Orally Effective Vanilloid Receptor 1 Antagonist with Analgesic Properties : II . In Vivo Characterization in Rat Models of Inflammatory and Neuropathic Pain

The vanilloid receptor 1 (VR1) is a cation channel expressed predominantly by nociceptive sensory neurons and is activated by a wide array of pain-producing stimuli, including capsaicin, noxious heat, and low pH. Although the behavioral effects of injected capsaicin and the VR1 antagonist capsazepine have indicated a potential role for VR1 in the generation and maintenance of persistent pain states, species differences in the molecular pharmacology of VR1 and a limited number of selective ligands have made VR1 difficult to study in vivo. N-(4-Tertiarybutylphenyl)-4-(3cholorphyridin-2-yl)tetrahydropryazine-1(2H)-carbox-amide (BCTC) is a recently described inhibitor of capsaicinand acidmediated currents at rat VR1. Here, we report the effects of BCTC on acute, inflammatory, and neuropathic pain in rats. Administration of BCTC (30 mg/kg p.o.) significantly reduced both mechanical and thermal hyperalgesia induced by intraplantar injection of 30 g of capsaicin. In rats with Freund’s complete adjuvantinduced inflammation, BCTC significantly reduced the accompanying thermal and mechanical hyperalgesia (3 mg/kg and 10 mg/kg p.o., respectively). BCTC also reduced mechanical hyperalgesia and tactile allodynia 2 weeks after partial sciatic nerve injury (10 and 30 mg/kg p.o.). BCTC did not affect motor performance on the rotarod after administration of doses up to 50 mg/kg p.o. These data suggest a role for VR1 in persistent and chronic pain arising from inflammation or nerve injury. The vanilloid receptor type 1 (VR1) is a pivotal molecular integrator of noxious stimuli that is expressed on somatic and autonomic primary afferent neurons. VR1 has been confirmed as a ligand-gated ion channel after its cloning from rat and human tissues, and has been shown to be highly expressed in small-diameter primary afferent neurons (Caterina et al., 1997; Hayes et al., 2000; McIntyre et al., 2001). In vitro studies have shown that, like the native vanilloid receptor, recombinant VR1 can be activated by a variety of chemical as well as physical stimuli. In vitro, VR1 responds to plant-derived compounds, including capsaicin, a pungent component of chili peppers, lipid mediators such as anandamide (Smart et al., 2000), the lipoxygenase product 12-(S)hydroperoxyeicosatetraenoic acid (Hwang et al., 2000), as well as noxious heat (Caterina et al., 1997) and low pH (Tominaga et al., 1998). A potential role for VR1 in nociception has been evident for some time because injection of the VR1 agonist capsaicin induces nocifensive and hyperalgesic behaviors in rodents and pain in humans (Szolcsanyi, 1977; Carpenter and Lynn, 1981; Simone et al., 1987, 1989; Gilchrist et al., 1996). Further support for VR1 as a therapeutic target arose from experiments involving capsazepine. Capsazepine is a VR1 antagonist that has been shown to competitively inhibit capsaicin-mediated responses in isolated dorsal root ganglion (DRG) neurons (Bevan et al., 1992a) and tissues from rat (Bevan et al., 1992b; Cholewinski et al., 1993; Maggi et al., 1993; Santicioli et al., 1993; Jerman et al., 2000), mouse (Urban and Dray, 1991), and guinea pig (Ellis and Undem, 1994; Fox et al., 1995; Auberson et al., 2000). In vivo, capsazepine has been shown to inhibit nocifensive and hyperalgesic responses to capsaicin in mice, rats, and guinea pigs Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.046268. ABBREVIATIONS: VR1, vanilloid receptor 1; DRG, dorsal root ganglion; BCTC, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; FCA, Freund’s complete adjuvant; PLSD, protected least significant difference; FW50, median fiber weight. 0022-3565/03/3061-387–393$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 1 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 46268/1076303 JPET 306:387–393, 2003 Printed in U.S.A. 387 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from (Santos and Calixto, 1997; Walker et al., 2003). However, both in vitro and in vivo studies have indicated that capsazepine also has species-dependent activity. In vitro, capsazepine has been shown to block low pH mediated activation of human or guinea pig, but not rat VR1 (Lou and Lundberg, 1992; Satoh et al., 1993; Fox et al., 1995; McIntyre et al., 2001; Savidge et al., 2002). These in vitro results correlate with the finding that capsazepine reverses inflammatory and neuropathic hyperalgesia in the guinea pig, but not in the rat (Walker et al., 2003). Together, these data suggest that blockade of low pH induced VR1 activation may be predictive of antihyperalgesic efficacy in vivo. If this hypothesis is valid, then molecules that inhibit pH induced activation of rat VR1, in vitro, would also produce a reduction in hyperalgesia in rat models of chronic pain. In the accompanying report, Valenzano et