Antihyperalgesic effects of (R,E)-N-(2-hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)phenyl)-acrylamide (AMG8562), a novel transient receptor potential vanilloid type 1 modulator that does not cause hyperthermia in rats.

Antagonists of the vanilloid receptor TRPV1 (transient receptor potential vanilloid type 1) have been reported to produce antihyperalgesic effects in animal models of pain. These antagonists, however, also caused concomitant hyperthermia in rodents, dogs, monkeys, and humans. Antagonist-induced hyperthermia was not observed in TRPV1 knockout mice, suggesting that the hyperthermic effect is exclusively mediated through TRPV1. Since antagonist-induced hyperthermia is considered a hurdle for developing TRPV1 antagonists as therapeutics, we investigated the possibility of eliminating hyperthermia while maintaining antihyperalgesia. Here, we report four potent and selective TRPV1 modulators with unique in vitro pharmacology profiles (profiles A through D) and their respective effects on body temperature. We found that profile C modulator, (R,E)-N-(2-hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)phenyl)acrylamide (AMG8562), blocks capsaicin activation of TRPV1, does not affect heat activation of TRPV1, potentiates pH 5 activation of TRPV1 in vitro, and does not cause hyperthermia in vivo in rats. We further profiled AMG8562 in an on-target (agonist) challenge model, rodent pain models, and tested for its side effects. We show that AMG8562 significantly blocks capsaicin-induced flinching behavior, produces statistically significant efficacy in complete Freund's adjuvant- and skin incision-induced thermal hyperalgesia, and acetic acid-induced writhing models, with no profound effects on locomotor activity. Based on the data shown here, we conclude that it is feasible to modulate TRPV1 in a manner that does not cause hyperthermia while maintaining efficacy in rodent pain models.

Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors.

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).

Repeated administration of vanilloid receptor TRPV1 antagonists attenuates hyperthermia elicited by TRPV1 blockade.

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 clinical investigations, AMG 517 was evaluated in a comprehensive panel of toxicology studies that included in vivo assessments in rodents, dogs, and monkeys. The toxicology studies indicated that AMG 517 was generally well tolerated; however, transient increases in body temperature (hyperthermia) were observed in all species after AMG 517 dosing. To further investigate this effect, we tested and showed that the antipyretic, acetaminophen, suppressed the hyperthermia caused by TRPV1 blockade. We also showed that repeated administration of TRPV1 antagonists attenuated the hyperthermia response, whereas the efficacy in capsaicin-induced flinch model was maintained. In conclusion, these studies suggest that the transient hyperthermia elicited by TRPV1 blockade may be manageable in the development of TRPV1 antagonists as therapeutic agents. However, the impact of TRPV1 antagonist-induced hyperthermia on their clinical utility is still unknown.

Antibodies to nerve growth factor reverse established tactile allodynia in rodent models of neuropathic pain without tolerance.

A considerable body of evidence implicates endogenous nerve growth factor (NGF) in conditions in which pain is a prominent feature, including neuropathic pain. However, previous studies of NGF antagonism in animal models of neuropathic pain have examined only the prevention of hyperalgesia and allodynia after injury, whereas the more relevant issue is whether treatment can provide relief of established pain, particularly without tolerance. In the current work, we studied the effects of potent, neutralizing anti-NGF antibodies on the reversal of tactile allodynia and thermal hyperalgesia in established models of neuropathic and inflammatory pain in rats and mice. In the complete Freund's adjuvant-induced hind-paw inflammation, spinal nerve ligation and streptozotocin-induced neuropathic pain models, a single intraperitoneal injection of a polyclonal anti-NGF antibody reversed established tactile allodynia from approximately day 3 to day 7 after treatment. Effects on thermal hyperalgesia were variable with a significant effect observed only in the spinal nerve ligation model. In the mouse chronic constriction injury (CCI) model, a mouse monoclonal anti-NGF antibody reversed tactile allodynia when administered 2 weeks after surgery. Repeated administration of this antibody to CCI mice for 3 weeks produced a sustained reversal (days 4 to 21) of tactile allodynia that returned 5 days after the end of dosing. In conclusion, NGF seems to play a critical role in models of established neuropathic and inflammatory pain in both rats and mice, with no development of tolerance to antagonism. Antagonists of NGF, such as fully human monoclonal anti-NGF antibodies, may have therapeutic utility in analogous human pain conditions.

The vanilloid receptor TRPV1 is tonically activated in vivo and involved in body temperature regulation.

