Pharmacologic Characterization of AMG 334, a Potent and Selective Human Monoclonal Antibody against the Calcitonin Gene-Related Peptide Receptor.

Therapeutic agents that block the calcitonin gene-related peptide (CGRP) signaling pathway are a highly anticipated and promising new drug class for migraine therapy, especially after reports that small-molecule CGRP-receptor antagonists are efficacious for both acute migraine treatment and migraine prevention. Using XenoMouse technology, we successfully generated AMG 334, a fully human monoclonal antibody against the CGRP receptor. Here we show that AMG 334 competes with [(125)I]-CGRP binding to the human CGRP receptor, with a Ki of 0.02 nM. AMG 334 fully inhibited CGRP-stimulated cAMP production with an IC50 of 2.3 nM in cell-based functional assays (human CGRP receptor) and was 5000-fold more selective for the CGRP receptor than other human calcitonin family receptors, including adrenomedullin, calcitonin, and amylin receptors. The potency of AMG 334 at the cynomolgus monkey (cyno) CGRP receptor was similar to that at the human receptor, with an IC50 of 5.7 nM, but its potency at dog, rabbit, and rat receptors was significantly reduced (>5000-fold). Therefore, in vivo target coverage of AMG 334 was assessed in cynos using the capsaicin-induced increase in dermal blood flow model. AMG 334 dose-dependently prevented capsaicin-induced increases in dermal blood flow on days 2 and 4 postdosing. These results indicate AMG 334 is a potent, selective, full antagonist of the CGRP receptor and show in vivo dose-dependent target coverage in cynos. AMG 334 is currently in clinical development for the prevention of migraine.

Voltage-gated sodium channel function and expression in injured and uninjured rat dorsal root ganglia neurons.

The nine members of the voltage-gated sodium channel (Nav) family mediate inward sodium currents that depolarize neurons and lead to action potential firing. Increased Nav expression and function in sensory ganglia may drive ectopic action potentials and result in neuropathic pain. Using patch-clamp electrophysiology and molecular biology techniques, experiments were performed to elucidate the contribution of Nav channels to sodium currents in rat dorsal root ganglion (DRG) neurons following the L5/L6 spinal nerve ligation (SNL) model of neuropathic pain. The abundance of DRG neurons with fast, tetrodotoxin sensitive (TTX-S) currents was seven-fold higher whereas the abundance of DRG neurons with slow, tetrodotoxin resistant (TTX-R) currents was nearly thirty-fold lower when comparing ipsilateral (injured) to contralateral (uninjured) neurons. TTX-S currents were elevated in larger neurons while TTX-R currents were reduced in both small and large neurons. Among Nav transcripts encoding TTX-R channels, Scn10a (Nav1.8) and Scn11a (Nav1.9) expression was twenty- to thirty-fold lower, while among Nav transcripts encoding TTX-S channels, Scn3a (Nav1.3) expression was four-fold higher in injured compared to uninjured DRG by qRT-PCR analysis. In summary, the SNL model of neuropathic pain induced a phenotypic switch in Nav expression from TTX-R to TTX-S channels in injured DRG neurons. Transcriptional reprogramming of Nav genes may drive ectopic action potential firing and contribute to neuropathic pain.

Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists.

Agonists of TRPA1 such as mustard oil and its key component AITC cause pain and neurogenic inflammation in humans and pain behavior in rodents. TRPA1 is activated by numerous reactive compounds making it a sensor for reactive compounds in the body. Failure of AITC, formalin and other reactive compounds to trigger pain behavior in TRPA1 knockout mice, as well as the ability of TRPA1 antisense to alleviate cold hyperalgesia after spinal nerve ligation, suggest that TRPA1 is a potential target for novel analgesic agents. Here, we have characterized CHO cells expressing human and rat TRPA1 driven by an inducible promoter. As reported previously, both human and rat TRPA1 are activated by AITC and inhibited by ruthenium red. We have also characterized noxious cold response of these cell lines and show that noxious cold activates both human and rat TRPA1. Further, we have used CHO cells expressing human TRPA1 to screen a small molecule compound library and discovered that 'trichloro(sulfanyl)ethyl benzamides' (AMG2504, AMG5445, AMG7160 and AMG9090) act as potent antagonists of human TRPA1 activated by AITC and noxious cold. However, trichloro(sulfanyl)ethyl benzamides' (TCEB compounds) displayed differential pharmacology at rat TRPA1. AMG2504 and AMG7160 marginally inhibited rat TRPA1 activation by AITC, whereas AMG5445 and AMG9090 acted as partial agonists. In summary, we conclude that both human and rat TRPA1 channels show similar AITC and noxious cold activation profiles, but TCEB compounds display species-specific differential pharmacology at TRPA1.

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.

The TRPV1 receptor and nociception.

The capsaicin receptor TRPV1 is an emerging target for the treatment of pain with a unique expression profile in peripheral nociceptors and the ability to show polymodal activation, TRPV1 is an important integrator of responses to inflammatory mediators. Sensitization of TRPV1 during chronic pain is believed to contribute to the transduction of noxious signaling for normally innocuous stimuli and consequently the search for novel TRPV1 therapeutics is intense. The current understanding of the physiological role the receptor, as well as the potential therapeutic utility and emerging liabilities of TRPV1 modulators are discussed.

ASIC3, an acid-sensing ion channel, is expressed in metaboreceptive sensory neurons.

BACKGROUND: ASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1) ASIC3 might trigger ischemic pain in heart and muscle; 2) it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses. RESULTS: Less than half (40%) of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small (<25 microm) sensory neurons that innervate muscle are more likely to express ASIC3 than those that innervate skin (51% of small muscle afferents vs. 28% of small skin afferents). Over 80% of ASIC3+ muscle afferents co-express CGRP (a vasodilatory peptide). Remarkably few (9%) ASIC3+ cells express P2X3 receptors (an ATP-gated ion channel), whereas 31% express TRPV1 (the noxious heat and capsaicin-activated ion channel also known as VR1). ASIC3+/CGRP+ sensory nerve endings were observed on muscle arterioles, the blood vessels that control vascular resistance; like the cell bodies, the endings are P2X3- and can be TRPV1+. The TrkC+/ASIC3+ cell bodies are uniformly large, possibly consistent with non-nociceptive mechanosensation. They are not proprioceptors because they fail two other tests: ASIC3+ cells do not express parvalbumin and they are absent from the mesencephalic trigeminal nucleus. CONCLUSION: Our data indicates that: 1) ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2) co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen.

Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade.

Acid-sensing ion channels (ASICs) open when extracellular pH drops and they are enhanced by lactate, making them specialized for detecting lactic acidosis. Highly expressed on cardiac nociceptors and some other sensory neurons, ASICs may help trigger pain caused by tissue ischemia. We report that H(+) opens ASIC3 by speeding release of Ca(2+) from a high-affinity binding site (K(Ca) = 150 nM) on the extracellular side of the pore. The bound Ca(2+) blocks permeation and the channel conducts when multiple H(+) ions relieve this block. Activation through Ca(2+) explains sensitivity to lactate, which decreases extracellular [Ca(2+)], and it may prove relevant in CNS pathologies (stroke, seizure) that simultaneously drop pH and Ca(2+).