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Purpose: Guanfacine is an α2A-adrenergic receptor agonist, FDA-approved to treat attention-deficit hyperactivity disorder and high blood pressure, typically as an extended-release formulation up to 7 mg/day. In our dysautonomia clinic, we observed that off-label use of short-acting guanfacine at 1 mg/day facilitated symptom relief in two families with multiple members presenting with severe generalized anxiety. We also noted anecdotal improvements in associated dysautonomia symptoms such as hyperhidrosis, cognitive impairment, and palpitations. We postulated that a genetic deficit existed in these patients that might augment guanfacine susceptibility. Methods: We used whole-exome sequencing to identify mutations in patients with shared generalized anxiety and dysautonomia symptoms. Guanfacine-induced changes in the function of voltage-gated Na+ channels were investigated using voltage-clamp electrophysiology. Results: Whole-exome sequencing uncovered the p.I739V mutation in SCN9A in the proband of two nonrelated families. Moreover, guanfacine inhibited ionic currents evoked by wild-type and mutant NaV1.7 encoded by SCN9A, as well as other NaV channel subtypes to a varying degree. Conclusion: Our study provides further evidence for a possible pathophysiological role of NaV1.7 in anxiety and dysautonomia. Combined with off-target effects on NaV channel function, daily administration of 1 mg short-acting guanfacine may be sufficient to normalize NaV channel mutation-induced changes in sympathetic activity, perhaps aided by partial inhibition of NaV1.7 or other channel subtypes. In a broader context, expanding genetic and functional data about ion channel aberrations may enable the prospect of stratifying patients in which mutation-induced increased sympathetic tone normalization by guanfacine can support treatment strategies for anxiety and dysautonomia symptoms. [ABSTRACT FROM AUTHOR]

Burn injuries are among the highly prevalent medical conditions worldwide that occur mainly in children, military veterans and victims of fire accidents. It is one of the leading causes of temporary as well as permanent disabilities in patients. Burn injuries are accompanied by pain that persists even after recovery from tissue damage which puts immense pressure on the healthcare system. The pathophysiology of burn pain is poorly understood due to its complex nature and lack of considerable preclinical and clinical shreds of evidence, that creates a substantial barrier to the development of new analgesics. Burns damage the skin layers supplied with nociceptors such as NAV1.7, TRPV1, and TRPA1. Burn injury-mediated co-localization and simultaneous activation of TRPA1 and TRPV1 in nociceptive primary afferent C-fibers which contributes to the development and maintenance of chronic pain. Burn injuries are accompanied by central sensitization, a key feature of pain pathophysiology mainly driven by a series of cascades involving aberrations in the glutamatergic system, microglial activation, release of neuropeptides, cytokines, and chemokines. Activation of p38 mitogen-activated protein kinase, altered endogenous opioid signaling, and distorted genomic expression are other pathophysiological factors responsible for the development and maintenance of burn pain. Here we discuss comprehensive literature on molecular mechanisms of burn pain and potential targets that could be translated into near future therapeutics. [ABSTRACT FROM AUTHOR]

At present, chronic post-surgical pain (CPSP) is difficult to prevent and cure clinically because of our lack of understanding of its mechanisms. Surgical injury induces the upregulation of voltage-gated sodium channel Nav1.7 in dorsal root ganglion (DRG) neurons, suggesting that Nav1.7 is involved in the development of CPSP. However, the mechanism leading to persistent dysregulation of Nav1.7 is largely unknown. Given that nerve growth factor (NGF) induces a long-term increase in the neuronal hyperexcitability after injury, we hypothesized that NGF might cause the long-term dysregulation of Nav1.7. In this study, we aimed to investigate whether Nav1.7 regulation by NGF is involved in CPSP and thus contributes to the specific mechanisms involved in the development of CPSP. Using conditional nociceptor-specific Nav1.7 knockout mice, we confirmed the involvement of Nav1.7 in NGF-induced pain and identified its role in the maintenance of pain behavior during long-term observations (up to 14 days). Using western blot analyses and immunostaining, we showed that NGF could trigger the upregulation of Nav1.7 expression and thus support the development of CPSP in rats. Using pharmacological approaches, we showed that the increase of Nav1.7 might be partly regulated by an NGF/TrkA-SGK1-Nedd4-2-mediated pathway. Furthermore, reversing the upregulation of Nav1.7 in DRG could alleviate spinal sensitization. Our results suggest that the maintained upregulation of Nav1.7 triggered by NGF contributes to the development of CPSP. Attenuating the dysregulation of Nav1.7 in peripheral nociceptors may be a strategy to prevent the transition from acute post-surgical pain to CPSP. [ABSTRACT FROM AUTHOR]

