heat was measured

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by a digital photocell timer to the nearest 0.1 s. The cutoff time of 10 swas used to minimize damage to the skin of the tail. The hot plate con-sisted of a thermally controlled 25.4-cm by 25.4-cm metal plate (50°Cand 55°C, respectively) surrounded by four Plexiglas walls (ColumbusInstruments). The time between placement of the mouse on the plateand the licking or lifting of a hindpaw or jumping was measured witha digital timer. Mice were removed from the hot plate immediately afterthe first response. The cutoff time of 30 s was imposed to prevent tissuedamage. All behavioral tests were performed at 10-min intervals. Theresponse latency was an average of three or four measurements. Thebaseline latencies were measured 1 day before drug injection. The re-sponse latencies for the animals were then retaken on hot plate andtail-flick tests 30 min after intraperitoneal injection of either saline orNB001.

Mechanical allodyniaMice were individually placed in a round, transparent container 20 cmin diameter and were allowed to acclimate for 30 min before testing.Mechanical threshold was assessed on the basis of the responsivenessof the hindpaw to the application of von Frey filaments (Stoelting) tothe point of bending. The filament was applied over the dorsum of thepaw while the animal was resting. Positive responses included licking,biting, and sudden withdrawal of the hindpaw. The baseline responseswere taken 1 day before intraperitoneal injection. Mechanical thresh-old was then reassessed 30 min after intraperitoneal injection of eithersaline or NB001.

Elevated plus mazeThe elevated plus maze (Med Associates) consisted of two open armsand two closed arms situated perpendicular to each other. The mazewas situated ~70 cm from the floor. For each test, mice were individ-ually placed in the center square and allowed to move freely for 5 min.The number of entries and time spent in each arm were recorded. Theanimals were given an intraperitoneal injection of NB001 (10.0 mg/kg) orsaline 30 min before testing. A video camera tracking system (Ethovision)was used to generate the traces.

Open-field testTo record locomotor activity, we used an open-field activity monitor(43.2 cm by 43.2 cm by 30.5 cm; Med Associates). Briefly, this systemuses paired sets of photo beams to detect movement in the open field,and movement is recorded as beam breaks. The open field is placedinside an isolation chamber with dim illumination and a fan. Eachsubject was given an intraperitoneal injection of NB001 (10.0 mg/kg)or saline 30 min before being placed in the center of the open field.Locomotor activity was then measured for 30 min.

RotaRod testTo test motor function, we used a RotaRod from Med Associates. TheRotaRod test was performed by measuring the time each animal wasable to maintain its balance while walking on a rotating drum. Onehour before testing, animals were trained on the RotaRod at a constantacceleration of 16 rpm until they could stay on for 30 s. For testing,the RotaRod was set to accelerate from 4 to 40 rpm over a 5-min pe-riod. Mice were given three trials with a maximum time of 300 s and a5-min intertrial rest interval. The latency to fall was taken as a mea-sure of motor function. Each subject was given an intraperitoneal in-jection of NB001 (10.0 mg/kg) or saline 30 min before testing.

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Fear memoryFear conditioning was performed in an isolated shock chamber (MedAssociates) and performed in a blind manner to the treatment. Theconditioned stimulus (CS) was an 85-dB sound at 2800 Hz, and theunconditioned stimulus (US) was a continuous scrambled footshockat 0.75 mA. After 2 min of habituation, animals received the CS-USpairing [a 30-s tone (CS) and a 2-s shock (US) starting at 28 s; threeshock-tone pairings were delivered at 30-s intervals], and the mice re-mained in the chamber for an additional 30 s for measurement of im-mediate freezing. At 1 day after training, each mouse was placed backinto the shock chamber and the freezing response was recorded for3 min (contextual conditioning). Two types of experiments were per-formed. To test the contribution of AC1 to the induction of fear mem-ory, we pretreated mice with NB001 (at 1 mg/kg ip) or saline. Bothgroups of mice then received fear conditioning 30 min after the in-jection. One day after the conditioning, we measured the freezing re-sponses. In the second type of experiments, we evaluated the effects ofNB001 on the expression of fear memory by injecting NB001 or saline1 day after the conditioning. NB001 or saline was injected 30 minbefore the measurement of freezing responses.

