Functional α6ß4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

The α6ß4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6ß4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6ß4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6ß4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6ß4 receptor assembly. Effects on α6ß4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6ß4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6ß4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6ß4 receptor.

Selective inhibition of peripheral cathepsin S reverses tactile allodynia following peripheral nerve injury in mouse.

Cathepsin S (CatS) is a cysteine protease found in lysosomes of hematopoietic and microglial cells and in secreted form in the extracellular space. While CatS has been shown to contribute significantly to neuropathic pain, the precise mechanisms remain unclear. In this report, we describe JNJ-39641160, a novel non-covalent, potent, selective and orally-available CatS inhibitor that is peripherally restricted (non-CNS penetrant) and may represent an innovative class of immunosuppressive and analgesic compounds and tools useful toward investigating peripheral mechanisms of CatS in neuropathic pain. In C57BL/6 mice, JNJ-39641160 dose-dependently blocked the proteolysis of the invariant chain, and inhibited both T-cell activation and antibody production to a vaccine antigen. In the spared nerve injury (SNI) model of chronic neuropathic pain, in which T-cell activation has previously been demonstrated to be a prerequisite for the development of pain hypersensitivity, JNJ-39641160 fully reversed tactile allodynia in wild-type mice but was completely ineffective in the same model in CatS knockout mice (which exhibited a delayed onset in allodynia). By contrast, in the acute mild thermal injury (MTI) model, JNJ-39641160 only weakly attenuated allodynia at the highest dose tested. These findings support the hypothesis that blockade of peripheral CatS alone is sufficient to fully reverse allodynia following peripheral nerve injury and suggest that the mechanism of action likely involves interruption of T-cell activation and peripheral cytokine release. In addition, they provide important insights toward the development of selective CatS inhibitors for the treatment of neuropathic pain in humans.

The discovery and preclinical characterization of 6-chloro-N-(2-(4,4-difluoropiperidin-1-yl)-2-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)quinoline-5-carboxamide based P2X7 antagonists.

The synthesis, SAR and preclinical characterization of a series of 6-chloro-N-(2-(4,4-difluoropiperidin-1-yl)-2-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)quinoline-5-carboxamide based P2X7 antagonists is described herein. The lead compounds are potent inhibitors in Ca(2+) flux and whole blood IL-1ß P2X7 release assays at both human and mouse isoforms. Compound 1e showed a robust reduction of IL-1ß release in a mouse ex vivo model with a 50mg/kg oral dose. Evaluation of compound 1e in the mouse SNI tactile allodynia, carrageenan-induced paw edema or CIA models resulted in no analgesic or anti-inflammatory effects.

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-µM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain.

Discovery of a novel series of selective HCN1 blockers.

The discovery of a series of novel, potent, and selective blockers of the cyclic nucleotide-modulated channel HCN1 is disclosed. Here we report an SAR study around a series of selective blockers of the HCN1 channel. Utilization of a high-throughput VIPR assay led to the identification of a novel series of 2,2-disubstituted indane derivatives, which had moderate selectivity and potency at HCN1. Optimization of this hit led to the identification of the potent, 1,1-disubstituted cyclohexane HCN1 blocker, 2-ethoxy-N-((1-(4-isopropylpiperazin-1-yl)cyclohexyl)methyl)benzamide. The work leading to the discovery of this compound is described herein.

Characterization of 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl]-(4-trifluoromethyl-phenyl)-amine (JNJ-39729209) as a novel TRPV1 antagonist.

As an integrator of multiple nociceptive and/or inflammatory stimuli, TRPV1 is an attractive therapeutic target for the treatment of various painful disorders. Several TRPV1 antagonists have been advanced into clinical trials and the initial observations suggest that TRPV1 antagonism may be associated with mild hyperthermia and thermal insensitivity in man. However, no clinical efficacy studies have been described to date, making an assessment of risk:benefit impossible. Furthermore, it is not clear whether these early observations are representative of all TRPV1 antagonists and whether additional clinical studies with novel TRPV1 antagonists are required in order to understand optimal compound characteristics. In the present study we describe 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl]-(4-trifluoromethyl-phenyl)-amine (JNJ-39729309) as a novel, TRPV1 antagonist. JNJ-39729209 displaced tritiated resiniferotoxin binding to TRPV1 and prevented TRPV1 activation by capsaicin, protons and heat. In-vivo, JNJ-39729209 blocked capsaicin-induced hypotension, induced a mild hyperthermia and inhibited capsaicin-induced hypothermia in a dose dependent manner. JNJ-39729209 showed significant efficacy against carrageenan- and CFA-evoked thermal hyperalgesia and exhibited significant anti-tussive activity in a guinea-pig model of capsaicin-induced cough. In pharmacokinetic studies, JNJ-39729209 was found to have low clearance, a moderate volume of distribution, good oral bioavailability and was brain penetrant. On the basis of these findings, JNJ-39729209 represents a structurally novel TRPV1 antagonist with potential for clinical development. The advancement of JNJ-39729209 into human clinical trials could be useful in further understanding the analgesic potential of TRPV1 antagonists.

Rodent models of persistent pain in drug discovery and development.