Purpose: This work describes a staged approach to the application of pharmacokinetic-pharmacodynamic (PK-PD) modeling in the voltage-gated sodium ion channel (NaV1.7) inhibitor drug discovery effort to address strategic questions regarding in vitro to in vivo translation of target modulation. Methods: PK-PD analysis was applied to data from a functional magnetic resonance imaging (fMRI) technique to non-invasively measure treatment mediated inhibition of olfaction signaling in non-human primates (NHPs). Initial exposure-response was evaluated using single time point data pooled across 27 compounds to inform on in vitro to in vivo correlation (IVIVC). More robust effect compartment PK-PD modeling was conducted for a subset of 10 compounds with additional PD and PK data to characterize hysteresis. Results: The pooled compound exposure-response facilitated an early exploration of IVIVC with a limited dataset for each individual compound, and it suggested a 2.4-fold in vitro to in vivo scaling factor for the NaV1.7 target. Accounting for hysteresis with an effect compartment PK-PD model as compounds advanced towards preclinical development provided a more robust determination of in vivo potency values, which resulted in a statistically significant positive IVIVC with a slope of 1.057 ± 0.210, R-squared of 0.7831, and p value of 0.006. Subsequent simulations with the PK-PD model informed the design of anti-nociception efficacy studies in NHPs. Conclusions: A staged approach to PK-PD modeling and simulation enabled integration of in vitro NaV1.7 potency, plasma protein binding, and pharmacokinetics to describe the exposure-response profile and inform future study design as the NaV1.7 inhibitor effort progressed through drug discovery. [ABSTRACT FROM AUTHOR]

Chronic pain is a global problem affecting up to 20% of the world's population and has a significant economic, social and personal cost to society. Sensory neurons of the dorsal root ganglia (DRG) detect noxious stimuli and transmit this sensory information to regions of the central nervous system (CNS) where activity is perceived as pain. DRG neurons express multiple voltage-gated sodium channels that underlie their excitability. Research over the last 20 years has provided valuable insights into the critical roles that two channels, NaV1.7 and NaV1.9, play in pain signalling in man. Gain of function mutations in NaV1.7 cause painful conditions while loss of function mutations cause complete insensitivity to pain. Only gain of function mutations have been reported for NaV1.9. However, while most NaV1.9 mutations lead to painful conditions, a few are reported to cause insensitivity to pain. The critical roles these channels play in pain along with their low expression in the CNS and heart muscle suggest they are valid targets for novel analgesic drugs. [ABSTRACT FROM AUTHOR]

The collapsin response mediator protein 2 (CRMP2) has emerged as a central node in assembling nociceptive signaling complexes involving voltage-gated ion channels. Concerted actions of post-translational modifications, phosphorylation and SUMOylation, of CRMP2 contribute to regulation of pathological pain states. In the present study, we demonstrate a novel role for CRMP2 in spinal nociceptive transmission. We found that, of six possible post-translational modifications, three phosphorylation sites on CRMP2 were critical for regulating calcium influx in dorsal root ganglion sensory neurons. Of these, only CRMP2 phosphorylated at serine 522 by cyclin-dependent kinase 5 (Cdk5) contributed to spinal neurotransmission in a bidirectional manner. Accordingly, expression of a non-phosphorylatable CRMP2 (S522A) decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), whereas expression of a constitutively phosphorylated CRMP2 (S522D) increased the frequency of sEPSCs. The presynaptic nature of CRMP2's actions was further confirmed by pharmacological antagonism of Cdk5-mediated CRMP2 phosphorylation with S-N-benzy-2-acetamido-3-methoxypropionamide ((S)-lacosamide; (S)-LCM) which (i) decreased sEPSC frequency, (ii) increased paired-pulse ratio, and (iii) reduced the presynaptic distribution of CaV2.2 and NaV1.7, two voltage-gated ion channels implicated in nociceptive signaling. (S)-LCM also inhibited depolarization-evoked release of the pro-nociceptive neurotransmitter calcitonin gene-related peptide (CGRP) in the spinal cord. Increased CRMP2 phosphorylation in rats with spared nerve injury (SNI) was decreased by intrathecal administration of (S)-LCM resulting in a loss of presynaptic localization of CaV2.2 and NaV1.7. Together, these findings indicate that CRMP2 regulates presynaptic excitatory neurotransmission in spinal cord and may play an important role in regulating pathological pain. Novel targeting strategies to inhibit CRMP2 phosphorylation by Cdk5 may have great potential for the treatment of chronic pain. [ABSTRACT FROM AUTHOR]

