Research report 137

Analgesic efficacy of small-molecule angiotensin II type 2 receptor antagonists in a rat model of antiretroviral toxic polyneuropathy Maree T. Smitha,b, Tanya Laua,b,*, Victoria C.J. Wallacec,*, Bruce D. Wysea,b and Andrew S.C. Ricec Individuals infected with the HIV and taking certain antiretroviral drugs to suppress viral replication have a high prevalence of neuropathic pain that is not alleviated by analgesic/adjuvant drugs that are often efficacious for the relief of other types of neuropathic pain. There is therefore a great need for new analgesics to alleviate the pain of antiretroviral toxic neuropathy (ATN). Small-molecule angiotensin II type 2 receptor (AT2R) antagonists, with Z 1000-fold selectivity over the angiotensin II type 1 receptor, produced analgesia in the chronic constriction injury of the sciatic nerve rat model of peripheral nerve trauma. Hence, the present study was designed to assess their analgesic efficacy in a rat model of ATN. The analgesic efficacy of small-molecule AT2R antagonists (EMA200 and EMA300) was assessed in a rat model of dideoxycytidine (ddC)-induced ATN. Single intraperitoneal bolus doses of EMA200 (0.3–10 mg/kg) induced dose-dependent analgesia in ddC-rats; the mean ED50 was 3.2 mg/kg. Twice-daily intraperitoneal administration of EMA300 at 30 mg/kg to ddC-rats for 3 days produced significant

analgesia on days 2 and 3 of the treatment period. Therefore, small-molecule AT2R antagonists should be investigated further as novel analgesics for the relief c 2014 of ATN. Behavioural Pharmacology 25:137–146  Wolters Kluwer Health | Lippincott Williams & Wilkins.

Introduction

Existing treatment options for analgesia in HIV-SN are limited. Only nerve growth factor (NGF), which is not approved by regulatory agencies and so is unavailable therapeutically (McArthur et al., 2000), topical application of high concentration capsaicin and cannabis have been shown to be efficacious (Phillips et al., 2010). However, cannabis is not a reasonable therapeutic option for some individuals, given the risk of psychiatric adverse events (Rice, 2008). A second randomized-controlled trial of topical capsaicin 8% (Clifford et al., 2012) did not replicate the efficacy observed in the first trial (Simpson et al., 2008) and there is not yet an evidence base confirming the long-term repeated application of this intervention. There is no evidence to support the use of opioids in HIV-SN, and drugs that are effective in other neuropathic pain conditions, such as tricyclic antidepressants and gabapentinoids, are known to be ineffective for the relief of neuropathic pain in the context of HIV-SN (Dworkin et al., 2007; Finnerup et al., 2010; Phillips et al., 2010). As a result, there is an unmet medical need for the discovery and development of novel analgesic agents that are efficacious and well tolerated for the relief of painful HIV-SN, which, in turn, would be expected to markedly improve the quality of life of HIV-infected individuals.

For individuals living with HIV, there is a 30–64% prevalence of distal sensory neuropathy (HIV-SN) (Cherry et al., 2012a, 2012b). Development of this usually painful neurological complication of HIV has two major aspects that are difficult to distinguish clinically: cytokine-driven peripheral nerve axonopathy associated with the HIV infection itself and a mitochondrial neuropathy because of antiretroviral toxic neuropathy (ATN) (Dalakas, 2001; Phillips et al., 2010). ATN is associated with the use of nucleoside reverse transcriptase inhibitors as part of combination antiretroviral therapy (cART) that was first introduced in 1996 (May et al., 2013) and that has led to a marked increase in the survival of individuals infected with HIV (Mocroft et al., 2003). Comparison of the prevalence rates of painful HIV-SN in the pre-cART and post-cART eras shows that it reduced from 42.5 to 34.4% after the commencement of cART, while suspected ATN increased from 13–20% to 31–42% during the same period (Maschke et al., 2000; Smyth et al., 2007). Hence, painful sensory neuropathy is an important cause of morbidity in HIV-infected individuals who are otherwise in reasonably good health (Ellis et al., 2010; Cherry et al., 2012a, 2012b). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0955-8810 

Behavioural Pharmacology 2014, 25:137–146 Keywords: analgesia, angiotensin II type 2 receptor antagonists, antiretroviral drug induced neuropathy, antiretroviral toxic neuropathy, EMA200, EMA300, HIV-associated sensory neuropathy, HIV-SN, mechanical hypersensitivity, neuropathic pain a

Centre for Integrated Preclinical Drug Development, bSchool of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia and cDepartment of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital Campus, London, UK Correspondence to Maree T. Smith, PhD, Centre for Integrated Preclinical Drug Development, The University of Queensland, St Lucia Campus, Brisbane, QLD 4072, Australia E-mail: [email protected] *Tanya Lau and Victoria C.J. Wallace contributed equally to the writing of this article. Received 18 June 2013 Accepted as revised 15 January 2014

DOI: 10.1097/FBP.0000000000000025

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Small-molecule angiotensin II type 2 receptor (AT2R) antagonists are a promising novel class of analgesic agents for the relief of neuropathic pain (Muralidharan et al., 2013; Smith et al., 2013a, 2013b). Specifically, in rats with a chronic constriction injury (CCI) of the sciatic nerve, a widely utilized rat model of traumatic nerve injury (Smith et al., 2013a), single bolus doses of three selective smallmolecule AT2R antagonists (EMA200, EMA300 and EMA400), with greater than 1000-fold specificity over the angiotensin II type 1 receptor (AT1R), produced dosedependent relief of hindpaw hypersensitivity. Ex-vivo investigation showed that the analgesic mode of action in CCI-rats involves blockade of augmented angiotensin II signalling through the AT2R in the ipsilateral lumbar dorsal root ganglias (DRGs) to inhibit p38 mitogen-activated protein kinase (MAPK) and p44/42 MAPK activation (Smith et al., 2013b). In addition, in a rat model of prostate cancer-induced bone pain (PCIBP), which is underpinned by both inflammatory and neuropathic mechanisms, the analgesic effects of EMA200 reduced augmented NGF [NGF/tyrosine kinase A (TrkA)] signalling in the lumbar DRGs, with the net result being inhibition of p38 MAPK and p44/p42 MAPK activation (Muralidharan et al., 2013). These enzymes are involved in the phosphorylation (activation) of multiple receptors and ion channels in sensory neuron hyperexcitability (Ji et al., 2002; Martin et al., 2006; Hudmon et al., 2008; Stamboulian et al., 2010) that contribute towards the pathobiology of peripheral neuropathic and inflammatory pain. In addition, a recent randomized placebo-controlled clinical trial in 183 patients with postherpetic neuralgia showed that administration of an orally active AT2R antagonist (EMA401) at 100 mg/twice daily for 4 weeks produced significant pain relief relative to placebo, and was well tolerated (Rice et al., 2014).

