A novel benzazepinone sodium channel blocker with oral efficacy in a rat model of neuropathic pain.

A series of benzazepinones were synthesized and evaluated for block of Nav1.7 sodium channels. Compound 30 from this series displayed potent channel block, good selectivity versus other targets, and dose-dependent oral efficacy in a rat model of neuropathic pain.

A potent and selective indole N-type calcium channel (Ca(v)2.2) blocker for the treatment of pain.

N-type calcium channels (Ca(v)2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca(v)2.2 blockers with good in vitro and in vivo potency.

Analgesic effects of a substituted N-triazole oxindole (TROX-1), a state-dependent, voltage-gated calcium channel 2 blocker.

Voltage-gated calcium channel (Ca(v))2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca(v)2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca(v)2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca(v)2.2 channels under depolarized conditions (IC(50) = 0.27 microM) compared with hyperpolarized conditions (IC(50) > 20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), indicating state-dependent Ca(v)2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca(v)2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca(v)2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca(v)2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca(v)2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.

Substituted biaryl pyrazoles as sodium channel blockers.

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. A series of low molecular weight biaryl substituted pyrazole carboxamides were identified with good in-vitro potency and in-vivo efficacy. Compound 26, a Nav1.7 blocker has excellent efficacy in the Chung model of neuropathic pain.

Substituted biaryl oxazoles, imidazoles, and thiazoles as sodium channel blockers.

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. With a goal to develop potent peripherally active sodium channel blockers, a series of low molecular weight biaryl substituted imidazoles, oxazoles, and thiazole carboxamides were identified with good in vitro and in vivo potency.

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain.

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.

Chemokines and pain mechanisms.

The development of new therapeutic approaches to the treatment of painful neuropathies requires a better understanding of the mechanisms that underlie the development of these chronic pain syndromes. It is now well established that astrocytic and microglial cells modulate the neuronal mechanisms of chronic pain in spinal cord and possibly in the brain. In animal models of neuropathic pain following peripheral nerve injury, several changes occur at the level of the first pain synapse between the central terminals of sensory neurons and second order neurons. These neuronal mechanisms can be modulated by pro-nociceptive mediators released by non neuronal cells such as microglia and astrocytes which become activated in the spinal cord following PNS injury. However, the signals that mediate the spread of nociceptive signaling from neurons to glial cells in the dorsal horn remain to be established. Herein we provide evidence for two emerging signaling pathways between injured sensory neurons and spinal microglia: chemotactic cytokine ligand 2 (CCL2)/CCR2 and cathepsin S/CX3CL1 (fractalkine)/CX3CR1. We discuss the plasticity of these two chemokine systems at the level of the dorsal root ganglia and spinal cord demonstrating that modulation of chemokines using selective antagonists decrease nociceptive behavior in rodent chronic pain models. Since up-regulation of chemokines and their receptors may be a mechanism that directly and/or indirectly contributes to the development and maintenance of chronic pain, these molecular molecules may represent novel targets for therapeutic intervention in sustained pain states.

Changes in mouse mu opioid receptor Exon 7/8-like immunoreactivity following food restriction and food deprivation in rats.

Opioid agonists and antagonists respectively increase and decrease food intake. That selective mu opioid antagonists are more effective than antisense probes directed against the mu opioid receptor (MOR-1) gene in reducing deprivation-induced feeding suggests a role for isoforms. Both food restriction and deprivation alter protein and mRNA levels of opioid peptides and receptors. Antisera directed against Exon 4 of the MOR-1-like immunoreactivity (LI) (Exon 4) clone or directed against mouse Exons 7/8 (mE7/8-LI) revealed high levels of immunoreactivity in brain nuclei related to feeding behavior. Therefore, the present study assessed MOR-1LI and mE7/8-LI in hypothalamic and extrahypothalamic sites in rats exposed to ad libitum feeding, food restriction (2, 7, 14 days), or food deprivation (24, 48 h). MOR-1-LI displayed robust reactivity, but was insensitive to food restriction or deprivation. mE7/8-LI, both in terms of cell counts and relative optical density, was significantly and selectively increased in the dorsal and ventral parvocellular subdivisions of the hypothalamic paraventricular nucleus in food-restricted (14 days) rats, but all other restriction or deprivation regimens were ineffective in other hypothalamic nuclei. In contrast, significant and site-specific decreases in relative optical density in the rostral part of the nucleus tractus solitarius (NTS) were observed in food-restricted (2, 7 days) or food-deprived (24, 48 h) animals, but these regimens were ineffective in the other extrahypothalamic sites. This study indicates the sensitivity of this mE7/8-LI probe in the hypothalamic parvocellular paraventricular nucleus and rostral NTS to food restriction and deprivation in rats.

