Shailen K. Joshi

A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat.

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Nav1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC50 = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Nav1.8 (IC50 = 8 nM) and was >100-fold selective vs. human Nav1.2, Nav1.3, Nav1.5, and Nav1.7 (IC50 values ≥1 μM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED50 = 47 mg/kg, i.p.), sciatic nerve injury (ED50 = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED50 ≈ 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freunds adjuvant injection (ED50 = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Nav1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation

&NA; Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A‐967079 potently blocks human (IC50: 51 nmol/L, electrophysiology, 67 nmol/L, Ca2+ assay) and rat TRPA1 (IC50: 101 nmol/L, electrophysiology, 289 nmol/L, Ca2+ assay). It is >1000‐fold selective over other TRP channels, and is >150‐fold selective over 75 other ion channels, enzymes, and G‐protein‐coupled receptors. Oral dosing of A‐967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate‐induced nocifensive response and osteoarthritic pain in rats (ED50: 23.2 mg/kg, p.o.). A‐967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A‐967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects. Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation.

Involvement of the TTX-resistant sodium channel Nav 1.8 in inflammatory and neuropathic, but not post-operative, pain states

Abstract Antisense (AS) oligodeoxynucleotides (ODNs) targeting the Nav 1.8 sodium channel have been reported to decrease inflammatory hyperalgesia and L5/L6 spinal nerve ligation‐induced mechanical allodynia in rats. The present studies were conducted to further characterize Nav 1.8 AS antinociceptive profile in rats to better understand the role of Nav 1.8 in different pain states. Consistent with earlier reports, chronic intrathecal Nav 1.8 AS, but not mismatch (MM), ODN decreased TTX‐resistant sodium current density (by 60.5 ± 10.2% relative to MM; p < 0.05) in neurons from L4 to L5 dorsal root ganglia and significantly attenuated mechanical allodynia following intraplantar complete Freund’s adjuvant. In addition, 10 days following chronic constriction injury of the sciatic nerve, Nav 1.8 AS, but not MM, ODN also attenuated mechanical allodynia (54.3 ± 8.2% effect, p < 0.05 vs. MM) 2 days after initiation of ODN treatment. The anti‐allodynic effects remained for the duration of the AS treatment, and CCI rats returned to an allodynic state 4 days after discontinuing AS. In contrast, Nav 1.8 AS ODN failed to reduce mechanical allodynia in the vincristine chemotherapy‐induced neuropathic pain model or a skin‐incision model of post‐operative pain. Finally, Nav 1.8 AS, but not MM, ODN treatment produced a small but significant attenuation of acute noxious mechanical sensitivity in naïve animals (17.6 ± 6.2% effect, p < 0.05 vs. MM). These data demonstrate a greater involvement of Nav 1.8 in frank nerve injury and inflammatory pain as compared to acute, post‐operative or chemotherapy‐induced neuropathic pain states.

Repeated dosing of ABT-102, a potent and selective TRPV1 antagonist, enhances TRPV1-mediated analgesic activity in rodents, but attenuates antagonist-induced hyperthermia

ABSTRACT Transient receptor potential vanilloid type 1 (TRPV1) is a ligand‐gated ion channel that functions as an integrator of multiple pain stimuli including heat, acid, capsaicin and a variety of putative endogenous lipid ligands. TRPV1 antagonists have been shown to decrease inflammatory pain in animal models and to produce limited hyperthermia at analgesic doses. Here, we report that ABT‐102, which is a potent and selective TRPV1 antagonist, is effective in blocking nociception in rodent models of inflammatory, post‐operative, osteoarthritic, and bone cancer pain. ABT‐102 decreased both spontaneous pain behaviors and those evoked by thermal and mechanical stimuli in these models. Moreover, we have found that repeated administration of ABT‐102 for 5–12 days increased its analgesic activity in models of post‐operative, osteoarthritic, and bone cancer pain without an associated accumulation of ABT‐102 concentration in plasma or brain. Similar effects were also observed with a structurally distinct TRPV1 antagonist, A‐993610. Although a single dose of ABT‐102 produced a self‐limiting increase in core body temperature that remained in the normal range, the hyperthermic effects of ABT‐102 effectively tolerated following twice‐daily dosing for 2 days. Therefore, the present data demonstrate that, following repeated administration, the analgesic activity of TRPV1 receptor antagonists is enhanced, while the associated hyperthermic effects are attenuated. The analgesic efficacy of ABT‐102 supports its advancement into clinical studies.