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 <5.7), 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.

A polyclonal antibody to the prepore loop of transient receptor potential vanilloid type 1 blocks channel activation.

Transient receptor potential vanilloid type 1 (TRPV1) can be activated by multiple chemical and physical stimuli such as capsaicin, anandamide, protons, and heat. Capsaicin interacts with the binding pocket constituted by transmembrane regions 3 and 4, whereas protons act through residues in the prepore loop of TRPV1. Here, we report on characterization of polyclonal and monoclonal antibodies to the prepore loop of TRPV1. A rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) acted as a full antagonist of proton activation (IC(50) values for pH 5 and 5.5 were 364.68 +/- 29.78 and 28.31 +/- 6.30 nM, respectively) and as a partial antagonist of capsaicin, heat, and pH 6 potentiated chemical ligand (anandamide and capsaicin) activation (50-79% inhibition). Ab-156H antagonism of TRPV1 is not affected by the conformation of the capsaicin-binding pocket because it is equally potent at wild-type (capsaicin-sensitive) rat TRPV1 and its T550I mutant (capsaicin-insensitive). With the goal of generating monoclonal antagonist antibodies to the prepore region of human TRPV1, we used a recently developed rabbit immunization protocol. Although rabbit polyclonal antiserum blocked human TRPV1 activation, rabbit monoclonal antibodies (identified on the basis of selective binding to Chinese hamster ovary cells expressing human TRPV1) did not block activation by either capsaicin or protons. Thus, rabbit polyclonal antibodies against rat and human TRPV1 prepore region seem to partially lock or stabilize the channel in the closed state, whereas rabbit anti-human TRPV1 monoclonal antibodies bind to the prepore region but do not lock or stabilize the channel conformation.

Proton activation does not alter antagonist interaction with the capsaicin-binding pocket of TRPV1.

Vanilloid receptor 1 (TRPV1) is activated by chemical ligands (e.g., capsaicin and protons) and heat. In this study, we show that (2E)-3-[2-piperidin-1-yl-6-(trifluoromethyl)pyridin-3-yl]-N-quinolin-7-ylacrylamide (AMG6880), 5-chloro-6-[(3R)-3-methyl-4-[6-(trifluoromethyl)-4-(3,4,5-trifluorophenyl)-1H-benzimidazol-2-yl]piperazin-1-yl]pyridin-3-yl)methanol (AMG7472), and N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) are potent antagonists of rat TRPV1 activation by either capsaicin or protons (pH 5) (defined here as group A antagonists), whereas (2E)-3-(6-tert-butyl-2-methylpyridin-3-yl)-N-(1H-indol-6-yl)acrylamide (AMG0610), capsazepine, and (2E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)acrylamide (SB-366791) are antagonists of capsaicin, but not proton, activation (defined here as group B antagonists). By using capsaicin-sensitive and insensitive rabbit TRPV1 channels, we show that antagonists require the same critical molecular determinants located in the transmembrane domain 3/4 region to block both capsaicin and proton activation, suggesting the presence of a single binding pocket. To determine whether the differential pharmacology is a result of proton activation-induced conformational changes in the capsaicin-binding pocket that alter group B antagonist affinities, we have developed a functional antagonist competition assay. We hypothesized that if group B antagonists bind at the same or an overlapping binding pocket of TRPV1 as group A antagonists, and proton activation does not alter the binding pocket, then group B antagonists should compete with and prevent group A antagonism of TRPV1 activation by protons. Indeed, we found that each of the group B antagonists competed with and prevented BCTC, AMG6880 or AMG7472 antagonism of rat TRPV1 activation by protons with pA2 values similar to those for blocking capsaicin, indicating that proton activation does not alter the conformation of the TRPV1 capsaicin-binding pocket. In conclusion, group A antagonists seem to lock the channel conformation in the closed state, blocking both capsaicin and proton activation.

AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide], a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties.

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, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide. AMG 9810 is a competitive antagonist of capsaicin activation (IC50 value for human TRPV1, 24.5 +/- 15.7 nM; rat TRPV1, 85.6 +/- 39.4 nM) and blocks all known modes of TRPV1 activation, including protons (IC50 value for rat TRPV1, 294 +/- 192 nM; human TRPV1, 92.7 +/- 72.8 nM), heat (IC50 value for rat TRPV1, 21 +/- 17 nM; human TRPV1, 15.8 +/- 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.

Molecular determinants of vanilloid sensitivity in TRPV1.

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.