Discovery of a novel class of isoxazoline voltage gated sodium channel blockers.

Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity, while non-aromatic heterocycle replacements were well tolerated.

Discovery of isoxazole voltage gated sodium channel blockers for treatment of chronic pain.

A series of novel isoxazole voltage gated sodium channel blockers have been synthesized and evaluated. Substitutions on the benzylic position of benzamide were investigated to determine their effect on Na(v)1.7 inhibitory potency. The spirocyclobutyl substitution had the most significant enhancement on Na(v)1.7 inhibitory activity.

A peripherally acting Na(v)1.7 sodium channel blocker reverses hyperalgesia and allodynia on rat models of inflammatory and neuropathic pain.

BACKGROUND: Voltage-gated sodium channels (Na(v)1) are expressed in primary sensory neurons where they influence excitability via their role in the generation and propagation of action potentials. Recently, human genetic data have shown that one sodium channel subtype, Na(v)1.7, plays a major role in pain. We performed these studies to characterize the antinociceptive effects of N-[(R)-1-((R)-7-chloro-1-isopropyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-ylcarbamoyl)-2-(2-fluorophenyl)-ethyl]-4-fluoro-2-trifluoromethyl-benzamide (BZP), a non-central nervous system (CNS) penetrant small molecule with high affinity and preferential selectivity for Na(v)1.7 over Na(v)1.8 and Na(v)1.5. METHODS: BZP was evaluated in rat preclinical models of inflammatory and neuropathic pain and compared with standard analgesics. Two models were used: the complete Freund's adjuvant model of inflammatory pain and the spinal nerve ligation model of neuropathic pain. BZP was also evaluated in a motor coordination assay to assess its propensity for CNS side effects. RESULTS: In preclinical models of chronic pain, BZP displayed efficacy comparable with that of leading analgesics. In the complete Freund's adjuvant model, BZP produced reversal of hyperalgesia comparable with nonsteroidal antiinflammatory drugs, and in the spinal nerve ligation model, BZP produced reversal of allodynia comparable with gabapentin and mexiletine. Unlike the CNS penetrant compounds gabapentin and mexiletine, BZP did not induce any impairment of motor coordination. CONCLUSIONS: These data suggest that a peripherally acting sodium channel blocker, preferentially acting through Na(v)1.7, could provide clinical relief of chronic pain without the CNS side effects typical of many existing pain treatments.

Imidazopyridines: a novel class of hNav1.7 channel blockers.

A series of imidazopyridines were evaluated as potential sodium channel blockers for the treatment of neuropathic pain. Several members were identified with good hNa(v)1.7 potency and excellent rat pharmacokinetic profiles. Compound 4 had good efficacy (52% and 41% reversal of allodynia at 2 and 4h post-dose, respectively) in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain when dosed orally at 10mg/kg.

Relationship between sodium channel NaV1.3 expression and neuropathic pain behavior in rats.

