Christopher Flores

TRPV2 Is Activated by Cannabidiol and Mediates CGRP Release in Cultured Rat Dorsal Root Ganglion Neurons

Transient receptor potential V2 (TRPV2) has been proposed to be a high-threshold thermosensor. However, further elucidation of the channel properties and physiological role of TRPV2 have been hindered by the lack of selective pharmacological tools as well as by the species-dependent differences in the activation of this channel. In the present study, we have used cell-based calcium mobilization and electrophysiological assays to identify and characterize several novel cannabinoid TRPV2 agonists. Among these, cannabidiol was found to be the most robust and potent (EC50 = 3.7 μm), followed by Δ9-tetrahydrocannabinol (EC50 = 14 μm) and cannabinol (EC50 = 77.7 μm). We also demonstrated that cannabidiol evoked a concentration-dependent release of calcitonin gene-related peptide (CGRP) from cultured rat dorsal root ganglion neurons in a cannabinoid receptor- and TRPV1-independent manner. Moreover, the cannabidiol-evoked CGRP release depended on extracellular calcium and was blocked by the nonselective TRP channel blocker, ruthenium red. We further provide evidence through the use of small interfering RNA knockdown and repetitive stimulation studies, to show that cannabidiol-evoked CGRP release is mediated, at least in part, by TRPV2. Together, these data suggest not only that TRPV2 may comprise a mechanism whereby cannabidiol exerts its clinically beneficial effects in vivo, but also that TRPV2 may constitute a viable, new drug target.

Peripheral CGRP release as a marker for neurogenic inflammation: a model system for the study of neuropeptide secretion in rat paw skin

&NA; The local release of pro‐inflammatory neuropeptides in the periphery has been associated with the development of neurogenic inflammation. However, there is an increasing number of reports demonstrating tissue‐dependent differences regarding the mechanisms engaged by these neuropeptides to initiate and maintain the inflammatory response in the target tissue. Since skin is often involved in tissue injury, the present studies were designed to develop a model for assessing cutaneous peptide secretion as a marker for neurogenic inflammation in skin tissue. Calcitonin gene‐related peptide (CGRP), as one of several neuropeptides known to be involved in neurogenic inflammation, was chosen to study capsaicin‐induced effects on peripheral neurosecretion. The corial surface of the hairy skin of a rat hindlimb was superfused in vitro, and the basal and capsaicin‐evoked peripheral release of immunoreactive CGRP (iCGRP) was measured using a radioimmunoassay. The main objectives of these studies were to characterize the various properties of this release including dose‐dependency, exocytosis and receptor‐mediation as well as the effects of acute and long‐term capsaicin desensitization. Capsaicin significantly and dose‐dependently increased the release of iCGRP at concentrations ranging from 3 to 300 &mgr;M. Omission of calcium ions or treatment with the competitive capsaicin receptor antagonist capsazepine completely inhibited the capsaicin‐induced iCGRP release. Superfusion of the skin with 100 &mgr;M capsaicin following a conditioning stimulation with capsaicin at concentrations ranging from 0.3 to 100 &mgr;M led to an acute, dose‐dependent desensitization of the CGRP response. In addition, chronic desensitization following the neonatal injection of capsaicin completely abolished the acute iCGRP response to capsaicin. The method described here should prove to be a valuable tool for the evaluation of the processes regulating the peripheral, cutaneous release of pro‐inflammatory neuropeptides. This strategy, therefore, may lead to a better understanding of the mechanisms involved in the development and maintenance of neurogenic inflammation, particularly in the skin.

