Accepted

The present series of studies examine the impact of systemically administered therapeutics on peripheral nerve injury (males; unilateral sciatic chronic constriction injury [CCI])-induced suppression of voluntary wheel running, across weeks after dosing cessation. Following CCI, active phase running distance and speed are suppressed throughout the 7-week observation period. A brief course of morphine, however, increased active phase running distance and speed throughout this same period, an effect apparent only in sham rats. For CCI rats, systemic co-administration of morphine with antagonists of either P2X7 (A438079) or TLR4 ((+)-naloxone) (receptors critical to the activation of NLRP3 inflammasomes and consequent inflammatory cascades) returned running behavior of CCI rats to that of shams through 5+ weeks after dosing ceased. This is a striking difference in effect compared to our prior CCI allodynia results using systemic morphine plus intrathecal delivery of these same antagonists, wherein a sustained albeit partial suppression of neuropathic pain was observed. This may point to actions of the systemic drugs at multiple sites along the neuraxis, modulating injury-induced, inflammasome-mediated effects at the injured sciatic nerve and/or dorsal root ganglia, spinal cord, and potentially higher levels. Given that our data to date point to morphine amplifying neuroinflammatory processes put into motion by nerve injury, it is intriguing to speculate that co-administration of TLR4 and/or P2X7 antagonists can intervene in these inflammatory processes in a beneficial way. That is, that systemic administration of such compounds may suppress inflammatory damage at multiple sites, rapidly and persistently returning neuropathic animals to sham levels of response.

Temporomandibular Disorder (TMD) patients report amplification of pain in the masticatory muscles after psychological trauma or stressful conditions. The mechanisms underlying this phenomenon are yet to be elucidated. This study combined immunohistochemistry with single cell in vivo electrophysiology recordings of masticatory muscle afferent fibers to investigate the role of α-adrenergic receptors in muscle nociception. It was found that a subset of trigeminal afferent fibers which innervate the masseter and temporal muscles expressed α, α and α receptors, including a smaller number of putative nociceptors which co-expressed TrpV receptors. Local injection of the selective α adrenergic receptor agonist phenylephrine into masticatory muscle decreased and increased the mechanical activation threshold of slow and fast conducting afferent fibers, respectively. This effect was reversed by co-administration of the α selective antagonist terazosin. To rule out the possibility that local ischemia was responsible for the observed effect of phenylephrine on masticatory muscle afferent fibers, additional experiments were conducted where blood flow to the masticatory muscle was reduced by common carotid artery occlusion. This investigation found that muscle blood flow occlusion increased the mechanical activation threshold of the majority of masticatory muscle afferent fibers unrelated to conduction velocity. These findings suggest that under conditions of increased sympathetic tone, such as those related to stress, noradrenaline may sensitize masticatory muscle nociceptors to increase pain and desensitize muscle proprioceptors to alter muscle tone, through activation of α receptors.

Pharmacological evidence indicated a functional interaction between neuropeptide FF (NPFF) and cannabinoid systems, and the cannabinoids combined with the NPFF receptor agonist neuropeptide VF (NPVF) produced antinociception without tolerance. In the present study, VF-13, a chimeric peptide containing the pharmacophores of the endogenous cannabinoid peptide VD-hemopressin(α) (VD-Hpα) and NPVF, was synthesized and pharmacologically evaluated. In vitro, VF-13 significantly upregulated the phosphorylated level of extracellular signal-regulated kinase 1/2 (ERK1/2) in CHO cells stably expressing CB1 receptors and inhibited forskolin-induced cAMP accumulation in HEK293 cells stably expressing NPFF or NPFF receptors. Moreover, VF-13 induced neurite outgrowth in Neuro 2A cells via CB1 and NPFF receptors. These results suggest that VF-13 exhibits multifunctional agonism at CB1, NPFF and NPFF receptors in vitro. Interestingly, intracerebroventricular VF-13 produced dose-dependent antinociception in mouse models of tail-flick and carrageenan-induced inflammatory pain via the TRPV1 receptor. In contrast, the reference compound (m)VD-Hpα-NH induced CB1 receptor-mediated supraspinal antinociception. Additionally, subcutaneous injection of (m)VD-Hpα-NH and VF-13 produced significant antinociception in carrageenan-induced inflammatory pain model. In the tetrad assay, our data demonstrated that VF-13 elicited hypothermia, but not catalepsy and hypoactivity after intracerebroventricular injection. Notably, VF-13 produced non-tolerance forming antinociception over 6 days treatment in both acute and inflammatory pain models. Furthermore, VF-13 had no apparent effects on gastrointestinal transit, pentobarbitone-induced sedation, food intake, and motor coordination at the supraspinal level. In summary, VF-13, a novel chimeric peptide of VD-Hpα and NPVF, produced non-tolerance forming antinociception in preclinical pain models with reduced cannabinoid-related side effects.

Compelling human genetic studies have identified the voltage-gated sodium channel Na1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa1.7 using patch-clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNa1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets Na1.4, Na1.5, and Na1.6. The Pn3a[D8N] mutant retained analgesic activity , significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na1.7 in pain.