A multitude of voltage-gated sodium channel subtypes (NaV1) are expressed in primary sensory neurons where they influence excitability via their role in the generation and propagation of action potentials. Peripheral nerve injury alters the expression of several NaV1subtypes, but among these only NaV1.3 is up-regulated in dorsal root ganglia (DRG) neurons. The increased expression of NaV1.3 implicates this subtype in the development and maintenance of neuropathic pain, but its contribution to neuropathic pain behavior has not been examined. Using the spared nerve injury (SNI) model, we found that peripheral nerve lesion increased NaV1.3-like immunoreactivity (-LI) in DRG neurons and that mechanical allodynia was partially alleviated following oral administration of two NaV1 blockers, mexiletine (30 and 100 mg/kg, p.o.) and lamotrigine (30 and 100 mg/kg, p.o.). Intrathecal administration of antisense oligonucleotides (4 days) selective for NaV1.3 decreased NaV1.3 immunostaining in the DRG by 50% in the SNI model, but did not attenuate mechanical or cold allodynia. Moreover, we found that only 18% of NaV1.3 positive neurons also expressed activated transcription factor-3 (ATF3), a marker of injured neurons. We then selectively axotomized a cutaneous nerve (sural) and a muscle nerve (gastrocnemius) in order to identify if NaV1.3 up-regulation is dependent on cutaneous and/or muscle afferent activation and found that the numbers of neurons expressing NaV1.3 was proportional to the magnitude of the injury, but independent of the nature of innervation. These results suggest that NaV1.3 increases in primary sensory neurons that are not directly damaged in response to injury. Thus, although NaV1.3 is up-regulated in a subpopulation of DRG neurons after injury, reduction in the expression of NaV1.3 subtype alone is not sufficient to influence the NaV1-dependent behavioral hypersensitivity associated with nerve injury.

Varicella zoster virus induces neuropathic changes in rat dorsal root ganglia and behavioral reflex sensitisation that is attenuated by gabapentin or sodium channel blocking drugs.

Reactivation of latent varicella zoster virus (VZV) within sensory trigeminal and dorsal root ganglia (DRG) neurons produces shingles (zoster), often accompanied by a chronic neuropathic pain state, post-herpetic neuralgia (PHN). PHN persists despite latency of the virus within human sensory ganglia and is often unresponsive to current analgesic or antiviral agents. To study the basis of varicella zoster-induced pain, we have utilised a recently developed model of chronic VZV infection in rodents. Immunohistochemical analysis of DRG following VZV infection showed the presence of a viral immediate early gene protein (IE62) co-expressed with markers of A- (neurofilament-200; NF-200) and C- (peripherin) afferent sensory neurons. There was increased expression of neuropeptide Y (NPY) in neurons co-expressing NF-200. In addition, there was an increased expression of alpha2delta1 calcium channel, Na(v)1.3 and Na(v)1.8 sodium channels, the neuropeptide galanin and the nerve injury marker, Activating Transcription Factor-3 (ATF-3) as determined by Western blotting in DRG of VZV-infected rats. VZV infection induced increased behavioral reflex responsiveness to both noxious thermal and mechanical stimuli ipsilateral to injection (lasting up to 10 weeks post-infection) that is mediated by spinal NMDA receptors. These changes were reversed by systemic administration of gabapentin or the sodium channel blockers, mexiletine and lamotrigine, but not by the non-steroidal anti-inflammatory agent, diclofenac. This is the first time that the profile of VZV infection-induced phenotypic changes in DRG has been shown in rodents and reveals that this profile appears to be broadly similar (but not identical) to changes in other neuropathic pain models.

Induction of CX3CL1 expression in astrocytes and CX3CR1 in microglia in the spinal cord of a rat model of neuropathic pain.

UNLABELLED: Previous studies have shown that chemokines might play a role in the pathology of chronic pain. The purpose of this study was to provide an immunohistochemical description of the distribution of CX3CL1 (fractalkine) and its receptor CX3CR1 in the rat spinal cord in a model of inflammatory pain induced by unilateral intraplantar complete Freund's adjuvant (CFA) and in a model of neuropathic pain induced by L5 spinal nerve ligation (modified Chung model or mSNL). In naïve rats, CX3CL1 is found in the cytoplasm of neurons as shown by colocalization of CX3XL1 and NeuN. Similar distribution of CX3CL1 was observed after CFA, whereas after mSNL, CX3CL1 was not only observed in neurons but also found in astrocytes, as shown by colocalization of CX3CL1 and GFAP. Weak immunoreactivity for the CX3CL1 receptor, CX3CR1, was found in microglia in the spinal cord of either naïve rats or rats with inflammation. However, after spinal nerve injury, CX3CR1-LI was upregulated in microglia throughout the dorsal horn. PERSPECTIVE: This study shows that spinal nerve injury, but not peripheral inflammation, induces the expression of a chemokine, CX3CL1 (fractalkine), in astrocytes and upregulates CX3CR1 in microglia in the spinal cord. This selective regulation of CX3CL1 and its receptor, CX3CR1, suggests that these chemokines may represent new targets for the treatment of neuropathic pain.