Discovery and biological evaluation of 5-aryl-2-furfuramides, potent and selective blockers of the Nav1.8 sodium channel with efficacy in models of neuropathic and inflammatory pain.

Nav1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons and has been implicated in the pathophysiology of inflammatory and neuropathic pain. Recent studies using an Nav1.8 antisense oligonucleotide in an animal model of chronic pain indicated that selective blockade of Nav1.8 was analgesic and could provide effective analgesia with a reduction in the adverse events associated with nonselective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 5-substituted 2-furfuramides, which are potent, voltage-dependent blockers (IC50 < 10 nM) of the human Nav1.8 channel. Selected derivatives, such as 7 and 27, also blocked TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons with comparable potency and displayed >100-fold selectivity versus human sodium (Nav1.2, Nav1.5, Nav1.7) and human ether-a-go-go (hERG) channels. Following systemic administration, compounds 7 and 27 dose-dependently reduced neuropathic and inflammatory pain in experimental rodent models.

Pharmacology of Modality-Specific Transient Receptor Potential Vanilloid-1 Antagonists That Do Not Alter Body Temperature

The transient receptor potential vanilloid-1 (TRPV1) channel is involved in the development and maintenance of pain and participates in the regulation of temperature. The channel is activated by diverse agents, including capsaicin, noxious heat (≥ 43°C), acidic pH (< 6), and endogenous lipids including N-arachidonoyl dopamine (NADA). Antagonists that block all modes of TRPV1 activation elicit hyperthermia. To identify efficacious TRPV1 antagonists that do not affect temperature antagonists representing multiple TRPV1 pharmacophores were evaluated at recombinant rat and human TRPV1 channels with Ca2+ flux assays, and two classes of antagonists were identified based on their differential ability to inhibit acid activation. Although both classes of antagonists completely blocked capsaicin- and NADA-induced activation of TRPV1, select compounds only partially inhibited activation of the channel by protons. Electrophysiology and calcitonin gene-related peptide release studies confirmed the differential pharmacology of these antagonists at native TRPV1 channels in the rat. Comparison of the in vitro pharmacological properties of these TRPV1 antagonists with their in vivo effects on core body temperature confirms and expands earlier observations that acid-sparing TRPV1 antagonists do not significantly increase core body temperature. Although both classes of compounds elicit equivalent analgesia in a rat model of knee joint pain, the acid-sparing antagonist tested is not effective in a mouse model of bone cancer pain.

A-995662 [(R)-8-(4-methyl-5-(4-(trifluoromethyl)phenyl)oxazol-2-ylamino)-1,2,3,4-tetrahydronaphthalen-2-ol], a novel, selective TRPV1 receptor antagonist, reduces spinal release of glutamate and CGRP in a rat knee joint pain model.

&NA; The TRPV1 antagonist A‐995662 demonstrates analgesic efficacy in monoiodoacetate‐induced osteoarthritic (OA) pain in rat, and repeated dosing results in increased in vivo potency and a prolonged duration of action. To identify possible mechanism(s) underlying these observations, release of neuropeptides and the neurotransmitter glutamate from isolated spinal cord was measured. In OA rats, basal release of glutamate, bradykinin and calcitonin gene‐related peptide (CGRP) was significantly elevated compared to naïve levels, whereas substance P (SP) levels were not changed. In vitro studies showed that capsaicin‐evoked TRPV1‐dependent CGRP release was 54.7 ± 7.7% higher in OA, relative to levels measured for naïve rats, suggesting that TRPV1 activity was higher under OA conditions. The efficacy of A‐995662 in OA corresponded with its ability to inhibit glutamate and CGRP release from the spinal cord. A single, fully efficacious dose of A‐995662, 100 &mgr;mol/kg, reduced spinal glutamate and CGRP release, while a single sub‐efficacious dose of A‐995662 (25 &mgr;mol/kg) was ineffective. Multiple dosing with A‐995662 increased the potency and duration of efficacy in OA rats. Changes in efficacy did not correlate with plasma concentrations of A‐995662, but were accompanied with reductions in spinal glutamate release. These findings suggest that repeated dosing of TRPV1 antagonists enhances therapeutic potency and duration of action against OA pain, at least in part, by the sustained reduction in release of glutamate and CGRP from the spinal cord.