al. (2003) describe N-(4-tertiarybutylphenyl)-4-(3-chloropyridin2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC) as a potent, selective, and orally bioavailable antagonist of rat VR1. In contrast to capsazepine, BCTC not only blocks the activation of rat VR1 by capsaicin but also by low pH at the native rat VR1 in a skin-nerve preparation. Thus, BCTC has provided us with an opportunity to test our hypothesis that the inhibition of low pH induced activation of VR1 confers in vivo efficacy in models of chronic pain. This report describes the effects of BCTC in models of inflammatory, neuropathic, and capsaicin-induced pain in the rat. The efficacy and side effect profile of BCTC in these models were compared with those of nonsteroidal anti-inflammatory drugs and antiepileptic drugs currently used for the clinical therapy of inflammatory and neuropathic pain, respectively. Materials and Methods Compounds and Administration Procedures. BCTC was synthesized according to known methods and was used in all experiments as its free base (molecular weight 372.89). Indomethacin (Sigma-Aldrich, St. Louis, MO) and BCTC were administered orally in 2% -cyclodextrin (Sigma-Aldrich) by gastric gavage in a dose volume of 10 ml/kg b.wt. Gabapentin (Kemprotec, Middlesborough, UK) was dissolved in saline and administered via i.p. injection in a dose volume of 2 ml/kg. Capsaicin (Sigma-Aldrich) was used in all experiments as its free base. The procedure of Gilchrist et al. (1996) was used to dissolve the capsaicin. Briefly, 6 mg of capsaicin was first dissolved in 0.14 ml of polyoxyethylene (20) sorbitan mono-oleate (Tween 80) by gently heating the solution to approximately 70°C. The solution was then diluted with 1.86 ml of 0.9% sodium chloride using an ultrasonic bath, and passed through a 0.20m filter. The final concentration of the capsaicin solution was 3 g/ l. Intraplantar injections of the capsaicin solution were given in a 10l volume using a 100l Hamilton syringe fitted with a 27-gauge needle. The Tween 80/saline vehicle was used for control injections. Animals. The Purdue Institutional Animal Care and Use Committee approved all animal procedures according to the guidelines of the Office of Laboratory Animal Welfare. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY), weighing 180 to 200 g at the start of acute and inflammatory experiments or 90 to 110 g at the start of nerve ligation experiments, were used. Animals were grouphoused and had free access to food and water at all times, except before oral administration of drugs when food was removed for 16 h before dosing. For comparison with compound-treated groups, animals treated with the appropriate drug vehicle were included in each experiment. The volume of administration was identical for vehicleand compound-treated rats, and rats were identical with respect to all other experimental procedures. Capsaicin-Induced Hyperalgesia. Intraplantar injection of capsaicin (30 g) was used to induce mechanical and thermal hyperalgesia, as described previously (Gilchrist et al., 1996). The paw pressure assay was used to assess capsaicin-induced mechanical hyperalgesia. For this assay, hind paw withdrawal thresholds (PWTs) to a noxious mechanical stimulus were determined using an analgesymeter (model 7200; Ugo Basile, Varese, Italy) (Walker et al., 2001). Cut-off was set at 250 g and the endpoint was taken as complete paw withdrawal. PWTs were determined once for each rat at each time point. All rats were tested for baseline PWT and 1 h later, animals received a single dose of 1, 3, 10, or 30 mg/kg BCTC or vehicle (p.o., volume 10 ml/kg, n 10/group). Thirty minutes later, under isofluorane/oxygen anesthesia, rats received a single intraplantar injection of 30 g of capsaicin or vehicle in a 10l volume. Ninety minutes after the intraplantar injection of capsaicin, PWTs were determined again. The plantar test was used to assess capsaicin-induced thermal hyperalgesia (n 8/group). For this test, hind paw withdrawal latencies (PWLs) to a noxious thermal stimulus were determined using the technique described by Hargreaves et al. (1988) using a plantar test apparatus (model 7370-371; Ugo Basile). Cut-off was set at 32 s, and any directed paw withdrawal from the heat source was taken as the endpoint. To assess the effects of BCTC on the development of capsaicin-induced thermal hyperalgesia rats were treated as described above for capsaicin-induced mechanical hyperalgesia, but PWLs were measured 30 min after capsaicin injection. Inflammatory Hyperalgesia. The efficacy of BCTC against hyperalgesia associated with inflammation was investigated using the Freund’s complete adjuvant (FCA) model (Walker et al., 2001). Mechanical and thermal hyperalgesia were measured in separate groups of rats (n 8–20/group), according to the procedure described above. Baseline PWTs or PWLs were determined, and rats were then anesthetized with isofluorane/oxygen and rec