The increasing debate regarding the predictiveness of rodent persistent pain models for clinical efficacy has spurred rapidly evolving numbers and types of novel models from which to choose. While several excellent reviews of these models have been published in recent years, few focus on their specific applications and particular challenges with the use of these models in the setting of drug discovery. Thus, in this review, how models of persistent pain may be used to: 1) screen molecules for in vivo efficacy, 2) advance lead compounds and 3) guide decision making for clinical trial design is discussed. Relative to other disease areas for potential drug discovery and development, chronic pain appears to be well-poised for drug discovery and development. This is in large part due to the advanced understanding of pain mechanisms and the upsurge in the development of novel, specialized rodent models of persistent pain and improvements in methods of pain assessment in animals.

Recent advances in the biology and medicinal chemistry of TRPA1.

The transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is a nonselective cation channel that is highly expressed in small-diameter sensory neurons, where it functions as a polymodal receptor, responsible for detecting potentially harmful chemicals, mechanical forces and temperatures. TRPA1 is also activated and/or sensitized by multiple endogenous inflammatory mediators. As such, TRPA1 likely mediates the pain and neurogenic inflammation caused by exposure to reactive chemicals. In addition, it is also possible that this channel may mediate some of the symptoms of chronic inflammatory conditions such as asthma. We review recent advances in the biology of TRPA1 and summarize the evidence for TRPA1 as a therapeutic drug target. In addition, we provide an update on TRPA1 medicinal chemistry and the progress in the search for novel TRPA1 antagonists.

Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis.

Mouse genetics has contributed significantly to our understanding of molecular mechanisms underlying tissue and nerve injury-induced persistent pain. To create a highly reproducible, relatively noninvasive model of neuropathic pain in the mouse, we examined the behavioral consequences of sparing each of the 3 distal branches of the sciatic nerve in wild-type mice after a model originally described in the rat. Sparing the tibial branch but sparing neither of the other branches produced robust mechanical allodynia while leaving heat sensibility intact. To assess the topographic organization of the IB4 population of afferents from each branch and to compare anatomic consistency across injury models, we examined loss of thiamine monophosphatase staining in the superficial dorsal horn after peripheral nerve injury. We found that each of the sciatic branches targets a distinct mediolateral location in inner lamina II and that each of the spared nerve injury models produced a more reproducible pattern of thiamine monophosphatase staining loss than did partial tight ligation. These results improve on previous nerve injury models in mouse, demonstrate similar behavioral changes as in rat, and provide novel information on the topographic organization of small diameter peripheral afferents in the mouse spinal cord.

Morphology and axonal arborization of rat spinal inner lamina II neurons hyperpolarized by mu-opioid-selective agonists.

The ventral or inner region of spinal substantia gelatinosa (SG; lamina II(i)) is a heterogeneous sublamina important for the generation and maintenance of hyperalgesia and neuropathic pain. To test whether II(i) neurons can be hyperpolarized by the mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO; 500 nM) and to address possible downstream consequences of mu-opioid-evoked inhibition of II(i) neurons, we combined in vitro whole-cell, tight-seal recording methods with fluorescent labeling of the intracellular tracer biocytin and confocal microscopy. Twenty-one of 23 neurons studied had identifiable axons. Nine possessed axons that projected ventrally into laminae III-V; six of these were hyperpolarized by DAMGO. Three of four neurons with identifiable axons that projected to lamina I were hyperpolarized by DAMGO. Most neurons could be classified as either islet cells or stalked cells. Five of nine labeled islet cells and only two of seven stalked cells were hyperpolarized by DAMGO. Three were stellate cells: one resembled a spiny cell and three could not be classified. DAMGO hyperpolarized each of the stellate cells, the spiny cell, and 1 of the unclassified cells. Our data support the hypothesis that part of the action of mu-opioid agonists involves the inhibition of interneurons that are part of a polysynaptic excitatory pathway from primary afferents to neurons in the deep and/or superficial dorsal horn.

Hyperpolarization of substantia gelatinosa neurons evoked by mu-, kappa-, delta 1-, and delta 2-selective opioids.

With whole-cell recordings of substantia gelatinosa (SG) neurons from rat spinal cord slices, we investigated the effects of bath application of highly selective delta(1), delta(2), kappa and mu opioid agonists on membrane potential and conductance. Each agonist was applied at 0.5 to 1 micromol/L and evoked robust hyperpolarizations and conductance increases in a subset of neurons. The response magnitude means were similar across agonists at several concentrations; no excitatory effects were observed. Nine of 55 (16%) were hyperpolarized by delta(1) opioids, 2 of 45 (4%) by delta(2), 8 of 59 (14%) by kappa, and 35 of 67 (52%) by mu opioids. To test the hypothesis that SG neurons may be hyperpolarized by multiple opioid subtype agonists, we applied 2, 3, or 4 selective agonists to individual neurons. Most neurons were hyperpolarized only by mu opioids; however, a minority were hyperpolarized by multiple subtype-selective agonists. These results indicate that delta(1)- and delta(2)-selective opioids can also evoke robust hyperpolarizations in spinal SG neurons, that the relative abundance of hyperpolarizing responses was mu > > delta (1) approximately equal kappa > delta(2), and that some SG neurons can be hyperpolarized by more than 1 opioid subtype-selective agonist. These powerful inhibitory postsynaptic responses likely contribute to analgesia evoked by spinally and systemically administered opioid subtype-selective agonists.