Discovery of a novel series of pyridone amides as NaV1.8 inhibitors.

The NaV1.8 channel, mainly found in the peripheral nervous system, is recognized as one of the key factors in chronic pain. The molecule VX-150 was initially promising in targeting this channel, but the phase II trials of VX-150 did not show expected pain relief results. By analyzing the interaction mode of VX-150 and NaV1.8, we developed two series with a total of 19 molecules and examined their binding affinity to NaV1.8 in vitro and analgesic effect in vivo. One compound, named 2j, stood out with notable activity against the NaV1.8 channel and showed effective pain relief in models of chronic inflammatory pain and neuropathic pain. Our research points to 2j as a strong contender for developing safer pain-relief treatments.

Pathogenic mutations perturb calmodulin regulation of Nav1.8 channel.

The voltage-gated sodium channels play a key role in the generation and propagation of the cardiac action potential. Emerging data indicate that the Nav1.8 channel, encoded by the SCN10A gene, is a modulator of cardiac conduction and variation in the gene has been associated with arrhythmias such as atrial fibrillation (AF) and Brugada syndrome (BrS). The voltage gated sodium channels contain a calmodulin (CaM)-binding IQ domain involved in channel slow inactivation, we here investigated the role of CaM regulation of Nav1.8 channel function, and showed that CaM enhanced slow inactivation of the Nav1.8 channel and hyperpolarized steady-state inactivation curve of sodium currents. The effects of CaM on the channel gating were disrupted in the Nav1.8 channel truncated IQ domain. We studied Nav1.8 IQ domain mutations associated with AF and BrS, and found that a BrS-linked mutation (R1863Q) reduced the CaM-induced hyperpolarization shift, AF-linked mutations (R1869C and R1869G) disrupted CaM-induced enhanced inactivation, and effects of CaM on both development and recovery from slow inactivation were attenuated in all pathogenic mutations. Our findings indicate a role of CaM in the regulation of Nav1.8 channel function in cardiac arrhythmias.

The nociceptive innervation of the normal and osteoarthritic mouse knee.

OBJECTIVES: To document the nociceptive innervation of the normal and osteoarthritic murine knee. METHODS: Knees were collected from naïve male C57BL/6 NaV1.8-tdTomato reporter mice aged 10, 26, and 52 weeks (n = 5/group). Destabilization of the medial meniscus (DMM) or sham surgeries (n = 5/group) were performed in the right knee of 10-week old male NaV1.8-tdTomato mice, and knees were harvested 16 weeks later. Twenty 20-µm frozen sections from a 400-µm mid-joint region were collected for confocal microscopy. Integrated density of the tdTomato signal was calculated using Image J by two independent observers blinded to the groups. Consecutive sections were stained with hematoxylin & eosin. C57BL/6-Pirt-GCaMP3 mice (n = 5/group) and protein gene product 9.5 (PGP9.5) immunostaining of C57BL/6 wild type (WT) mice (n = 5/group) were used to confirm innervation patterns. RESULTS: In naive 10-week old mice, nociceptive innervation was most dense in bone marrow cavities, lateral synovium and at the insertions of the cruciate ligaments. By age 26 weeks, unoperated knees showed a marked decline in nociceptors in the lateral synovium and cruciate ligament insertions. No further decline was observed by age 1 year. Sixteen weeks after DMM, the medial compartment of OA knees exhibited striking changes in NaV1.8+ innervation, including increased innervation of the medial synovium and meniscus, and nociceptors in subchondral bone channels. All results were confirmed through quantification, also in Pirt-GCaMP3 and PGP9.5-immunostained WT mice. CONCLUSIONS: Nociceptive innervation of the mouse knee markedly declines by age 26 weeks, before onset of spontaneous OA. Late-stage surgically induced OA is associated with striking plasticity of joint afferents in the medial compartment of the knee.

The discovery and optimization of benzimidazoles as selective NaV1.8 blockers for the treatment of pain.

