RESUMEN
The transient receptor potential (subfamily M, member 8; TRPM8) is a nonselective cation channel localized in primary sensory neurons, and is a candidate for cold thermosensing, mediation of cold pain, and bladder overactivity. Studies with TRPM8 knockout mice and selective TRPM8 channel blockers demonstrate a lack of cold sensitivity and reduced cold pain in various rodent models. Furthermore, TRPM8 blockers significantly lower body temperature. We have identified a moderately potent (IC50 = 103 nM), selective TRPM8 antagonist, PF-05105679 [(R)-3-[(1-(4-fluorophenyl)ethyl)(quinolin-3-ylcarbonyl)amino]methylbenzoic acid]. It demonstrated activity in vivo in the guinea pig bladder ice water and menthol challenge tests with an IC50 of 200 nM and reduced core body temperature in the rat (at concentrations >1219 nM). PF-05105679 was suitable for acute administration to humans and was evaluated for effects on core body temperature and experimentally induced cold pain, using the cold pressor test. Unbound plasma concentrations greater than the IC50 were achieved with 600- and 900-mg doses. The compound displayed a significant inhibition of pain in the cold pressor test, with efficacy equivalent to oxycodone (20 mg) at 1.5 hours postdose. No effect on core body temperature was observed. An unexpected adverse event (hot feeling) was reported, predominantly periorally, in 23 and 36% of volunteers (600- and 900-mg dose, respectively), which in two volunteers was nontolerable. In conclusion, this study supports a role for TRPM8 in acute cold pain signaling at doses that do not cause hypothermia.
Asunto(s)
Dolor/metabolismo , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/metabolismo , Animales , Temperatura Corporal/efectos de los fármacos , Frío , Estudios Cruzados , Método Doble Ciego , Cobayas , Células HEK293 , Humanos , Masculino , Moduladores del Transporte de Membrana/farmacología , Oxicodona/farmacología , Dolor/tratamiento farmacológico , Ratas , Ratas WistarRESUMEN
INTRODUCTION: In patients with detrusor hyperreflexia, intravesical instillation of ice-cold water results in the development of involuntary bladder contractions at volumes less than normal cystometric capacity. This is referred to as a positive ice-water test (+IWT) and can be reversed by vanilloid receptor agonists and potentiated by menthol. The present study was designed to investigate the existence of an analogous cooling reflex in the guinea-pig bladder that could be used as a small animal model in order to test the effects of drugs on the reflex. METHODS: Bladder pressure and external urethral sphincter electromyogram (EUS EMG) were recorded in alpha-chloralose/urethane anaesthetised guinea-pigs during rapid infusion of cold or warm saline into the bladder with or without prior intravesical exposure to menthol or resiniferatoxin (RTX). RESULTS: The mean control micturition threshold volume (TV) of 2.58 ml at 38 degrees C was reduced to 1.52 ml in response to saline infusion at 3 degrees C (P=0.001). The cold-induced reduction in TV was reproducible during several subsequent repeat infusions at 38 degrees C and 3 degrees C and was accompanied by decreases in bladder voiding pressure. The duration of the micturition reflex was markedly increased following cold compared with warm saline infusion (mean 24.5 s at 3 degrees C, 10.2 s at 38 degrees C, P=0.001) and was associated with oscillations in bladder pressure and concomitant bursting activity in the EUS EMG. During step-wise decreases in infusate temperature from 38 degrees C to 23 degrees C, 15 degrees C, 7 degrees C and 3 degrees C, the threshold infusate temperature to elicit a significant reduction in TV was 15 degrees C. The reduction in TV at 3 degrees C was potentiated by intravesical infusion of 0.6 mM menthol whilst intravesical infusion of 500 nM RTX reversed the reduction in TV at 3 degrees C. DISCUSSION: These data suggest that a bladder-cooling reflex is present in the anaesthetised guinea-pig and represents a useful small animal model of the clinical +IWT.
Asunto(s)
Anestesia , Modelos Animales de Enfermedad , Contracción Muscular/fisiología , Reflejo/fisiología , Vejiga Urinaria/fisiología , Agua , Animales , Frío , Diterpenos/farmacología , Electromiografía/métodos , Femenino , Cobayas , Mentol/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Cloruro de Sodio/administración & dosificación , Cloruro de Sodio/farmacología , Temperatura , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/inervación , Micción/efectos de los fármacos , Micción/fisiologíaRESUMEN
Patients with overactive bladder often exhibit abnormal bladder contractions in response to intravesical cold saline (positive ice-water test). The molecular entity involved in cold sensation within the urinary bladder is unknown, but a potential candidate is the ion channel, transient receptor potential (melastatin)-8 (TRPM8). The objective of the present study was to investigate the role of TRPM8 in a bladder-cooling reflex evoked in anaesthetised guinea-pigs that is comparable to the positive ice-water test seen in patients. Guinea-pig TRPM8 was cloned from L6 dorsal root ganglia (DRG) and expressed in HEK293 cells. Functional agonist- and cold-induced Ca2+ influx and electrophysiology assays were performed in these cells, and for comparison in HEK293 cells expressing human TRPM8, using a novel TRPM8 antagonist, the S-enantiomer of 1-phenylethyl 4-(benzyloxy)-3-methoxybenzyl (2-aminoethyl) carbamate hydrochloride (PBMC). Potency data from these assays was used to calculate intravenous infusion protocols for targeted plasma concentrations of PBMC in studies on micturition reflexes evoked by intravesical infusion of menthol or cold saline in anaesthetised guinea-pigs. Tissue expression of TRPM8 in guinea-pig bladder, urethra and in dorsal root ganglia neurones traced from the bladder was also investigated. TRPM8 mRNA and protein were detected in L6 dorsal root ganglia, bladder urothelium and smooth muscle. PBMC antagonised in vitro activation of human and guinea-pig TRPM8 and reversed menthol and cold-induced facilitation of the micturition reflex at plasma concentrations consistent with in vitro potencies. The present data suggest that the bladder-cooling reflex in the guinea-pig involves TRPM8. The potential significance of TRPM8 in bladder disease states deserves future investigation.