Methods Study design

We assessed the analgesic efficacy of two selective smallmolecule AT2R antagonists, EMA200 and EMA300 (Fig. 1), in a rat model of ATN. Specifically, two independent studies were carried out at The University of Queensland (UQ) and at Imperial College London (ICL) to test the reproducibility of the findings and to test the effectiveness of two treatment regimens. The design parameters for each of these two studies are described in the following sections and summarized in Table 1. Subjects

Approval was obtained from The University of Queensland (UQ) Animal Ethics Committee for the in-vivo experiments conducted at UQ and these experiments complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th ed., 2004). In-vivo experimentation performed at ICL was conducted under Home Office approval (Project Licence number 70/6321). Male Sprague–Dawley (SD) rats used in the experiments conducted at UQ were purchased from the UQ Biological Resources (Brisbane, Australia). SD rats were housed in a facility with a mean (±SD) temperature of 21 (±2)1C and with artificial lighting maintained on a 12-h/12-h light–dark cycle. For the experiments conducted at ICL, male Wistar rats were purchased from B&K Universal Ltd (Hull, UK). Wistar rats were housed in a facility at ICL with a mean (±SD) temperature of 24 (±1)1C and a 14-h/10-h light–dark cycle. All experiments in both laboratories were conducted during the light phase, and rodent food pellets and water were available ad libitum. Rat model of ddC-induced neuropathic pain

Hence, the present study was designed to assess the analgesic efficacy of two selective small-molecule AT2R antagonists, EMA200 and EMA300, in a rat model of ATN. In particular, two independent studies were carried out to test the reproducibility of the findings and to test the effectiveness of two treatment regimens. Here, we show for the first time that single bolus doses of EMA200 produced dose-dependent relief of mechanical hypersensitivity in the bilateral hindpaws of the ddC-rat model of ATN. We also show that the analgesic effects are generalizable to other members of the AT2R antagonist drug class. Furthermore, we have provided crucial replication data, in that the same effects were robustly observed between two independent laboratories operating distinct experimental paradigms and different treatment regimens in different rat strains. Twicedaily administration of EMA300 at 30 mg/kg for 3 days alleviated bilateral mechanical hypersensitivity in the hindpaws of ddC-rats, whereas vehicle was inactive. The orally active small-molecule AT2R antagonist, EMA401, is in clinical development as a novel analgesic for the relief of neuropathic pain (Rice et al., 2014).

Male SD rats (n = 24) at UQ or male Wistar rats (n = 132) at ICL in the weight range 200–225 g were administered Fig. 1

(b) Me

(a) Me

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.2CF3CO2H Chemical structures of (a) EMA200 (also known as PD123319) and (b) EMA300 (also known as PD121981).

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Table 1

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Comparative summary of study design parameters for experiments conducted at UQ and ICL

Study design parameters Rat strain/sex Rat weight at study initiation Protocol for induction of ddC-neuropathy AT2R antagonist Dosing route Dosing regimen Positive control (gabapentin) dosing regimen Blinded behavioural assessment Pain behavioural endpoints AT2R antagonist dosing: pain behaviour assessment times Other behaviours assessed in normal rats

Smith lab (UQ)

Rice lab (ICL)

Male Sprague–Dawley 200–225 g ddC at 50 mg/kg intraperitoneally three times per week for 3 weeks EMA200 Intraperitoneally Single bolus doses (0.3–10 mg/kg)

Male Wistar 200–225 g ddC at 50 mg/kg intraperitoneally three times per week for 3 weeks EMA300a Intraperitoneally Twice-daily doses at 1, 10 & 30 mg/kg for three days Twice-daily at 30 mg/kg for 3 days Yes Punctate mechanical stimulation using an electronic Von Frey device Predose and at 0.75 h postdosing on each dosing day Thigmotaxis in an open field

Single bolus doses at 100 mg/kg Yes Von Frey paw withdrawal thresholds in the bilateral hindpaws Predose and at 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2 and 3 h postdosing NA

AT2R, angiotensin II type 2 receptor; ddC, dideoxycytidine; ICL, Imperial College London; NA, not applicable; UQ, The University of Queensland. EMA300 is a structural analogue of EMA200; see Fig. 1.

a

the neurotoxic antiretroviral drug, ddC, at 50 mg/kg, by intraperitoneal injection, three times per week for 3 weeks, as described previously (Wallace et al., 2007b, 2008; Maratou et al., 2009). Although ddC is no longer used clinically because of inferior pharmacokinetics requiring thrice-daily dosing and adverse effects because of mitochondrial toxicity (Lee et al., 2003), it is representative of this class of antiretroviral drugs, used as part of cART to treat HIV-1 infection in humans and there are still a large number of patients who have been exposed to ddC. Mechanical hypersensitivity: non-noxious stimuli

The time-course for the development of mechanical hypersensitivity to applied non-noxious stimuli in the hindpaws of conscious male SD rats was determined using Von Frey filaments (2–20 g; Stoelting Co., Wood Dale, Illinois, USA) (Suzuki et al., 2000). Animals were placed in wire mesh cages and acclimatized for at least 15–20 min before Von Frey testing. Initially the 6 g filament was applied to the plantar surface of each hindpaw until the fibre just began to bend. If there was no paw withdrawal response observed after 3 s, the next filament in the ascending force sequence was used. The paw withdrawal threshold (PWT) was determined when a brisk paw withdrawal reflex was elicited. Any response to forces of 6 g or less was considered as fully developed mechanical hypersensitivity. A score of 20 g was assigned to rats that did not respond to any of the Von Frey filaments. Baseline PWT values were measured in all rats before the first ddC injection. SD rats that did not show fully developed mechanical hypersensitivity in the hindpaws, by 4 weeks after initiation of the ddCtreatment regimen, were excluded from further experimentation. Thermal hypersensitivity