Chronic treatments with aspirin or acetaminophen reduce both the development of polyarthritis and Fos-like immunoreactivity in rat lumbar spinal cord.

We have previously shown that during the development of adjuvant-induced arthritis (AIA), and without any peripheral stimulation, the number of Fos-like immunoreactive (Fos-LI) neurons in lumbar spinal cord increases in parallel with the clinical and behavioral signs of the disease and peaks 3 weeks after the inoculation which corresponds to the maximal stage of hyperalgesia (Abbadie and Besson 1992a). The aim of this study was to evaluate the suitability of the Fos-LI technique to gauge the effects of the two most prescribed analgesics, aspirin and acetaminophen (paracetamol), on spinal cord neurons of polyarthritic rats. The effects of the two drugs were tested on the "evoked" Fos-LI induced by peripheral mechanical noxious stimulus, as well as the effects of a chronic treatment on "basal" Fos-LI appearing during the development of polyarthritis in the absence of any intentional stimulation. We showed that: (1) Fos-LI evoked by ankle stimulation was not modified by either aspirin (150 mg/kg i.v.) or pro-acetaminophen (300 mg/kg i.v.) injection or by a 10-day chronic treatment with acetaminophen (250 or 500 mg/kg/day). (2) Despite the fact that the clinical signs of arthritis were reduced, basal Fos-LI induced by AIA disease was not changed after a 2-week chronic treatment with either aspirin (300 mg/kg/day) or acetaminophen (500 mg/kg/day) starting 3 weeks after AIA inoculation, i.e., at the maximal stage of hyperalgesia and when Fos-LI is maximal. This observation questions the suitability of Fos-LI technique to gauge the effects of mild analgesics. (3) In contrast, when the same chronic treatment was applied during the development of AIA, i.e., 1 week after inoculation, the number of Fos-LI nuclei was significantly decreased (about 50%) in aspirin- and acetaminophen-treated groups as compared to vehicle-treated groups. In parallel, the clinical signs of AIA disease were blocked by the two drug treatments. In addition, 2 weeks after the end of treatment, neither the clinical signs nor the number of Fos-LI increased again. The fact that the two drugs are able to prevent c-fos expression during development of arthritis, but not to interfere with already existing c-fos expression, suggests that for pharmacological investigations this technique should be used with caution. Thus, the potential use of Fos-LI to gauge the effects of non-steroidal antinociceptive drugs and other mild analgesics during chronic disease such as arthritis is discussed.

Effects of morphine and naloxone on basal and evoked Fos-like immunoreactivity in lumbar spinal cord neurons of arthritic rats.

We have previously shown, on the one hand, that the number of Fos-like immunoreactive (Fos-LI) neurons in the lumbar spinal cord observed during the development of adjuvant-induced arthritis in the rat correlates with the clinical and behavioral scores and, on the other hand, that the number of Fos-LI neurons induced by repeated mechanical pressure to the ankle was greater in arthritic animals than in healthy ones. In non-stimulated arthritic rats, Fos-LI neurons were mainly present in the neck of the dorsal horn and in the ventral horn of L3-L5, whereas following stimulation they were numerous in the superficial laminae. The aim of this study was to evaluate Fos-LI following morphine injection (1) in arthritic animals in the absence of any stimulation, (2) in arthritic rats after ankle stimulation pretreated with morphine or by the combination of morphine and naloxone, and (3) following naloxone treatment in non-stimulated and stimulated polyarthritic rats. In non-stimulated arthritic rats, a single morphine injection (1-9 mg/kg, i.v.) or a single naloxone injection (1-3 mg/kg, i.v.) induced no change in the basal Fos-LI present in lumbar spinal neurons. In contrast, Fos-LI evoked by noxious pressure was strongly depressed by morphine. In the superficial laminae pretreatment with a single morphine injection of either 0.5 or 1 mg/kg, i.v., reduced by more than 50% the number of Fos-LI neurons and at 3 mg/kg completely abolished the labeling evoked by the stimulation. Similar effects were obtained in the neck of the dorsal horn. These effects were reversed by naloxone (morphine 3 mg/kg and naloxone 0.3 mg/kg). Pretreatment with naloxone (1 mg/kg) did not change Fos labeling. This study which is based on mechanical stimulation in arthritic rats confirms and extends previous investigations and demonstrates that the use of Fos-LI is a suitable method to reveal the efficacy of opioid analgesic. However, the lack of effects of opioids on basal labeling suggests that long-term drug treatment should be used to study the effects of various putative analgesics on chronic pain models.

