The vanilloid receptor 1 (VR1 or TRPV1) is a membrane-bound, nonselective cation channel expressed by peripheral sensory neurons. TRPV1 antagonists produce antihyperalgesic effects in animal models of inflammatory and neuropathic pain. Here, we describe the in vitro and in vivo pharmacology of a novel TRPV1 antagonist, AMG 9810,AMG 9810 is a competitive antagonist of capsaicin activation (IC 50 value for human TRPV1, 24.5 Ϯ 15.7 nM; rat TRPV1, 85.6 Ϯ 39.4 nM) and blocks all known modes of TRPV1 activation, including protons (IC 50 value for rat TRPV1, 294 Ϯ 192 nM; human TRPV1, 92.7 Ϯ 72.8 nM), heat (IC 50 value for rat TRPV1, 21 Ϯ 17 nM; human TRPV1, 15.8 Ϯ 10.8 nM), and endogenous ligands, such as anandamide, N-arachidonyl dopamine, and oleoyldopamine. AMG 9810 blocks capsaicin-evoked depolarization and calcitonin gene-related peptide release in cultures of rat dorsal root ganglion primary neurons. Screening of AMG 9810 against a panel of G protein-coupled receptors and ion channels indicated selectivity toward TRPV1. In vivo, AMG 9810 is effective at preventing capsaicin-induced eye wiping in a dose-dependent manner, and it reverses thermal and mechanical hyperalgesia in a model of inflammatory pain induced by intraplantar injection of complete Freund's adjuvant. At effective doses, AMG 9810 did not show any significant effects on motor function, as measured by open field locomotor activity and motor coordination tests. AMG 9810 is the first cinnamide TRPV1 antagonist reported to block capsaicin-induced eye wiping behavior and reverse hyperalgesia in an animal model of inflammatory pain.Activation of peripheral nociceptors in humans by capsaicin results in burning pain (Park et al., 1995). Capsaicin, and its ultrapotent analog resiniferatoxin, aided the identification and characterization of the vanilloid receptor 1 (aka VR1 and TRPV1). TRPV1 is a nonselective cation channel with high permeability to calcium (Caterina et al., 1997) and belongs to a superfamily of ion channels known as the transient receptor potential channels or TRPs (Clapham et al., 2001). In addition to activation by exogenous agonists such as capsaicin and resiniferatoxin, TRPV1 can be activated by physical stimuli, such as heat (Ͼ42°C) and protons (pH 5). Based on their structural similarity to capsaicin, several endogenous ligands have been proposed that include anandamide (AEA), 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid,N-arachidonyl dopamine (NADA), N-oleoyldopamine (OLDA), and products of lipoxygenases (Hwang et al., 2000;Olah et al., 2001;Huang et al., 2002;Chu et al., 2003). TRPV1 is up-regulated during inflammation (Ji et al., 2002), and channel activity is modulated by the action of inflammaArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org.doi :

An involvement of the transient receptor potential vanilloid (TRPV) 1 channel in the regulation of body temperature (T b ) has not been established decisively. To provide decisive evidence for such an involvement and determine its mechanisms were the aims of the present study. We synthesized a new TRPV1 antagonist, AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide], and characterized it in vitro. We then found that this drug is the most potent TRPV1 antagonist known to increase T b of rats and mice and showed (by using knock-out mice) that the entire hyperthermic effect of AMG0347 is TRPV1 dependent. AMG0347-induced hyperthermia was brought about by one or both of the two major autonomic cold-defense effector mechanisms (tail-skin vasoconstriction and/or thermogenesis), but it did not involve warmth-seeking behavior. The magnitude of the hyperthermic response depended on neither T b nor tail-skin temperature at the time of AMG0347 administration, thus indicating that AMG0347-induced hyperthermia results from blockade of tonic TRPV1 activation by nonthermal factors. AMG0347 was no more effective in causing hyperthermia when administered into the brain (intracerebroventricularly) or spinal cord (intrathecally) than when given systemically (intravenously), which indicates a peripheral site of action. We then established that localized intra-abdominal desensitization of TRPV1 channels with intraperitoneal resiniferatoxin blocks the T b response to systemic AMG0347; the extent of desensitization was determined by using a comprehensive battery of functional tests. We conclude that tonic activation of TRPV1 channels in the abdominal viscera by yet unidentified nonthermal factors inhibits skin vasoconstriction and thermogenesis, thus having a suppressive effect on T b .