Noxious heat hypersensitivity in the hindpaws of conscious male SD rats administered the ddC-treatment regimen was assessed using the Hargreaves test. Rats were placed in a clear plastic chamber with a glass floor

for at least a 15-min acclimatization period. A radiant heat source was positioned under the glass floor beneath each of the hindpaws in turn. A photoelectric cell was used to detect the paw withdrawal latency to the nearest 0.1 s. Measurements were performed in triplicate for each hindpaw with at least a 3-min interval between each assessment. Mechanical hypersensitivity: noxious mechanical stimuli (ICL)

In conscious rats, hindpaw withdrawal thresholds were measured in response to punctate noxious mechanical stimulation, applied using an electronic ‘Von Frey’ device (Somedic Sales AB, Ho¨rby, Sweden) as described previously (Moller et al., 1998; Hasnie et al., 2007; Wallace et al., 2007b; Huang et al., 2013). Briefly, a metal probe (tip area 0.5 mm2) was applied manually at a rate of 8–15 g/s to the mid-plantar surface of the hindpaw. The withdrawal threshold was defined as the mean force that induced an active limb withdrawal response over five applications. Baseline measurements were obtained for all animals over the course of a week before the initial ddC injection. Animals were then tested once between days 19 and 23 following the initial ddC injection to identify the development of hindpaw reflex sensitivity. Only animals that developed significant hypersensitivity to mechanical stimulation (Z 30% decrease from baseline) were included in the study. All testing was performed by a ‘blinded’ investigator. Test compound administration EMA200

On each dosing occasion, male SD ddC-rats with fully developed mechanical hypersensitivity in the hindpaws were randomized to receive an intraperitoneal bolus dose of EMA200 at 0.3, 3 and 10 mg/kg or vehicle (negative control), or single subcutaneous bolus doses of gabapentin at 100 mg/kg as the positive control. Baseline PWT values were measured just before dosing and at 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2 and 3 h after dosing. Von Frey assessments were masked and performed by a ‘blinded’ tester. Specifically,

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dosing solutions of the test compounds in the desired concentrations as well as vehicle solutions were prepared by a first individual. These dosing solutions were coded by a second individual, who then administered them to ddCrats in a randomized manner. Von Frey testing was performed by the first individual in a ‘blinded’ manner. Dosing was performed according to a ‘washout’ protocol such that each ddC-rat received up to five bolus doses of EMA200, gabapentin or vehicle, with a 2–3-day ‘washout’ period between successive doses. EMA300

Male Wistar rats with fully developed mechanical hypersensitivity in the hindpaws were randomized into six groups (minimum of n = 10 per group) and administered intraperitoneal bolus doses of EMA300 at 1, 10 and 30 mg/kg, gabapentin at 30 mg/kg or vehicle (water or sterile saline, respectively) twice daily (8:00 and 18:00 h) for 3 days. Baseline PWT values were measured just before dosing and at 0.75 h after dosing following the first injection on each of the 3 treatment days. All PWTs were measured by a tester who was blinded to the treatment administered. Specifically, a first individual prepared the test compounds and control solutions, which were coded and administered to the ddC-rats in a randomized manner by a second individual. A third individual, unaware of which drug had been prepared and administered to each rat, performed the testing. Only animals that developed significant hypersensitivity to mechanical stimulation (at least 30% decrease from baseline) were included in the study.

arena was tracked over a 15-min period using a Sanyo VCB 3372 high-resolution monochrome camera (Tracksys, Notts, UK). These data were stored and analysed using Ethovision software v.3 (Tracksys). The total distance moved, time spent in the inner zone and the number of entries into the inner zone were calculated and presented as mean±SEM. The tester performing the open-field experiments was blinded to the treatment administered. As above, a first individual prepared the test and vehicle solutions, which were coded and administered to the ddC-rats in a randomized manner by a second individual. A third individual, unaware of which drug had been prepared and administered to each rat, performed the testing. Drugs and reagents

EMA200 (also known as PD123319), as the ditrifluoroacetate salt, was purchased from Tocris Biosciences (Bristol, UK). EMA300 (also known as PD121981), as the sodium salt, was synthesized by Glycosynirl (Lower Hutt, New Zealand) and supplied by Spinifex Pharmaceuticals Pty Ltd (Melbourne, Australia). Gabapentin was supplied by Dr Ben Ross (School of Pharmacy, UQ) or purchased from Sigma-Aldrich (Poole, UK). Distilled water was from Invitrogen (Paisley, UK). Water for injection ampoules was from Pfizer (West Ryde, Australia). 20 -30 dideoxycytidine (ddC also known as zalcitabine) was purchased from Sigma-Aldrich (Sydney, Australia and Poole, UK). Data and statistical analysis

The effect of EMA300 at 30 mg/kg on spontaneous exploratory activity in the open field was assessed as a surrogate measure of spontaneous pain-related behaviour that does not rely on reflex thresholds. This test has been used extensively for the assessment of anxiolytic agents in rodents (Carli et al., 1989; Holmes, 2001; Cryan and Holmes, 2005). Thigmotactic (wall-hugging) behaviour in this test has been reported previously to be a feature of rodent models of neuropathic pain (Hasnie et al., 2007; Wallace et al., 2007a), including rats with neurotoxic antiretroviral neuropathy (Wallace et al., 2008; Huang et al., 2013).