Distribution of the voltage gated sodium channel Na(v)1.3-like immunoreactivity in the adult rat central nervous system.

Voltage-gated sodium channels are transmembrane proteins responsible for the initiation and propagation of action potentials. One subtype, Na(v)1.3 (brain type III) is tetrodotoxin sensitive and fast inactivated. Na(v)1.3 has been shown to be expressed at low levels in the adult rat, but to be upregulated after sciatic nerve axotomy in the dorsal root ganglia. In the present study, we used immunohistochemistry to look at the distribution of Na(v)1.3 in the adult rat central nervous system. We used a polyclonal antibody, raised against residues 511-524. This epitope corresponds to the sequence located in the intracellular loop between domains I and II of Na(v)1.3 and is specific for this sodium channel subtype. We found Na(v)1.3-like immunoreactivity (-LI) neurons in the cerebral cortex, hippocampal formation, colliculi, and mesencephalic reticular formation. Na(v)1.3-LI was observed in fiber tracts such as the corpus callosum, anterior commissure, corticofugal fibers, lateral lemniscus, and cerebellar peduncles. Na(v)1.3-LI was particularly intense in sensory nerve tracts such as the mesencephalic trigeminal tract, vestibulospinal tract, or spinal trigeminal tract. In the spinal cord, Na(v)1.3-LI was intense throughout the white matter and the dorsal roots. In the spinal cord grey matter, Na(v)1.3-LI fibers terminate in the deep laminae of the dorsal horn and in the ventral horn. Na(v)1.3-LI was also found in motoneurons as well as in ventral roots. This study shows that Na(v)1.3 is present at the protein level in the adult rat.

Mu and delta opioid receptor-like immunoreactivity in the cervical spinal cord of the rat after dorsal rhizotomy or neonatal capsaicin: an analysis of pre- and postsynaptic receptor distributions.

Opioid compounds have powerful analgesic properties when administered to the spinal cord. These effects are exerted through mu and delta opioid receptors, and both pre- and postsynaptic mechanisms have been implicated. To specifically address the relative pre- and postsynaptic contribution to spinal opioid analgesia, we have quantitatively assessed the pre- vs. postsynaptic distribution of the mu-opioid (MOR-1, MOP(1)) and delta-opioid receptors (DOR-1, DOP(1)). We also examined the rostro-caudal arborization of MOR-1 and DOR-1 immunoreactive primary sensory neurons, using an isolated dorsal root preparation. These results were compared to those obtained by labeling for calcitonin gene-related peptide (CGRP), a neuropeptide whose expression in the spinal cord is restricted to the terminals of small diameter primary sensory neurons. We estimate that approximately one half of MOR-1 and two thirds of DOR-1 immunoreactivity in the cervical spinal cord is located on primary afferent fibers. These fibers have a broad rostro-caudal distribution, extending at least three segments rostral and caudal to their segment of entry. Regardless of marker used, the rostral projection was greatest, however, the distribution of CGRP-immunoreactive fibers differed somewhat in that they had a much smaller projection to the most caudal segments examined. Our results suggest that presynaptic delta opioid actions predominate, but that there are mixed pre- and postsynaptic inhibitory effects exerted by opioid analgesics that act at the spinal cord mu opioid receptor.

Discovery of MK-4409, a Novel Oxazole FAAH Inhibitor for the Treatment of Inflammatory and Neuropathic Pain.

We report herein the identification of MK-4409, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing of FAAH inhibition in vitro potency, improving the pharmacokinetic (PK) profile, and increasing in vivo efficacy in rodent inflammatory and neuropathic pain assays.