Microinjection of NB001 into the ACCTo determine the effects of NB001 on the ACC, we performed localmicroinjection of NB001 into the ACC as reported previously (14).Briefly, under ketamine and xylazine anesthesia, mice were placed ina stereotaxic instrument (Kopf Instruments). Guide cannulas (24-gauge)were implanted bilaterally into the ACC (0.7 mm anterior to bregma,0.3 mm lateral from the midline, and 1.7 mm beneath the surface of theskull). Mice were given at least 2 weeks to recover after cannula implan-tation. Thirty-gauge injection cannulas that were 0.1 and 0.6 mm longerthan the guide were used for intra-ACC injections. The microinjectionapparatus consisted of a Hamilton glass syringe (5 ml). NB001 (10 mg/mlin saline) was infused into each side of the ACC at a rate of 0.05 ml/min;an equivalent volume of saline was used as a control. To access the dor-sal and ventral ACC, we administered two injections per side, the firstwith the 0.1-mm-longer cannula, followed by the 0.6-mm-longer can-nula. After each injection, the microinjection needle was left in place forat least 2 min.

Intrathecal injection of NB001A 30-gauge needle attached to a 25-ml Hamilton syringe was used forthe intrathecal injection of drugs. The needle was inserted into theintervertebral space between the fifth and the sixth spinal vertebraein conscious mice, as described (57). Accurate placement of the needlewas confirmed by a quick flick of the tail. NB001 (189.0 nmol) wasadministered slowly in a volume of 5 ml via the intrathecal route.

Whole-cell patch-clamp recordingsCoronal brain slices (300 mm) at the level of the ACC were preparedby standard methods (15, 46). Slices were transferred to a submergedrecovery chamber with oxygenated (95% O2/5% CO2) ACSF (contain-ing 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mMMgSO4, 25 mMNaHCO3, 1 mM NaH2PO4, and 10 mM glucose) at room tempera-ture for at least 1 hour. Experiments were performed in a recordingchamber on the stage of a BX51W1 microscope equipped with in-frared DIC (differential interference contrast) optics for visualization.EPSCs were recorded from layer II/III neurons with an Axon 200Bamplifier (Axon Instruments), and the stimulations were delivered by




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a bipolar tungsten stimulating electrode placed in layer V of the ACC.AMPA receptor–mediated EPSCs were induced by repetitive stimula-tions at 0.05 Hz, and neurons were voltage-clamped at −70 mV in thepresence of (AP5) [(2R)-amino-5-phosphonovaleric acid] (50 mM). Therecording electrodes were filled with a solution containing (pipettes,3 to 5 megohms) 145 mM K-gluconate, 5 mM NaCl, 1 mM MgCl2,0.2mMEGTA, 10mMHepes, 2 mMMg-ATP, 0.1 mMNa3GTP, and10 mM phosphocreatine disodium (adjusted to pH 7.2 with KOH). Forminiature EPSC (mEPSC) recording, 0.5 mM tetrodotoxin was addedto the perfusion solution. Picrotoxin (100 mM) was always present toblock GABAA (g-aminobutyric acid type A) receptor–mediated inhib-itory synaptic currents in all experiments, and the access resistance was15 to 30 megohms throughout the experiment. Data were discarded ifaccess resistance changed more than 15% during an experiment. Datawere filtered at 1 kHz and digitized at 10 kHz.