For male SD rats, changes in individual PWT (DPWT) values were calculated by subtracting baseline PWTs from the postdosing PWT values. The DPWT values were plotted against time and the area under the DPWT versus time curve for individual rats (DPWT AUC value) was calculated using trapezoidal integration in the GraphPad Prism software package (v5.03; GraphPad Software Inc., San Diego, California, USA). The DPWT AUC values for EMA200 were converted into the per cent maximum DPWT AUC for each rat and a dose–response curve was generated by plotting mean (±SEM) per cent maximum DPWT AUC values against log dose. The ED50 was estimated using nonlinear regression (GraphPad Prism v5.03). The criterion of statistical significance was a Pvalue of less than 0.05.

On the day following establishment of mechanical hypersensitivity (a decrease of at least 30% from baseline) in the hindpaws of Wistar rats using the electronic ‘Von Frey’ device, drug-naive ddC-rats were randomized into one of four treatment groups (n = 11–12 per group). These animals were administered single bolus doses of EMA300 at 30 mg/kg (n = 10), gabapentin at 30 mg/kg (n = 10) or vehicle (distilled water or sterile saline, respectively; n = 10 per group). At 40 min after dosing, rats were placed into a 1 m  1 m arena illuminated to 4 lux with a defined inner zone of 40 cm  40 cm. Locomotion of the rats within the

In male Wistar rats, PWTs in response to applied noxious mechanical stimuli are reported as mean (±SEM) at each time-point. Significant differences between treatment groups for PWTS at each time of assessment for the chronic dosing treatment schedule were assessed using a two-way analysis of variance with either Dunnett’s (relative to vehicle) or Tukey’s (relative to gabapentin) multiple comparison test, as appropriate. For the openfield test, significant differences between the treatment groups for number of entries into the inner zone, duration spent in the inner zone and total distance moved were

Open-field activity

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AT2R antagonists: novel analgesics for HIV-SN Smith et al. 141

Results Time course for development of mechanical hypersensitivity in the hindpaws of SD rats

There was temporal development of bilateral mechanical hypersensitivity in the hindpaws of male SD rats. At day 28 after the initiation of the ddC-treatment regimen, hindpaw PWTs were r 6 g in 20 out of 24 animals (B80%) (Fig. 2a). The remaining four rats showing only partial hindpaw hypersensitivity to ddC were excluded from further experimentation and euthanized.

Fig. 2

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Mechanical allodynia

14 Paw withdrawal thresholds (g)

determined by one-way analysis of variance with Dunn’s all-pairwise multiple-comparisons test. The criterion of statistical significance was a P-value of less than 0.05.

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Hindpaw withdrawal latencies in response to applied noxious thermal (heat) stimuli did not change significantly throughout the 3-week ddC-treatment regimen, in contrast to the previously mentioned development of bilateral mechanical hypersensitivity in the hindpaws of the same animals (Fig. 2b), in agreement with a previous report for this model (Wallace et al., 2007a).

EMA200: dose-dependent relief of mechanical hypersensitivity in the hindpaws

Single intraperitoneal bolus doses of EMA200 at 0.3 (n = 6), 3.0 (n = 14) and 10 mg/kg (n = 14) produced dose-dependent relief of mechanical hypersensitivity in the bilateral hindpaws of ddC-rats (Fig. 3). The onset of action was within 30 min of dose administration and the mean peak effect was observed at 1.5 h after dosing (Fig. 3a). The mean duration of action was approximately 3 h (Fig. 3a). The estimated mean ED50 for single intraperitoneal bolus doses of EMA200 is 3.2 [95% confidence interval (CI): 1.43–7.0] mg/kg (Fig. 3b). Single intraperitoneal bolus doses of EMA200 at 10 mg/kg induced a comparable extent and duration of antihypersensitivity to that produced by subcutaneous bolus doses of gabapentin at 100 mg/kg (n = 6) (Fig. 3a). Single bolus doses of vehicle (n = 14) did not alleviate mechanical hypersensitivity in the hindpaws of ddC-rats (Fig. 3a), consistent with expectations.

Development of mechanical hypersensitivity in the hindpaws of Wistar rats

Following administration of the 3-week ddC-treatment regime, B75% of male Wistar rats (100 from 132) developed significant mechanical hypersensitivity (> 30% fall from baseline) in the bilateral hindpaws by days 19–23 from ddC-treatment initiation, relative to pretreatment values. The remaining 32 out of 132 rats not fulfilling these criteria were removed from the study.

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(a) Following the initiation of the ddC-treatment regimen in male Sprague– Dawley (SD) rats, mechanical hypersensitivity in the bilateral hindpaws, assessed using Von Frey filaments, developed in a temporal manner such that it was fully developed (paw withdrawal thresholds r 6 g) in 20 out of 24 rats by day 28. (b) Thermal (heat) hypersensitivity assessed using the Hargreaves apparatus did not develop in the bilateral hindpaws of male SD rats administered the ddC-treatment regimen. ddC, dideoxycytidine.

EMA300 attenuates mechanical hypersensitivity in the hindpaws of ddC-rats

Twice-daily administration of EMA300 at 30 mg/kg attenuated mechanical hypersensitivity in the bilateral hindpaws when assessed at 0.75 h after dosing on days 2 and 3 of the 3-day treatment regimen compared with vehicle (Fig. 4). However, twice-daily administration of EMA300 at 1.0 and 10 mg/kg for 3 days did not significantly (P > 0.05) alleviate mechanical hypersensitivity in the hindpaws when assessed at 0.75 h after dosing in a manner similar to vehicle (Fig. 4). By contrast, twice-daily administration of gabapentin at 30 mg/kg significantly attenuated (P < 0.05) mechanical hypersensitivity in the bilateral hindpaws of ddC-rats on days 1, 2 and 3 of the 3-day treatment regimen compared with vehicle (Fig. 4). On all days of test compound administration, the effect of EMA300 at 30 mg/kg did not differ significantly (P > 0.05) from that of gabapentin at

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30 mg/kg (Fig. 4). One week after the last day of administration of either EMA300 at 30 mg/kg or gabapentin at 30 mg/kg, bilateral hindpaw withdrawal thresholds to applied noxious mechanical stimuli were not significantly different from pretreatment baseline values (P > 0.05). EMA300 does not affect measures of thigmotaxis in the open field

Single intraperitoneal bolus doses of EMA300 at 30 mg/kg did not significantly (P > 0.05) affect thigmotactic behaviour as measured by the number of entries into the inner

