AMG2850, a potent and selective TRPM8 antagonist, is not effective in rat models of inflammatory mechanical hypersensitivity and neuropathic tactile allodynia

Abstract

TRPM8 has been implicated in pain and migraine based on dorsal root- and trigeminal ganglion-enriched expression, upregulation in preclinical models of pain, knockout mouse studies, and human genetics. Here, we evaluated the therapeutic potential in pain of AMG2850 ((R)-8-(4-(trifluoromethyl)phenyl)-N-((S)-1,1,1-trifluoropropan-2-yl)-5,6-dihydro-1,7-naphthyridine-7(8H)-carboxamide), a small molecule antagonist of TRPM8 by in vitro and in vivo characterization. AMG2850 is potent in vitro at rat TRPM8 (IC90 against icilin activation of 204 ± 28 nM), highly selective (>100-fold IC90 over TRPV1 and TRPA1 channels), and orally bioavailable (F po > 40 %). When tested in a skin-nerve preparation, AMG2850 blocked menthol-induced action potentials but not mechanical activation in C fibers. AMG2850 exhibited significant target coverage in vivo in a TRPM8-mediated icilin-induced wet-dog shake (WDS) model in rats (at 10 mg/kg p.o.). However, AMG2850 did not produce a significant therapeutic effect in rat models of inflammatory mechanical hypersensitivity or neuropathic tactile allodynia at doses up to 100 mg/kg. The lack of efficacy suggests that either TRPM8 does not play a role in mediating pain in these models or that a higher level of target coverage is required. The potential of TRPM8 antagonists as migraine therapeutics is yet to be determined.

Fig. 1

a AMG2850 potently inhibits both cold temperature- and icilin-induced increase in luminescence believed to reflect intracellular calcium in CHO cells stably expressing rat TRPM8. After a 2-h incubation with coelenterazine, cells were incubated with compounds for 2.5 min prior to the addition of either icilin (1 μM) or cold buffer (12 °C). Luminescence was measured by a CCD camera-based FLASH-luminometer. Concentration-response curves were generated using GraphPad Prism 4.01. Each point in the graphs is an average ± SEM of an experiment conducted in triplicate. Responses of agonists used were normalized to 100 % of maximum. b In a skin-nerve preparation, total percentage of C fibers responding to 300 μM menthol after incubation with vehicle or AMG2850. A response to menthol was defined as three action potentials above baseline over a 2-min interval. Incubation with AMG2850 significantly decreased the percentage of C fibers responding to menthol as compared to vehicle controls (**p < 0.005). c In a skin-nerve preparation, AMG2850 has no effect on C fiber firing in response to sustained mechanical force of increasing intensities. Note an increasing number of action potentials with increased force that is not altered by preincubation of AMG2850

Fig. 2

a Intraperitoneal injection of icilin induces wet-dog shakes (WDS) in a dose-dependent manner. b AMG2850 reduces WDS in a dose- and plasma concentration-dependent manner. c AMG2850 (10 mg/kg, p.o.) significantly and fully blocked the cold pressor effect (***p < 0.001)

Fig. 3

a AMG2850 did not affect the total distance traveled in reversed light cycle rat open field boxes (p > 0.05) while the positive control compound chlordiazepoxide (CDP; 5.6 mg/kg) significantly reduced distance traveled (*p < 0.05). b Unilateral hindpaw injection of complete Freund’s adjuvant (CFA) causes a reduction in rearing behavior. The positive control indomethacin (indo; 1 mg/kg) significantly (⁺⁺⁺⁺ p < 0.0001) reversed the CFA-induced reduction in rearing behavior. AMG2850 (100 mg/kg) produced no significant effect (*p > 0.05). c SNL produces a reduction in tactile threshold from approximately 15 g to approximately 5 g. The positive control, gabapentin (200 mg/kg), significantly reversed the reduction in tactile threshold (**p < 0.01). AMG2850 (100 mg/kg) produced no significant effect at 2.5 h post treatment (p > 0.05)