Contributions of Central and Peripheral TRPV1 Receptors to Mechanically Evoked and Spontaneous Firing of Spinal Neurons in Inflamed Rats

TRPV1 receptors are activated and/or modulated by noxious heat, capsaicin, protons and other endogenous agents released following tissue injury. There is a growing appreciation that this molecular integrator may also have a role in mechanosensation. To further understand this role, we investigated the systemic and site-specific effects of a selective TRPV1 receptor antagonist, A-889425, on low-intensity mechanical stimulation in inflamed rats. Systemic administration of A-889425 (30 and 100 micromol/kg po) reduced mechanical allodynia in complete Freunds adjuvant (CFA)-inflamed rats. Systemic A-889425 (3 and 10 micromol/kg iv) also decreased the responses of spinal wide dynamic range (WDR) neurons to low-intensity mechanical stimulation in CFA-inflamed but not uninjured rats. This effect of A-889425 was likely mediated via multiple sites since local injection of A-889425 into the spinal cord (1-3 nmol), ipsilateral hindpaw (200 nmol), and cerebral ventricles (30-300 nmol) all attenuated WDR responses to low-intensity mechanical stimulation. In addition to an effect on mechanotransmission, systemic administration of A-889425 reduced the spontaneous firing of WDR neurons in inflamed but not uninjured rats. Spontaneous firing is elevated after injury and may reflect ongoing pain in the animal. Local injection experiments indicated that this effect of A-889425 on spontaneous firing was mainly mediated via TRPV1 receptors in the spinal cord. Thus the current data demonstrate that TRPV1 receptors have an enhanced role after an inflammatory injury, impacting both low-intensity mechanotransmission and possibly spontaneous pain. Furthermore this study delineates the differential contribution of central and peripheral TRPV1 receptors to affect spontaneous or mechanically evoked firing of WDR neurons.

TRPV1-related modulation of spinal neuronal activity and behavior in a rat model of osteoarthritic pain

The TRPV1 receptor functions as a molecular integrator, and blockade of this receptor modulates enhanced somatosensitivity across several animal models of pathological pain, including models of osteoarthritic (OA) pain. In order to further characterize the contributions of TRPV1 to OA-related pain, we investigated the systemic effects of a selective TRPV1 receptor antagonist, A-889425, on grip force behavior, and on the evoked and spontaneous firing of spinal wide dynamic range (WDR) and nociceptive specific (NS) neurons in the monoiodoacetate (MIA) model of OA. Administration of A-889425 (10-300 μmol/kg, p.o.) alleviated grip force impairment in OA rats 3 weeks after the MIA injection. Also at 3 weeks post-MIA injection, the responses of WDR and NS neurons to 300 g von Frey hair stimulation of the knee joint were significantly reduced by A-889425 administration (10 and 30 μmol/kg, i.v.) in OA, but not sham-OA rats. Spontaneous firing of WDR neurons was elevated in the OA rats compared to sham-OA rats and may reflect ongoing discomfort in the OA animal. In addition to an effect on mechanotransmission, systemic administration of A-889425 reduced the elevated spontaneous firing of WDR neurons in OA rats but did not alter spontaneous firing in sham rats. The present data demonstrate that blockade of TRPV1 receptors modulates the firing of two important classes of spinal nociceptive neurons in a rat model of OA. The effect of A-889425 on neuronal responses to intense mechanical stimulation of the knee and on the spontaneous firing of WDR neurons adds to the growing appreciation for the role of TRPV1 receptors in pathological mechanotransmission and possibly non-evoked discomfort, respectively.

Chroman and tetrahydroquinoline ureas as potent TRPV1 antagonists.

Novel chroman and tetrahydroquinoline ureas were synthesized and evaluated for their activity as TRPV1 antagonists. It was found that aryl substituents on the 7- or 8-position of both bicyclic scaffolds imparted the best in vitro potency at TRPV1. The most potent chroman ureas were assessed in chronic and acute pain models, and compounds with the ability to cross the blood-brain barrier were shown to be highly efficacious. The tetrahydroquinoline ureas were found to be potent CYP3A4 inhibitors, but replacement of bulky substituents at the nitrogen atom of the tetrahydroisoquinoline moiety with small groups such as methyl can minimize the inhibition.