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Mean (±SEM) PWTs for the hindpaws of ddC-rats (Wistar strain), assessed using punctate mechanical stimulation with an electronic Von Frey device, before initiation of the ddC-treatment regimen and at the time of peak behavioural change (19–23 days later). Thereafter, rats received twice-daily (i) gabapentin (30 mg/kg intraperitoneally; n = 12), (ii) EMA300 (1, 10 and 30 mg/kg intraperitoneally; n = 10 per dose) or vehicle (n = 10) for 3 successive days. PWTs were measured at 0.75 h after dosing after doses 1, 3 and 5, and again 7 days after dosing cessation. Significant differences (**P < 0.05) between gabapentin and vehicle or between EMA300 and vehicle at each assessment time were determined using two-way ANOVA with Dunnett’s test for multiple comparisons. Significant differences (P < 0.05) between gabapentin and EMA300 at each assessment time were determined by two-way ANOVA with Tukey’s test for multiple comparisons. ANOVA, analysis of variance; ddC, dideoxycytidine; PWT, paw withdrawal threshold.

zone (Fig. 5a) or the time spent in the inner zone (Fig. 5c) relative to the corresponding vehicle-treated control animals. By contrast, single intraperitoneal bolus doses of gabapentin at 30 mg/kg significantly increased (P < 0.05) the number of entries into the inner zone (Fig. 5a) as well as the time spent in the inner zone (Fig. 5c) as reported previously (Wallace et al., 2008). As an indicator of normal locomotion in response to all treatments, there was no

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AT2R antagonists: novel analgesics for HIV-SN Smith et al. 143

Fig. 5

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In ddC-rats, the potency of EMA200 to alleviate mechanical hypersensitivity in the bilateral hindpaws was B10-fold higher than that of gabapentin. The mean time to peak analgesia was 1.5 h for both EMA200 and gabapentin at 100 mg/kg and the corresponding durations of action were more than 3 h. Our present findings for gabapentin in ddC-rats complement previous work showing that gabapentin alleviated bilateral mechanical hypersensitivity that had been induced in the hindpaws of adult male Wistar rats by previous administration of ddC three times per week for 3 weeks, either alone or in combination with the HIV envelope protein, glycoprotein 120 (Wallace et al., 2007a, 2007b).

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Here, we show for the first time that the small-molecule AT2R antagonists, EMA200 and EMA300, which have a greater than 1000-fold selectivity over AT1R (Smith et al., 2013a), produced analgesia in the ddC-rat model of ATN. Specifically, single intraperitoneal bolus doses of EMA200 produced dose-dependent relief of mechanical hypersensitivity in the bilateral hindpaws of ddC-rats and the analgesic potency (ED50 at 3.2 mg/kg) matched that for CCI-rats, a widely utilized rat model of mechanical nerve trauma-induced neuropathic pain (Smith et al., 2013a). The mean time to peak antihypersensitivity produced by single bolus doses of EMA200 in ddC-rats was 1.5 h after dosing whereas in CCI-rats the mean time to peak effect was shorter at 0.75 h (Smith et al., 2013a). At the highest dose tested (10 mg/kg) in ddC-rats, the mean duration of analgesia was more than 3 h in a manner similar to that observed previously for EMA200 at 10 mg/kg in CCI-rats (Smith et al., 2013a).

Open field - entries 10 Number of entries

significant difference (P > 0.05) in the total distance moved by rats in any treatment group (Fig. 5b). Audible vocalization, likely because of the acidity of the injection solution, was induced by the intraperitoneal injection of EMA300 at 30 mg/kg, but not at the lower doses.

EMA300 is a structural analogue of EMA200, with the dimethylamino substituent in EMA200 replaced by a methoxy group in EMA300 (Smith et al., 2013a). In the present study in ddC-rats, twice-daily administration of EMA300 at 30 mg/kg, but not 1 or 10 mg/kg, for 3 successive days, produced significant relief of mechanical hypersensitivity in the bilateral hindpaws at 0.75 h after dosing on days 2 and 3 of the treatment regimen. On each treatment day, the anti-hyperalgesic potency of EMA300 at 30 mg/kg did not differ significantly from that of gabapentin at 30 mg/kg in ddC-rats. The anti-hyperalgesic effects induced by each of EMA300 and gabapentin in ddC-rats were fully reversed by the time of the final measurement, 1 week after completion of the 3-day dosing regimen, showing that the effects of treatment were reversible.

Thigmotactic behaviour in ddC-rats (Wistar strain) is attenuated by a single intraperitoneal bolus dose of gabapentin (30 mg/kg; n = 10) but not EMA300 (30 mg/kg; n = 10) as measured in an open-field arena. (a) The number of entries into the inner zone (40  40 cm) and (b) time spent in the inner zone of the open-field arena were significantly increased (P < 0.05) in rats administered gabapentin (30 mg/kg) relative to saline-treated ddC-rats (control group; n = 10). EMA300 at 30 mg/kg did not significantly (P > 0.05) alter either measure relative to ddC-rats administered water for injection (n = 10); both measures were significantly lower than those for the gabapentin group. Significant differences between the groups (**P < 0.05) were determined using one-way analysis of variance with Dunn’s all-pairwise multiple-comparisons test. (c) The total distance moved within the open-field arena (1 m  1 m) was measured over a 15 min period and it did not differ significantly (P > 0.05) between the groups. Data are shown as mean (±SEM). ddC, dideoxycytidine.

