Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

BackgroundSpinal Toll-like receptors (TLRs) and signaling intermediaries have been implicated in persistent pain states. We examined the roles of two major TLR signaling pathways and selected TLRs in a mononeuropathic allodynia.MethodsL5 spinal nerve ligation (SNL) was performed in wild type (WT, C57BL/6) male and female mice and in male Tlr2 -/- Tlr3 -/- , Tlr4 -/- , Tlr5 -/- , Myd88 -/- , Trif lps2 , Myd88/Trif lps2 , Tnf -/- , and Ifnar1 -/- mice. We also examined L5 ligation in Tlr4 -/- female mice. We examined tactile allodynia using von Frey hairs. Iba-1 (microglia) and GFAP (astrocytes) were assessed in spinal cords by immunostaining. Tactile thresholds were analyzed by 1- and 2-way ANOVA and the Bonferroni post hoc test was used.ResultsIn WT male and female mice, SNL lesions resulted in a persistent and robust ipsilateral, tactile allodynia. In males with TLR2, 3, 4, or 5 deficiencies, tactile allodynia was significantly, but incompletely, reversed (approximately 50%) as compared to WT. This effect was not seen in female Tlr4 -/- mice. Increases in ipsilateral lumbar Iba-1 and GFAP were seen in mutant and WT mice. Mice deficient in MyD88, or MyD88 and TRIF, showed an approximately 50% reduction in withdrawal thresholds and reduced ipsilateral Iba-1. In contrast, TRIF and interferon receptor null mice developed a profound ipsilateral and contralateral tactile allodynia. In lumbar sections of the spinal cords, we observed a greater increase in Iba-1 immunoreactivity in the TRIF-signaling deficient mice as compared to WT, but no significant increase in GFAP. Removing MyD88 abrogated the contralateral allodynia in the TRIF signaling-deficient mice. Conversely, IFNβ, released downstream to TRIF signaling, administered intrathecally, temporarily reversed the tactile allodynia.ConclusionsThese observations suggest a critical role for the MyD88 pathway in initiating neuropathic pain, but a distinct role for the TRIF pathway and interferon in regulating neuropathic pain phenotypes in male mice.

We addressed the hypothesis that intraplantar Botulinum toxin B (rimabotulinumtoxin B: BoNT-B) has an early local effect upon peripheral afferent terminal releasing function and over time will be transported to the central terminals of the primary afferent. Once in the terminals it will cleave synaptic protein, block spinal afferent transmitter release and thereby prevent spinal nociceptive excitation and behavior. In mice, C57Bl/6 males, intraplantar BoNT-B (1U), given unilaterally into the hind paw had no effect upon survival or motor function but ipsilaterally decreased: i) intraplantar formalin evoked flinching; ii) intraplantar capsaicin evoked plasma extravasation in the hindpaw measured by Evans blue in the paw; iii) intraplantar formalin evoked dorsal horn SP release (NK1 receptor internalization); iv) intraplantar formalin evoked dorsal horn neuronal activation (cFos); v) ipsilateral DRG VAMP; vi) ipsilateral SP release otherwise evoked bilaterally by intrathecal capsaicin; vii) ipsilateral activation of cFos otherwise evoked bilaterally by intrathecal substance P. These results indicate that BoNT-B after unilateral intraplantar delivery is taken up by the peripheral terminal, is locally active (blocking plasma extravasation), is transported to the ipsilateral DRG to cleave VAMP and is acting presynaptically to block release from the spinal peptidergic terminal. The observations following intrathecal SP offer evidence for a possible transsynaptic effect of intraplantar BoNT. These results provide robust evidence that peripheral BoNT-B can alter peripheral and central terminal release from a nociceptor and attenuate downstream nociceptive processing via a presynaptic effect, with further evidence suggesting a possible postsynaptic effect.

Objective Pain resulting from local tissue injury or inflammation typically resolves with time. Frequently, however, this pain may unexpectedly persist, becoming a pathological chronic state. Increasingly, the innate and adaptive immune systems are being implicated in the initiation and maintenance of these persistent conditions. In particular, Toll-like receptor 4 (TLR4) signaling has been shown to mediate the transition to a persistent pain state in a sex-dependent manner. In the present work, we explored this contribution using the TLR4 antagonist, TAK-242. Methods Male and female C57Bl/6 mice were given intravenous (IV), intrathecal (IT), or intraperitoneal (IP) TAK-242 prior to IT delivery of lipopolysaccharide (LPS), and tactile reactivity was assessed at regular intervals over 72-hours. Additional groups of mice were treated with IP TAK-242 prior to intraplantar formalin, and flinching was monitored for 1-hour. Tactile reactivity was assessed at 7-days after formalin delivery. Results LPS evoked TNF release from male and female macrophages and RAW267.4 cells, which was blocked in a concentration dependent fashion by TAK-242. In vivo, IT LPS evoked tactile allodynia to a greater degree in male than female mice. TAK-242, given by all routes, prevented development of IT LPS-induced tactile allodynia in male animals, but did not reverse their established allodynia. TLR4 deficiency and TAK-242 treatment attenuated IT LPS-induced allodynia in male, but not female mice. In the formalin model, pre-treatment with TAK-242 did not affect Phase 1 or Phase 2 flinching, but prevented the delayed tactile allodynia in both male and unexpectedly in female mice (Phase 3). Conclusions Together, these results suggest that TAK-242 is a TLR4 antagonist that has efficacy after systemic and intrathecal delivery and confirms the role of endogenous TLR4 signaling in triggering the development of a delayed allodynia in both male and female mice.