The voltage gated sodium channel NaV1.8 has been postulated to play a key role in the transmission of pain signals. Core hopping from our previously reported phenylimidazole leads has allowed the identification of a novel series of benzimidazole NaV1.8 blockers. Subsequent optimization allowed the identification of compound 9, PF-06305591, as a potent, highly selective blocker with an excellent preclinical in vitro ADME and safety profile.

Molecular mechanisms and signaling by comenic acid in nociceptive neurons influence the pathophysiology of neuropathic pain.

Comenic acid (CA), a specific agonist of opioid-like receptors, effectively and safely relieves neuropathic pain by decreasing the NaV1.8 channel voltage sensitivity in the primary sensory neuron membrane. CA triggers downstream signaling cascades, in which the Na,K-ATPase/Src complex plays a key role. After leaving the complex, the signal diverges 'tangentially' and 'radially'. It is directed 'tangentially' along the neuron membrane to NaV1.8 channels, decreasing the effective charge of their activation gating system. In the radial direction moving towards the cell genome, the signal activates the downstream signaling pathway involving PKC and ERK1/2. A remarkable feature of CA is its ability to modulate NaV1.8 channels, which relieves neuropathic pain while simultaneously stimulating neurite growth via the receptor-coupled activation of the ERK1/2-dependent signaling pathway.

Neonatal Injury Modulates Incisional Pain Sensitivity in Adulthood: An Animal Study.

Neonatal pain experiences including traumatic injury influence negatively on development of nociceptive circuits, resulting in persistent pain hypersensitivity in adults. However, the detailed mechanism is not yet well understood. In the present study, to clarify the pathogenesis of orofacial pain hypersensitivity associated with neonatal injury, the involvement of the voltage-gated sodium channel (Nav) 1.8 and the C-C chemokine ligand 2 (CCL2)/C-C chemokine receptor 2 (CCR2) signaling in the trigeminal ganglion (TG) in facial skin incisional pain hypersensitivity was examined in 190 neonatal facial-injured and sham male rats. The whisker pad skin was incised on postnatal day 4 and week 7 (Incision-Incision group). Compared to the group without neonatal incision (Sham-Incision group), mechanical hypersensitivity in the whisker pad skin was enhanced in Incision-Incision group. The number of Nav1.8-immunoreactive TG neurons and the amount of CCL2 expressed in the macrophages and satellite glial cells in the TG were increased on day 14 after re-incision in the Incision-Incision group, compared with Sham-Incision group. Blockages of Nav1.8 in the incised region and CCR2 in the TG suppressed the enhancement of mechanical hypersensitivity in the Incision-Incision group. Administration of CCL2 into the TG enhanced mechanical hypersensitivity in the Sham-Sham, Incision-Sham and Sham-Incision group. Our results suggest that neonatal facial injury accelerates the TG neuronal hyperexcitability following orofacial skin injury in adult in association with Nav1.8 overexpression via CCL2 signaling, resulting in the enhancement of orofacial incisional pain hypersensitivity in the adulthood.

Discovery of selective NaV1.8 inhibitors based on 5-chloro-2-(4,4-difluoroazepan-1-yl)-6-methyl nicotinamide scaffold for the treatment of pain.

The NaV1.8 channel is a genetically validated target for pain and it is mostly expressed in the peripheral nervous system. Based on the disclosed structures of NaV1.8-selective inhibitors, we designed and synthesized a series of compounds by introducing bicyclic aromatic fragments based on the nicotinamide scaffold. In this research, a systematic structure-activity relationship study was carried out. While compound 2c possessed moderate inhibitory activity (IC50 = 50.18 ± 0.04 nM) in HEK293 cells stably expressing human NaV1.8 channels, it showed potent inhibitory activity in DRG neurons and isoform selectivity (>200-fold against human NaV1.1, NaV1.5 and NaV1.7 channels). Moreover, the analgesic potency of compound 2c was identified in a post-surgical mouse model. These data demonstrate that compound 2c can be further evaluated as a non-addictive analgesic agent with reduced cardiac liabilities.

Influence of combined voltage-gated sodium channel NaV1.7 and NaV1.8 inhibitors on cough in a guinea pig model.