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Using an ethologically relevant behavioural test as a surrogate indicator of an on-going spontaneous pain state (Hasnie et al., 2007; Wallace et al., 2007a, 2007b, 2008; Huang et al., 2013), single bolus intraperitoneal doses of gabapentin at 30 mg/kg significantly reduced (P < 0.05) thigmotactic (wall-hugging) behaviour in the open field, but this was not the case for single intraperitoneal bolus doses of EMA300 at 30 mg/kg when assessed at 40–60 min after dosing in ddC-rats. However, it was observed that injection (intraperitoneal) of EMA300 at 30 mg/kg induced audible vocalization. This may indicate the presence of stress as a confounding factor as thigmotaxis is increased by anxiogenic stimuli (Carli et al., 1989; Holmes, 2001; Cryan and Holmes, 2005). Such a confound would have impacted on this behaviour in the direction opposite to that expected of an analgesic drug, thus obscuring any effect of EMA300 in these experiments. This observation may also reflect the fact that gabapentin, with its predominantly central site of action and well-described anxiolytic properties, may be expected to perform differently in a thigmotaxis-based testing paradigm compared with EMA300, which has a peripheral site of action (Anand et al., 2013; Smith et al., 2013b). In adult male SD rats, pharmacokinetic studies show that the systemic exposure of EMA300 is approximately twice that of EMA200 after both intravenous and oral bolus dose administration (Smith et al., 2013a). In CCI-rats, the antiallodynic potency of single bolus intraperitoneal doses of EMA300 was approximately four-fold higher than that for EMA200 and the mean time to peak effect for both test compounds was 0.75 h (Smith et al., 2013a). By contrast, the mean time to peak effect for EMA200 and gabapentin in ddC-rats in the present study was later, at 1.5 h after dosing. Hence, the apparent lack of efficacy of EMA300 at 30 mg/kg to alleviate mechanical hypersensitivity in the hindpaws of ddC-rats after administration of the first dose on day 1 of the 3-day dosing period may have been because testing was performed too early, at 0.75 h after dosing (time of peak effect in CCI-rats), rather than at the likely time of peak effect at 1.5 h after dosing that was observed in ddC-rats for both EMA200 and gabapentin. This may have also contributed, at least in part, towards the apparent lack of efficacy, in the present study, of EMA300 at 1 and 10 mg/kg for the relief of mechanical hypersensitivity in ddC-rats, but this remains for future investigation. Although the potency of EMA300 to alleviate mechanical hypersensitivity in the hindpaws of ddC-rats here appears to be approximately three-fold lower than that for CCI-rats (Smith et al., 2013a), these potencies are not directly comparable as different pain modalities were assessed in the two studies. Specifically, in ddC-rats, the ability of EMA300 to alleviate mechanical hypersensitivity in response to applied noxious stimuli in the hindpaws was assessed, whereas the ability of EMA300 to alleviate

mechanical hypersensitivity to applied non-noxious stimuli was assessed previously in CCI-rats (Smith et al., 2013a). Recent high-profile publications by Bayer and Amgen in Nature Reviews Drug Discovery (Prinz et al., 2011) and in Nature (Begley and Hill, 2012), respectively, highlighted their companies’ inability to reproduce the majority of data reported by academic groups in peer-reviewed publications. To address this problem, these authors recommended that key experiments be replicated by independent groups. One approach would be to carry out an exact replication study. An alternative approach would be to allow some flexibility in the replication study design as was done in the present study (Table 1), where we tested two closely related structural analogues and evaluated the effectiveness of two treatment regimens, with our findings collectively showing that both analogues and dosing regimens produced efficacy. Clearly, a limitation of allowing some flexibility in the design of replication studies is that it increases the risk that the replication may fail. Conversely, a strength of allowing some flexibility in the replication study design is that a positive outcome confers a sense of robustness to the findings. This latter notion is supported by the fact that another small-molecule AT2R antagonist (EMA401) has since been taken forward successfully to a positive phase 2 clinical trial in patients with postherpetic neuralgia (Rice et al., 2014), a type of neuropathic pain notoriously difficult to treat. Over the past decade, many other new ‘targets’ have been reported from preclinical studies that have not been replicated independently and almost none in the pain field have been verified in a phase 2 clinical trial. In humans with HIV-SN and rodent models of ATN, structural changes in peripheral nerves include lengthdependent degeneration of myelinated and unmyelinated nerve fibres, characterized by distal axonal degeneration and reduced epidermal nerve fibre density, DRG neuronal loss and die-back of the distal terminals of primary afferents (Kamerman et al., 2012). Although the precise cellular events leading to the development of HIV-SN in humans are unclear, ex-vivo work in the ddCrat model of ATN implicates upregulated expression of the CXCR4 chemokine receptor by both satellite cells and neurons in the DRGs (Bhangoo et al., 2007). The net result is significantly increased signalling of the chemokine, CXCL12, through its CXCR4 receptor to produce DRG neuron hyperexcitability (Bhangoo et al., 2007, 2009), with the latter being a hallmark feature of a range of neuropathic pain states (Oh et al., 2001; Miller et al., 2009; Payne et al., 2011). In cultured adult rat and human DRG neurons, angiotensin II signalling through the AT2R augmented capsaicininduced neuronal excitability, which was inhibited in a concentration-dependent manner by the selective AT2R antagonist, EMA401 (Anand et al., 2013). In nerve-injured mice with genetic deletion of the AT2R, the pain-relieving

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AT2R antagonists: novel analgesics for HIV-SN Smith et al. 145