Mechanosensory fibers are enveloped by myelin, a unique multilamellar membrane permitting salutatory neuronal conduction. Damage to myelin is thought to contribute to severe pain evoked by innocuous tactile stimulation (i.e. mechanical allodynia). Our earlier (Liu et al, J. Neuroinflammation, 9 (1): 119, 2012) and present data demonstrate that a single injection of a myelin basic protein-derived peptide (MBP84–104) into an intact sciatic nerve produces a robust and long-lasting (>30 days) mechanical allodynia in female rats. The MBP84-104 peptide represents the immunodominant epitope and requires T cells to maintain allodynia. Surprisingly, only systemic gabapentin (a ligand of voltage-gated calcium channel α2δ1), but not ketorolac (COX inhibitor), lidocaine (sodium channel blocker) or MK801 (NMDA antagonist) reverse allodynia induced by the intrasciatic MBP84-104. The genome-wide transcriptional profiling of the sciatic nerve followed by the bioinformatics analyses of the expression changes identified interleukin (IL)-6 as the major cytokine induced by MBP84-104 in both the control and athymic T cell-deficient nude rats. The intrasciatic MBP84-104 injection resulted in both unilateral allodynia and unilateral IL-6 increase the segmental spinal cord (neurons and astrocytes). An intrathecal delivery of a function-blocking IL-6 antibody reduced the allodynia in part by the transcriptional effects in large-diameter primary afferents in DRG. Our data suggest that MBP regulates IL-6 expression in the nervous system and that the spinal IL-6 activity mediates nociceptive processing stimulated by the MBP epitopes released after damage or disease of the somatosensory nervous system.

In the peripheral nerve, mechanosensitive axons are insulated by myelin, a multilamellar membrane formed by Schwann cells. Here, we offer first evidence that a myelin degradation product induces mechanical hypersensitivity and global transcriptomics changes in a sex-specific manner. Focusing on downstream signaling events of the functionally active 84-104 myelin basic protein (MBP84-104) fragment released after nerve injury, we demonstrate that exposing the sciatic nerve to MBP84-104 via endoneurial injection produces robust mechanical hypersensitivity in female, but not in male, mice. RNA-Seq and systems biology analyses revealed a striking sexual dimorphism in molecular signatures of the dorsal root ganglia (DRG) and spinal cord response, not observed at the nerve injection site. Mechanistically, intra-sciatic MBP84-104 induced phospholipase C (PLC)-driven (females) and phosphoinositide 3-kinase-driven (males) phospholipid metabolism (tier 1). PLC/inositol trisphosphate receptor (IP3R) and estrogen receptor co-regulation in spinal cord yielded Ca2+-dependent nociceptive signaling induction in females that was suppressed in males (tier 2). IP3R inactivation by intrathecal xestospongin C attenuated the female-specific hypersensitivity induced by MBP84-104. According to sustained sensitization in tiers 1–2, T cell–related signaling spreads to the DRG and spinal cord in females, but remains localized to the sciatic nerve in males (tier 3). These results are consistent with our previous finding that MBP84-104–induced pain is T cell–dependent. In summary, an autoantigenic peptide endogenously released in nerve injury triggers multi-site, sex-specific transcriptome changes, leading to neuropathic pain only in female mice. MBP84-104 acts through sustained co-activation of metabolic, estrogen receptor–mediated nociceptive and autoimmune signaling programs.

Pain is the primary motivation for seeking medical care. Although pain may subside as inflammation resolves or an injury heals, it is increasingly evident that persistency of the pain state can occur with significant regularity. Chronic pain requires aggressive management to minimize its physiological consequences and diminish its impact on quality of life. Although opioids commonly are prescribed for intractable pain, concerns regarding reduced efficacy, as well as risks of tolerance and dependence, misuse, diversion, and overdose mortality rates limit their utility. Advances in development of nonopioid interventions hinge on our appreciation of underlying mechanisms of pain hypersensitivity. For instance, the contributory role of immunity and the associated presence of autoimmune syndromes has become of particular interest. Males and females exhibit fundamental differences in innate and adaptive immune responses, some of which are present throughout life, whereas others manifest with reproductive maturation. In general, the incidence of chronic pain conditions, particularly those with likely autoimmune covariates, is significantly higher in women. Accordingly, evidence is now accruing in support of neuroimmune interactions driving sex differences in the development and maintenance of pain hypersensitivity and chronicity. This review highlights known sexual dimorphisms of neuroimmune signaling in pain states modeled in rodents, which may yield potential high-value sex-specific targets to inform future analgesic drug discovery efforts.

Neurogenic inflammation results from the release of biologically active agents from the peripheral primary afferent terminal. This release reflects the presence of releasable pools of active product and depolarization-exocytotic coupling mechanisms in the distal afferent terminal and serves to alter the physiologic function of innervated organ systems ranging from the skin and meninges to muscle, bone, and viscera. Aside from direct stimulation, this biologically important release from the peripheral afferent terminal can be initiated by antidromic activity arising from five anatomically distinct points of origin: (i) afferent collaterals at the peripheral-target organ level, (ii) afferent collaterals arising proximal to the target organ, (iii) from mid-axon where afferents lacking myelin sheaths (C fibers and others following demyelinating injuries) may display crosstalk and respond to local irritation, (iv) the dorsal root ganglion itself, and (v) the central terminals of the afferent in the dorsal horn where local circuits and bulbospinal projections can initiate the so-called dorsal root reflexes, i.e., antidromic traffic in the sensory afferent.