Pathological excessive cough is a serious clinical problem in many patients. It is no doubt that an increased activation and sensitization of airway vagal C-fibres in disease stems from dysregulation of the neural pathways that control cough. Due to the limited efficacy and unwanted side effects of current antitussives, there is a continual demand for the development of a novel more effective antitussive. Since voltage-gated sodium channels (NaVs) are absolutely required for action potentials initiation and conduction irrespective of the stimulus, NaVs became a promising and attractive neural target. Current studies establish that NaV1.7 and NaV1.8 inhibitors have the potential to suppress cough. In this study, we demonstrated that inhaled aerosol of NaV1.7 inhibitor PF-05089771 (10 µM) and NaV1.8 inhibitor A-803467 (1 mM) mixture inhibited the capsaicin-induced cough by ≈ 60 % and citric acid-induced cough by ≈ 65 % at doses that did not modify respiratory rate. Our previous and present studies indicate that NaV1.7 and NaV1.8 may present promising therapeutic targets for antitussive therapy.

Neurotrophin-4 is essential for survival of the majority of vagal afferents to the mucosa of the small intestine, but not the stomach.

Vagal afferents form the primary gut-to-brain neural axis, communicating signals that regulate gastrointestinal (GI) function and promote satiation, appetition and reward. Neurotrophin-4 (NT-4) is essential for the survival of vagal smooth muscle afferents of the small intestine, but not the stomach. Here we took advantage of near-complete labeling of GI vagal mucosal afferents in Nav1.8cre-Rosa26tdTomato transgenic mice to determine whether these afferents depend on NT-4 for survival. We quantified the density and distribution of vagal afferent terminals in the stomach and small intestine mucosa and their central terminals in the solitary tract nucleus (NTS) and area postrema in NT-4 knockout (KO) and control mice. NT-4KO mice exhibited a 75% reduction in vagal afferent terminals in proximal duodenal villi and a 55% decrease in the distal ileum, whereas, those in the stomach glands remained intact. Vagal crypt afferents were also reduced in some regions of the small intestine, but to a lesser degree. Surprisingly, NT-4KO mice exhibited an increase in labeled terminals in the medial NTS. These findings, combined with previous results, suggest NT-4 is essential for survival of a large proportion of all classes of vagal afferents that innervate the small intestine, but not those that supply the stomach. Thus, NT-4KO mice could be valuable for distinguishing gastric and intestinal vagal afferent regulation of GI function and feeding. The apparent plasticity of central vagal afferent terminals - an increase in their density - could have compensated for loss of peripheral terminals by maintaining near-normal levels of satiety signaling.

MicroRNA-96 is required to prevent allodynia by repressing voltage-gated sodium channels in spinal cord.

Voltage-gated sodium channels (Navs) 1.7, 1.8, and 1.9 are predominately expressed in peripheral sensory neurons and are critical for action potential propagation in nociceptors. Unexpectedly, we found that expression of SCN9A, SCN10A, SCN11A, and SCN2A, the alpha subunit of Nav1.7, Nav1.8, Nav1.9 and Nav1.2, respectively, are up-regulated in spinal dorsal horn (SDH) neurons of miR-96 knockout mice. These mice also have de-repression of CACNA2D1/2 in DRG and display thermal and mechanical allodynia that could be attenuated by intrathecal or intraperitoneal injection of Nav1.7 or Nav1.8 blockers or Gabapentin. Moreover, Gad2::CreERT2 conditional miR-96 knockout mice phenocopied global knockout mice, implicating inhibitory neurons; nerve injury induced significant loss of miR-96 in SDH GABAergic and Glutamatergic neurons in mice which negatively correlated to up-regulation of Nav1.7, Nav1.8, Nav1.9 and Scn2a, this dis-regulation of miR-96 and Navs in SDH neurons contributed to neuropathic pain which can be alleviated by intrathecal injection of Nav1.7 or Nav1.8 blockers. In conclusion, miR-96 is required to avoid allodynia through limiting the expression of VGCCs and Navs in DRG and Navs in SDH in naïve and nerve injury-induced neuropathic pain mice. Our findings suggest that central nervous system penetrating Nav1.7 and Nav1.8 blockers may be efficacious for pain relief.