effects of EMA300 in nerve-injured CCI-mice were abolished, confirming the AT2R as the target mediating EMA300 pain relief (Smith et al., 2013b). In the CCI-rat model of neuropathic pain, augmented angiotensin II signalling through the AT2R in the ipsilateral lumbar DRGs was blocked at the time of peak effect of an analgesic dose of EMA300, resulting in inhibition of p38 MAPK and p42/ p44 MAPK activation (Smith et al., 2013b), a mechanism implicated in the pathobiology of neuropathic pain (Devor, 2009). In more recent work using a rat model of PCIBP, intravenous bolus doses of EMA200 were shown to produce dose-dependent analgesia (Muralidharan et al., 2013). In PCIBP rats, lumbar DRG levels of angiotensin II, NGF, TrkA, pp38 MAPK and pp42/pp44 MAPK, but not the AT2R, were increased significantly (P < 0.05) compared with the corresponding levels for sham controls (Muralidharan et al., 2013). At the time of peak EMA200 analgesia in PCIBP rats, elevated lumbar DRG levels of angiotensin II were reduced to attenuate augmented angiotensin II/AT2R signalling. This in turn reduced augmented NGF/TrkA signalling in the lumbar DRGs, with the net effect being inhibition of p38 MAPK and p44/ p42 MAPK activation (Muralidharan et al., 2013). Administration of small-molecule AT2R antagonists with a greater than 1000-fold selectivity over AT1R (Smith et al., 2013a), as a strategy to block augmented angiotensin II/ AT2R signalling to inhibit peripheral nerve injury-induced activation of p38 MAPK and p42/p44 MAPK in the DRGs (Smith et al., 2013b), would be expected to reduce phosphorylation of multiple receptors and ion channels implicated in DRG neuronal hyperexcitability and neuropathic pain. The introduction of cART for the chronic suppression of viral replication in HIV-infected individuals means that this infectious disease has become a chronic manageable condition rather than an acute fatal illness (Cherry et al., 2012a, 2012b). Although cART has markedly reduced mortality, HIV-SN results in neuropathic pain, disturbed sleep and impaired patient quality of life in 30–64% of individuals infected with HIV, and commonly used analgesic/adjuvant drugs do not effectively alleviate this painful condition. There is therefore a large unmet medical need for a new generation of novel analgesic agents that are highly efficacious and well tolerated for the alleviation of HIV-SN. Our present findings in the ddC-rat model of ATN suggest that highly selective small-molecule AT2R antagonists should be further investigated as novel analgesics for the relief of painful sensory neuropathy in HIV-infected individuals receiving cART for disease prophylaxis.

Acknowledgements This research used infrastructure purchased using investment funds from the Queensland Government Smart State Research Facilities Fund (SSRFF).

Conflicts of interest

M.T.S. and B.D.W. are named inventors on a University of Queensland (UQ) patent for the use of AT2R antagonists as analgesics in neuropathic pain. This technology is being commercialized by Spinifex Pharmaceuticals Pty Ltd, a venture capital-funded biotechnology company, formed in 2005 by UniQuest Pty Ltd. In addition, M.T.S. and B.D.W. have undertaken a large number of contract R&D studies for a broad range of biopharmaceutical companies. The research performed at UQ was supported financially by UQ research funds. Research performed at Imperial College London was supported financially by Spinifex Pharmaceuticals Pty Ltd. A.S.C.R. is a paid consultant, member of the Spinifex scientific advisory board and owns share options in Spinifex Pharmaceuticals Pty Ltd. For the remaining authors there are no conflicts of interest.

References Anand U, Facer P, Yiangou Y, Sinisi M, Fox M, McCarthy T, et al. (2013). Angiotensin II type 2 receptor (AT(2)R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons. Eur J Pain 17:1012–1026. Begley CG, Hill LM (2012). Raise standards for preclinical cancer research. Nature 483:531–533. Bhangoo SK, Ren D, Miller RJ, Chan DM, Ripsch MS, Weiss C, et al. (2007). CXCR4 chemokine receptor signaling mediates pain hypersensitivity in association with antiretroviral toxic neuropathy. Brain Behav Immun 21:581–591. Bhangoo SK, Ripsch MS, Buchanan DJ, Miller RJ, White FA (2009). Increased chemokine signaling in a model of HIV1-associated peripheral neuropathy. Mol Pain 5:48. Carli M, Prontera C, Samanin R (1989). Effect of 5-HT1A agonists on stressinduced deficit in open field locomotor activity of rats: evidence that this model identifies anxiolytic-like activity. Neuropharmacology 28:471–476. Cherry CL, Kamerman PR, Bennett DLH, Rice ASC (2012a). HIV-associated sensory neuropathy: still a problem in the post-stavudine era? Future Virology 7:1–6. Cherry CL, Wadley AL, Kamerman PR (2012b). Painful HIV-associated sensory neuropathy. Pain Management 2:43–52. Clifford DB, Simpson DM, Brown S, Moyle G, Brew BJ, Conway B, et al. NGX4010 C119 Study Group (2012). A randomized, double-blind, controlled study of NGX-4010, a capsaicin 8% dermal patch, for the treatment of painful HIV-associated distal sensory polyneuropathy. J Acquir Immune Defic Syndr 59:126–133. Cryan JF, Holmes A (2005). The ascent of mouse: advances in modeling human depression and anxiety. Nat Rev Drug Discov 4:775–790. Dalakas MC (2001). Peripheral neuropathy and antiretroviral drugs. J Peripher Nerv Syst 6:14–20. Devor M (2009). Ectopic discharge in Abeta afferents as a source of neuropathic pain. Exp Brain Res 196:115–128. Dworkin RH, O’Connor AB, Backonja M, Farrar JT, Finnerup NB, Jensen TS, et al. (2007). Pharmacologic management of neuropathic pain: evidence-based recommendations. Pain 132:237–251. Ellis RJ, Rosario D, Clifford DB, McArthur JC, Simpson D, Alexander T, et al. (2010). Continued high prevalence and adverse clinical impact of human immunodeficiency virus-associated sensory neuropathy in the era of combination antiretroviral therapy: the CHARTER Study. Arch Neurol 67:552–558. Finnerup NB, Sindrup SH, Jensen TS (2010). The evidence for pharmacological treatment of neuropathic pain. Pain 150:573–581. Hasnie FS, Breuer J, Parker S, Wallace V, Blackbeard J, Lever I, et al. (2007). Further characterization of a rat model of varicella zoster virus-associated pain: relationship between mechanical hypersensitivity and anxiety-related behavior and the influence of analgesic drugs. Neuroscience 144:1495–1508. Holmes A (2001). Targeted gene mutation approaches to the anxiety-like behavior in mice. Neurosci Biobehav Rev 25:261–273. Huang W, Calvo M, Karu K, Olausen HR, Bathgate G, Okuse K, et al. (2013). A clinically relevant rodent model of the HIV antiretroviral drug stavudine induced painful peripheral neuropathy. Pain 154:560–575. Hudmon A, Choi JS, Tyrrell L, Black JA, Rush AM, Waxman SG, et al. (2008). Phosphorylation of sodium channel Na(v)1.8 by p38 mitogen-activated