Capsaicin-evoked CGRP release from rat buccal mucosa: development of a model system for studying trigeminal mechanisms of neurogenic inflammation: CGRP neurosecretion in the trigeminal system

Many of the physiological hallmarks associated with neurogenic inflammatory processes in cutaneous tissues are similarly present within orofacial structures. Such attributes include the dependence upon capsaicin‐sensitive sensory neurons and the involvement of certain inflammatory mediators derived therein, including calcitonin gene‐related peptide (CGRP). However, there are also important differences between the trigeminal and spinal nervous systems, and the potential contributions of neurogenic processes to inflammatory disease within the trigeminal system have yet to be fully elucidated. We present here a model system that affords the ability to study mechanisms regulating the efferent functions of peptidergic terminals that may subserve neurogenic inflammation within the oral cavity. Freshly dissected buccal mucosa tissue from adult, male, Sprague–Dawley rats was placed into chambers and superfused with oxygenated, Krebs buffer. Serial aliquots of the egressing superfusate were acquired and analysed by radioimmunoassay for immunoreactive CGRP (iCGRP). Addition of the selective excitotoxin, capsaicin (10–300 µm), to the superfusion buffer resulted in a significant, concentration‐dependent increase in superfusate levels of iCGRP. Similarly, release of iCGRP from the buccal mucosa could also be evoked by a depolarizing concentration of potassium chloride (50 mm) or by the calcium ionophore A23187 (1 µm). The specific, capsaicin receptor antagonist, capsazepine (300 µm), completely abolished the capsaicin‐evoked release of iCGRP while having no effect whatsoever on the potassium‐evoked release. Moreover, capsaicin‐evoked release was dependent upon the presence of extracellular calcium ions and was significantly, though incompletely, attenuated by neonatal capsaicin denervation. Collectively, these data indicate that the evoked neurosecretion of iCGRP in response to capsaicin occurs via a vanilloid receptor‐mediated, exocytotic mechanism. The model system described here should greatly facilitate future investigations designed to identify and characterize the stimuli that regulate the release of CGRP or other neurosecretory substances in isolated tissues. This system may also be used to elucidate the role of these mediators in the aetiology of inflammatory processes within the trigeminal field of innervation.

Dynamic Mass Redistribution as a Means to Measure and Differentiate Signaling via Opioid and Cannabinoid Receptors

Classically, G protein-coupled receptor activation by a ligand has been viewed as producing a defined response such as activation of a G protein, activation or inhibition of adenylyl cyclase, or stimulation of phospholipase C and/or alteration in calcium flux. Newer concepts of ligand-directed signaling recognize that different ligands, ostensibly acting at the same receptors, may induce different downstream effects, complicating the selection of a screening assay. Dynamic mass redistribution (DMR), a label-free technology that uses light to measure ligand-induced changes in the mass of cells proximate to the biosensor, provides an integrated cellular response comprising multiple pathways and cellular events. Using DMR, signals induced by opioid or cannabinoid agonists in cells transfected with these receptors were blocked by pharmacologically appropriate receptor antagonists as well as by pertussis toxin. Differences among compounds in relative potencies at DMR versus ligand-stimulated GTPγS or receptor binding endpoints, suggesting functional selectivity, were observed. Preliminary evidence indicates that inhibitors of intermediate steps in the cell signaling cascade, such as receptor recycling inhibitors, mitogen-activated protein kinase kinase/p38 mitogen-activated protein kinase inhibitors, or cytoskeletal disruptors, altered or attenuated the cannabinoid-induced response. Notable is the finding that mitogen-activated protein kinase kinase 1/2 inhibitors attenuated signaling induced by the cannabinoid type 2 receptor inverse agonist AM630 but not that stimulated by the agonist CP 55,940. Thus, DMR has the potential to not only identify ligands that activate a given G protein-coupled receptor, but also ascertain the signaling pathways engaged by a specific ligand, making DMR a useful tool in the identification of biased ligands, which may ultimately exhibit improved therapeutic profiles.