Hippocampal field LTP recordingsMale adult mice (7 to 10 weeks old) were anesthetized with inhaledisoflurane. Transverse slices (400 mm) of hippocampus were rapidlyprepared and maintained in an interface chamber at 28°C, where theywere perfused with ACSF consisting of 124 mM NaCl, 4.4 mM KCl,1.2 mM CaCl2, 1.0 mMMgSO4, 25 mMNaHCO3, 1.0 mM NaH2PO4,and 10 mM glucose bubbled with 95% O2 and 5% CO2. The protocolof electrical stimulation and recordings has been described previously(22, 24, 58). Slices were kept in the recording chamber for at least2 hours before the experiments. A bipolar tungsten stimulating electrodewas placed in the stratum radiatum in the CA1 region, and extracellularfield potentials were also recorded in the stratum radiatum with a glassmicroelectrode (3 to 12 megohms) filled with ACSF. Stimulus inten-sity was adjusted to produce a response of ~1-mV amplitude. Testresponses were elicited at 0.02 Hz. LTP was induced with two tetanictrain stimulations (100 Hz, 1-s trains at 20-s intervals). To study theeffects of NB001 on LTP in the CA1 region of the hippocampus, weperfused 50 mM NB001 for at least 30 min before and throughoutexperiments in the NB001 group.

Data analysisResults were expressed as means ± SEM. Statistical comparisons wereperformed with two-way ANOVA and Student’s t test. Analysis ofmEPSCs was performed with cumulative probability plots. The resultof hippocampal LTP in each slice was calculated as the average of thepercentage of responses between 40 and 45 min after stimulationcompared with baseline. Student’s t test was used for statistical anal-ysis. The level of significance was set at P < 0.05.

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/3/65/65ra3/DC1Fig. S1. The effects of local administrations of NB001 into the periphery, spinal, or the ACC onmechanical allodynia in mice after nerve injury.Fig. S2. The effects of NB001 on classic contextual fear memory.Table S1. Screening of AC1 inhibitors in HEK293 cells stably expressing AC1.

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59. Acknowledgments: We thank L. Muglia and D. R. Storm for providing AC1 knockout mice,R. Taussig and A. G. Gilman (University of Texas Southwestern Medical Center, Dallas, TX)for providing the expression plasmid for AC1 (pcDNA3-AC1), and D. M. F. Cooper (Departmentof Pharmacology, University of Cambridge, Cambridge, UK) for providing expression plasmidsfor AC5, 6, 7, and 8. Funding: M.Z. was supported by grants from the EJLB Foundation–Canadian Institutes of Health Research (CIHR) Michael Smith Chair in Neurosciences andMental Health, Canada Research Chair, and a CIHR operating grant (CIHR81086) andproof-of-concept grant (CIHR PPP-81425). H.W. is supported by a postdoctoral fellowshipfrom Fragile X Research Foundation of Canada. B.K. is supported by National Creative ResearchInitiative, Korea. Author contributions: H.W. carried out biochemical screening experimentsand drafted the manuscript, H.X., L.-J.W., T.C., K.K., B.G., and X.Z. participated in electrophysio-logical experiments. S.S.K., G.D., H.X., X.Z., H.W., and K.I.V. participated in behavioralexperiments. B.-K.K. helped to design the cyclic AMP screening experiments. M.Z. designed,drafted, and finished the final manuscript. All authors contributed to the preliminary writingof the manuscript and approved the final manuscript. Competing interests:M.Z. has filed aninternational patent application (USPTO Patent Application 20090233922, “Method fortreating neuronal and non-neuronal pain”) based on the results reported in this paper.

Submitted 7 May 2010Accepted 8 December 2010Published 12 January 201110.1126/scitranslmed.3001269

Citation: H. Wang, H. Xu, L.-J. Wu, S. S. Kim, T. Chen, K. Koga, G. Descalzi, B. Gong,K. I. Vadakkan, X. Zhang, B.-K. Kaang, M. Zhuo, Identification of an adenylyl cyclase inhibitorfor treating neuropathic and inflammatory pain. Sci. Transl. Med. 3, 65ra3 (2011).

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