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

146

Behavioural Pharmacology 2014, Vol 25 No 2

protein kinase increases current density in dorsal root ganglion neurons. J Neurosci 28:3190–3201. Ji RR, Samad TA, Jin SX, Schmoll R, Woolf CJ (2002). p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 36:57–68. Kamerman PR, Moss PJ, Weber J, Wallace VC, Rice AS, Huang W (2012). Pathogenesis of HIV-associated sensory neuropathy: evidence from in vivo and in vitro experimental models. J Peripher Nerv Syst 17:19–31. Lee H, Hanes J, Johnson KA (2003). Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. Biochemistry 42:14711–14719. Maratou K, Wallace VC, Hasnie FS, Okuse K, Hosseini R, Jina N, et al. (2009). Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIV-associated neuropathic pain. Eur J Pain 13:387–398. Martin SW, Butcher AJ, Berrow NS, Richards MW, Paddon RE, Turner DJ, et al. (2006). Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels. Cell Calcium 39:275–292. Maschke M, Kastrup O, Esser S, Ross B, Hengge U, et al. (2000). Incidence and prevalence of neurological disorders associated with HIV since the introduction of highly active antiretroviral therapy (HAART). J Neurol Neurosurg Psychiatry 69:376–380. May MT, Ingle SM, Costagliola D, Justice AC, de Wolf F, Cavassini M; The Antiretroviral Cohort Collaboration (2013). Cohort profile: Antiretroviral Therapy Cohort Collaboration (ART-CC). Int J Epidemiol [Epub ahead of print]. McArthur JC, Yiannoutsos C, Simpson DM, Adornato BT, Singer EJ, Hollander H, et al. (2000). A phase II trial of nerve growth factor for sensory neuropathy associated with HIV infection. AIDS Clinical Trials Group Team 291. Neurology 54:1080–1088. Miller RJ, Jung H, Bhangoo SK, White FA (2009). Cytokine and chemokine regulation of sensory neuron function. Handb Exp Pharmacol 194:417–449. Mocroft A, Ledergerber B, Katlama C, Kirk O, Reiss P, d’Arminio Monforte A, et al.; EuroSIDA study group (2003). Decline in the AIDS and death rates in the EuroSIDA study: an observational study. Lancet 362:22–29. Moller KA, Johansson B, Berge OG (1998). Assessing mechanical allodynia in the rat paw with a new electronic algometer. J Neurosci Methods 84:41–47. Muralidharan A, Wyse BD, Smith MT (2013). Analgesic efficacy and mode of action of a selective small molecule angiotensin II type 2 receptor antagonist, in a rat model of prostate cancer-induced bone pain. Pain Med 15:93–110. Oh JW, Drabik K, Kutsch O, Choi C, Tousson A, Benveniste EN (2011). CXC chemokine receptor 4 expression and function in human astroglioma cells. J Immunol 166:2695–2704. Payne BA, Wilson IJ, Hateley CA, Horvath R, Santibanez-Koref M, Samuels DC, et al. (2011). Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations. Nat Genet 43:806–810.

Phillips TJ, Cherry CL, Cox S, Marshall SJ, Rice AS (2010). Pharmacological treatment of painful HIV-associated sensory neuropathy: a systematic review and meta-analysis of randomised controlled trials. PLoS One 5:e14433. Prinz F, Schlange T, Asadullah K (2011). Believe it or not: how much can we rely on published data on potential drug targets? Nat Rev Drug Discov 10:712–713. Rice ASC (2008). Should cannabinoids be used as analgesics for neuropathic pain? Nat Clin Pract Neurol 4:654–655. Rice ASC, Dworkin RH, McCarthy TD, Anand P, Bountra C, McCloud PI, et al. (2014). A novel target and treatment for post-herpetic neuralgia: EMA401, an orally administered highly selective angiotensin II type 2 receptor antagonist studied in a double-blind, placebo-controlled, randomised Phase 2 clinical trial. Lancet (in press). Simpson DM, Brown S, Tobias J (2008). Controlled trial of high-concentration capsaicin patch for treatment of painful HIV neuropathy. Neurology 70: 2305–2313. Smith MT, Woodruff TM, Wyse BD, Muralidharan A, Walther T (2013a). A small molecule angiotensin II type 2 receptor (AT2R) antagonist produces analgesia in a rat model of neuropathic pain by inhibition of p38 mitogen activated protein kinase (MAPK) and p44/p42 MAPK activation in the dorsal root ganglia. Pain Med 14:1557–1568. Smith MT, Wyse BD, Edwards SR (2013b). Small molecule angiotensin II type 2 receptor (AT2R) antagonists as novel analgesics for neuropathic pain: comparative pharmacokinetics, radioligand binding and efficacy in rats. Pain Med 14:692–705. Smyth K, Affandi JS, McArthur JC, Bowtell-Harris C, Mijch AM, Watson K, et al. (2007). Prevalence of and risk factors for HIV-associated neuropathy in Melbourne, Australia 1993-2006. HIV Med 8:367–373. Stamboulian S, Choi JS, Ahn HS, Chang YW, Tyrrell L, Black JA, et al. (2010). ERK1/2 mitogen-activated protein kinase phosphorylates sodium channel Na(v)1.7 and alters its gating properties. J Neurosci 30:1637–1647. Suzuki R, Kontinen VK, Matthews E, Williams E, Dickenson AH (2000). Enlargement of the receptive field size to low intensity mechanical stimulation in the rat spinal nerve ligation model of neuropathy. Exp Neurol 163:408–413. Wallace VC, Blackbeard J, Pheby T, Segerdahl AR, Davies M, Hasnie F, et al. (2007a). Pharmacological, behavioural and mechanistic analysis of HIV-1 gp120 induced painful neuropathy. Pain 133:47–63. Wallace VC, Blackbeard J, Segerdahl AR, Hasnie F, Pheby T, McMahon SB, Rice AS (2007b). Characterization of rodent models of HIV-gp120 and anti-retroviralassociated neuropathic pain. Brain 130:2688–2702. Wallace VC, Segerdahl AR, Blackbeard J, Pheby T, Rice AS (2008). Anxiety-like behavior is attenuated by gabapentin, morphine and diazepam in a rodent model of HIV anti-retroviral-associated neuropathic pain. Neurosci Lett 448:153–156.

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