Benzo[d]imidazole Transient Receptor Potential Vanilloid 1 Antagonists for the Treatment of Pain: Discovery of trans-2-(2-{2-[2-(4-Trifluoromethyl-phenyl)-vinyl]-1H-benzimidazol-5-yl}-phenyl)-propan-2-ol (Mavatrep)

Reported herein is the design, synthesis, and pharmacologic characterization of a class of TRPV1 antagonists constructed on a benzo[d]imidazole platform that evolved from a biaryl amide lead. This design composes three sections: a 2-substituted 5-phenyl headgroup attached to the benzo[d]imidazole platform, which is tethered at the two position to a phenyl tail group. Optimization of this design led to the identification of 4 (mavatrep), comprising a trifluoromethyl-phenyl-vinyl tail. In a TRPV1 functional assay, using cells expressing recombinant human TRPV1 channels, 4 antagonized capsaicin-induced Ca(2+) influx, with an IC50 value of 4.6 nM. In the complete Freunds adjuvant- and carrageenan-induced thermal hypersensitivity models, 4 exhibited full efficacy, with ED80 values of 7.8 and 0.5 mg/kg, respectively, corresponding to plasma levels of 270.8 and 9.2 ng/mL, respectively. On the basis of its superior pharmacologic and safety profile, 4 (mavatrep) was selected for clinical development for the treatment of pain.

Development and Validation of a Secondary Screening Assay for TRPM8 Antagonists Using QPatch HT

QPatch HT is an automated patch clamp system with high data quality/content and greatly increased throughput over conventional patch clamp methods. To determine whether this platform is suitable for secondary screening of antagonists of TRPM8, a cold- and menthol-activated ion channel that belongs to the transient receptor potential channel family, we used QPatch HT to test a set of chemically diverse compounds identified as TRPM8 antagonists by FLIPR and conventional patch clamp. We found that most compounds exhibited slower inhibition kinetics compared with conventional patch clamp, requiring multiple applications to reach steady-state inhibition. For most compounds, there was a relatively small (< or =4-fold) right shift in potency compared with conventional patch clamp. Nonetheless, the compound potencies obtained from QPatch HT exhibited a highly significant correlation with those from either conventional patch clamp (r(2) = 0.98) or FLIPR (r(2) = 0.97), over a wide range of concentrations and cLogP values (approximately 4 orders of magnitude) and with virtually identical rank-order potency. The throughput by QPatch HT was at least 10-fold higher than that obtained by conventional patch clamp. Our results validate the use of QPatch HT for secondary screening of TRPM8 antagonists and, along with other recent studies, illustrate its utility as an important tool for ion channel drug discovery.

TRPV1 antagonist JNJ-39439335 (mavatrep) demonstrates proof of pharmacology in healthy men: a first-in-human, double-blind, placebo-controlled, randomized, sequential group study

Abstract This double-blind, randomized, placebo-controlled, sequential group, phase 1 study was designed to assess in healthy men, the safety, tolerability, pharmacokinetics, and translational pharmacodynamics of JNJ-39439335 (mavatrep), a transient receptor potential vanilloid subtype 1 antagonist; it was preceded by a translational preclinical study which assessed the ability of JNJ-39439335 to block capsaicin-induced flare in rats, providing predictive pharmacokinetic and pharmacodynamic data that informed the subsequent phase 1 clinical study. The clinical study consisted of 2 parts: part 1 assessed pharmacokinetics and pharmacodynamics, including heat pain detection threshold and heat pain tolerance, of JNJ-39439335, and part 2 assessed pharmacodynamic effect of JNJ-39439335 on capsaicin-induced flare and sensory testing on naïve and UVB-sensitized skin in humans. Plasma concentrations of JNJ-39439335 peaked at approximately 2 to 4 hours postdose, then declined multiexponentially, with a prolonged terminal phase (half-life: 30–86 hours). Renal clearance of JNJ-39439335 was negligible. JNJ-39439335 treatment resulted in clear, consistent dose-related increases in heat pain detection threshold, heat pain tolerance, and heat pain latency. JNJ-39439335 reduced the capsaicin-induced flare area and flare intensity, with complete blocking observed in the 50-mg dose group at 144 hours postdose. This was consistent with the capsaicin flare results observed with JNJ-39439335 in rats. The most common adverse events observed in the clinical study were related to increases in body temperature after JNJ-39439335 treatment; these were predominately mild to moderate in severity with no evidence of exposure dependence up to 225 mg. JNJ-39439335 was well tolerated at single doses up to 225 mg, recommending its suitability for further clinical development.

Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.