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 α6β4 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 α6β4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6β4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6β4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6β4 receptor assembly. Effects on α6β4 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 α6β4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6β4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6β4 receptor.

Innate lymphoid cell (ILC) cytokine signatures mirror those of T helper subsets, but ILCs differ substantially as far as biological role. Tissue-resident and antigen-independent, ILCs mediate induction and resolution of inflammation, tissue homeostasis, and barrier maintenance. Although extensively character-ized in periphery, ILCs were newly identified as resident in central nervous system (CNS) meninges, and are less well-understood within this novel context. Here we show that ILC-deficient mice exhibit enhanced microglial reactivity, amplified neuroinflammatory responses and blood-brain barrier (BBB) permeability. Accordingly, transcriptomic and functional examination revealed meningeal ILC2s as vigorous interleukin (IL)-10 producers. Mechanistic relevance was demonstrated by amelioration of neuroinflammation following meningeal engraftment of adoptively-transferred wild type — but not IL-10-deficient — ILC2s. Notably, ILC2s from murine bone marrow and human blood also showed IL-10 competency, suggesting a previously-unappreciated immunoregulatory role for canonical ILC2s. Collectively, these findings reveal meningeal ILC2s as suppressors of neuroinflammation — suggesting potential for ILC2-based cell therapies in CNS pathology.

Toreforant is a potent and selective histamine H4 receptor (H4R) antagonist with a Ki at the human receptor of 8.4 ± 2.2 nM and excellent selectivity over other receptors including the other histamine receptors. The compound acts as an antagonist in all species tested and inhibits histamine-induced eosinophil shape change in vitro. Toreforant was anti-inflammatory in mouse models of asthma and arthritis. However, it was not able to inhibit histamine-induced scratching in mice or block neuropathic pain in rats. The lack of effect in these models may be related to low exposure levels in the central nervous system. Preclinical toxicity studies of up to 6 months in rats and 9 months in monkeys indicated an excellent safety profile with the exception of QT prolongation seen in vivo due to inhibition of the human ether-à-go-go-related gene (hERG) channel. Toreforant was studied in phase 1 human clinical studies to assess safety, pharmacokinetics and pharmacodynamics. The compound was well-tolerated at all doses tested and no safety issues were noted in the phase 1 studies with the exception of QT prolongation observed at the highest dose. Toreforant exhibited good pharmacokinetics upon oral dosing with a plasma half-life consistent with once a day dosing. In addition, dose-dependent inhibition of histamine-induced eosinophil shape change was detected suggesting that the H4R was inhibited in vivo.

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-1β P2X7 release assays at both human and mouse isoforms. Compound 1e showed a robust reduction of IL-1β 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.

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.

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.

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.

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. Keywords: Animal models, behavioral assays, behavioral pharmacology, drug discovery, in vivo efficacy models, pain models, predictive validity, translational research, rodent persistent pain models, clinical efficacy, persistent pain, upsurge, nerve injury, chronic inflammation

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.

Purinergic receptor subtype 7 (P2X7) is expressed on immune/glial cells and is hypothesized to modulate both inflammatory and neuropathic pain as seen by genetic disruption and pharmacological intervention. In this study, we report the characterization of a novel P2X7 receptor antagonist discovered by GlaxoSmithKline (compound 16; Chambers et al., 2009; RSC‐SCI Medicinal Chemistry Symposium, Cambridge, UK). The compound blocked human, rat and mouse recombinant P2X7 receptors with mean pIC50 value of 6.8, 5.3 and 5.8 in FLIPR assay of intracellular calcium mobilization by Bz‐ATP. Compound 16 also attenuated Bz‐ATP dependent IL‐1β release in human and mouse whole blood with mean pIC50 value of 6.2 and 5.5, respectively. The block induced by compound 16 was surmountable in human whole blood and it displaced [3H]‐A804598 in membrane preparations from human P2X7‐1321N1 cells. In whole‐cell patch clamp assay, 1 μM of the compound blocked 300 μM Bz‐ATP‐induced inward current in a reversible manner. The compound was orally bioavailable when dosed in mice and produced a dose dependent inhibition of IL‐1β at 2 hours post oral dose in a blood ex‐vivo assay (ED50 ~ 1–3 mg/kg). At 15 mg/kg (oral) the compound also reversed mechanical allodynia in a mouse model of neuropathic pain (SNI). In addition, the compound also reversed CFA‐induced spontaneous inflammatory pain (weight bearing) in rats at 10 mg/kg tested at 1 and 2 hours post oral dose. Compound 16 is an efficacious P2X7 antagonist that maybe used as a tool to better understand the role of P2X7 in animal models of pain.

Our previous recordings from dorsal root ganglion and spinal lamina V neurons from TRPV1-mutant mice showed dramatic decreases in responses to temperatures near the activation threshold of this channel (43–49°C). Somewhat unexpectedly, we only observed behavioral deficits in these mice at higher temperatures (50–58°C). In the present study, we tested the hypothesis that the noxious heat-evoked pain behavior that persists in TRPV1-mutant mice reflects residual responsiveness of neurons in the superficial, but not deep, dorsal horn. To this end, we performed in vivo extracellular recordings of spinal nociresponsive neurons in laminae I and V in wild type (WT) and TRPV1 mutant mice. Neurons in WT and mutant mice from both laminae did not differ in their spontaneous activity or evoked responses to mechanical or cold stimuli. By contrast, most lamina I neurons from mutant mice responded to noxious heat with significantly higher thresholds than in WT mice. In contrast, lamina V neurons from mutant mice were virtually unresponsive to noxious heat before and after topical mustard oil-induced tissue injury. Interestingly, lamina I neurons in mutant mice displayed thermal sensitization following tissue injury, comparable in magnitude, but of shorter duration, than in WT mice. We conclude that TRPV1 is necessary for noxious heat-evoked responses of lamina V neurons, both before and after tissue injury. It is also an essential contributor to the normal activation threshold of lamina I neurons to noxious heat and for the full duration of thermal sensitization of lamina I neurons following injury. Finally, our results suggest that the processing of noxious thermal messages by neurons in lamina I involves convergent inputs from a heterogeneous population of primary afferent thermal nociceptors.

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 μ‐opioid agonist [D‐Ala 2 , N‐Me‐Phe 4 , Gly 5 ‐ol]‐enkephalin (DAMGO; 500 nM) and to address possible downstream consequences of μ‐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 μ‐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. J. Comp. Neurol. 458:240–256, 2003. © 2003 Wiley‐Liss, Inc.