The vanilloid receptor TRPV1 (transient receptor potential vanilloid 1) is a cation channel that serves as a polymodal detector of pain-producing stimuli such as capsaicin, protons (pH Ͻ5.7), and heat. TRPV1 antagonists block pain behaviors in rodent models of inflammatory, neuropathic, and cancer pain, suggesting their utility as analgesics. Here, we report that TRPV1 antagonists representing various chemotypes cause an increase in body temperature (hyperthermia), identifying a potential issue for their clinical development. Peripheral restriction of antagonists did not eliminate hyperthermia, suggesting that the site of action is predominantly outside of the blood-brain barrier. Antagonists that are ineffective against proton activation also caused hyperthermia, indicating that blocking capsaicin and heat activation of TRPV1 is sufficient to produce hyperthermia. All TRPV1 antagonists evaluated here caused hyperthermia, suggesting that TRPV1 is tonically activated in vivo and that TRPV1 antagonism and hyperthermia are not separable. TRPV1 antagonists caused hyperthermia in multiple species (rats, dogs, and monkeys), demonstrating that TRPV1 function in thermoregulation is conserved from rodents to primates. Together, these results indicate that tonic TRPV1 activation regulates body temperature.

Development of analgesic agents for the treatment of severe pain requires the identification of compounds that are devoid of opioid receptor liabilities. A potent (inhibition constant = 37 picomolar) neuronal nicotinic acetylcholine receptor (nAChR) ligand called ABT-594 was developed that has antinociceptive properties equal in efficacy to those of morphine across a series of diverse animal models of acute thermal, persistent chemical, and neuropathic pain states. These effects were blocked by the nAChR antagonist mecamylamine. In contrast to morphine, repeated treatment with ABT-594 did not appear to elicit opioid-like withdrawal or physical dependence. Thus, ABT-594 may be an analgesic that lacks the problems associated with opioid analgesia.

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Experimental models of pain include tests of response thesholds to high intensity stimuli (acute pain tests) and changes in spontaneous or evoked behavioral responses in animals with peripheral injury or inflammation (persistent pain models). Acute thermal pain is modeled by the hot-plate and tail-flick test, while persistent pain can be modeled by the formalin test. This unit presents protocols for all three of these tests, including preparation of animals (rats or mice), administration of a compound being tested for its analgesic properties and data collection.

Capsaicin, the active ingredient in some pain-relieving creams, is an agonist of a nonselective cation channel known as the transient receptor potential vanilloid type 1 (TRPV1). The pain-relieving mechanism of capsaicin includes desensitization of the channel, suggesting that TRPV1 antagonism may be a viable pain therapy approach. In agreement with the above notion, several TRPV1 antagonists have been reported to act as antihyperalgesics. Here, we report the in vitro and in vivo characterization of a novel and selective TRPV1 antagonist, N-(4-[6-(4-trifluoromethyl-phenyl)-pyrimidin-4-yloxy]-benzothiazol-2-yl)-acetamide I (AMG 517), and compare its pharmacology with that of a closely related analog, tert-butyl-2- (6-([2-(acetylamino)-1,3-benzothiazol-4-yl]oxy)pyrimidin-4-yl)-5-(trifluoromethyl)phenylcarbamate (AMG8163). Both AMG 517 and AMG8163 potently and completely antagonized capsaicin, proton, and heat activation of TRPV1 in vitro and blocked capsaicin-induced flinch in rats in vivo. To support initial