Abstract: The voltage‐gated sodium channel NaV1.7 plays a critical role in pain pathways. We generated an epitope‐tagged NaV1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of NaV1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo. The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the NaV1.7 channel. [ABSTRACT FROM AUTHOR]

Purpose: The pathomechanisms of pain resulting from lumbar disc herniation have not been fully elucidated. Prostaglandins and cytokines generated at the inflammatory site produce associated pain; however, non-steroidal anti-inflammatory drugs and steroids are sometimes ineffective in patients. Tetrodotoxin-sensitive voltage-gated sodium (NaV) channels are related to sensory transmission in primary sensory nerves. The sodium channel NaV1.7 has emerged as an attractive analgesic target. The purpose of this study was to evaluate pain-related behavior and expression of NaV1.7 in dorsal root ganglia (DRG) after combined sciatic nerve compression and nucleus pulposus (NP) application in rats. Methods: Rats were divided into three groups and underwent either sciatic nerve compression with NP for 2 s using forceps ( n = 20), sham operation with neither compression nor NP ( n = 20), or no operation (controls, n = 20). Mechanical hyperalgesia was measured every second day for three weeks using von Frey filaments. NaV1.7 expression in L5 DRG was examined 7 and 14 days after surgery using immunohistochemistry. The number of neurons immunoreactive for NaV1.7 was compared among the three groups. Results: Mechanical hyperalgesia was found over the 14-day observation in the nerve compression plus NP application group, but not in the sham-operated or control groups ( P < 0.05). NaV1.7 expression in L5 DRG was up-regulated in the nerve compression plus NP application group, compared with sham-operated and control rats ( P < 0.01). Conclusions: Our results indicate that nerve compression plus NP application produces pain-related behavior. We conclude that NaV1.7 expression in DRG neurons may play an important role in mediating pain from sciatic nerves after compression injury and exposure to NP. [ABSTRACT FROM AUTHOR]

Summary: Voltage-gated sodium channels are essential for electrogenesis in excitable cells. The isoform Nav1.7 is primarily expressed in nociceptors. Mutations of the SCN9A gene, which codes for the α-subunit of Nav1.7, are the cause of primary erythromelalgia and paroxysmal extreme pain disorder, two rare neuropathic pain conditions. Recent studies have shown that mutations in the SCN9A gene are the cause of a subgroup of idiopathic small fiber neuropathies and that polymorphisms of SCN9A are associated with an increase in susceptibility to pain. These findings not only contribute to the understanding of the pathophysiology of neuropathic pain but also offer targets for a more specific pain therapy. [ABSTRACT FROM AUTHOR]

A number of agents from diverse pharmacological classes are used to treat neuropathic pain associated with diabetic peripheral neuropathy. Only three of these have regulatory approval for this indication in the U.S. In this focused article, I will discuss selected drugs, newly approved or in development, to treat neuropathic pain in patients with diabetic neuropathy. These will include agonists and antagonists of the transient receptor potential channels, a family of receptor proteins that play a role in the transduction of physical stress; sodium channel isoform specific antagonists; a recently approved dual-action opioid receptor agonist-norepinephrine reuptake inhibitor; gene therapy for neuropathic pain; and anti-nerve growth factor molecules. Mechanisms of action, preclinical supporting data, clinical trial evidence, and adverse effects will be reviewed. [ABSTRACT FROM AUTHOR]

We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 ( DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype-phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM. [ABSTRACT FROM AUTHOR]

Purpose: In vivo and ex vivo inhibition of ectopic activity of clinically used and newly developed sodium channel (NaV) blockers were quantified in the rat spinal nerve ligation (SNL) model using a pharmacokinetic-pharmacodynamic (PKPD) approach and correlated to in vitro NaV1.7 channel inhibition and clinical effective concentrations. Methods: In vivo, drug exposure and inhibition of ectopic activity were assessed in anaesthetized SNL rats at two dose levels. Ex vivo, compounds were applied at increasing concentrations to dorsal root ganglias isolated from SNL rats. The inhibitory potency ( IC) was estimated using PKPD analysis. In vitro IC was estimated using an electrophysiology-based assay using recombinant rat and human NaV1.7 expressing HEK293 cells. Results: In vivo and ex vivo inhibition of ectopic activity correlated well with the in vitro inhibition on the rat NaV1.7 channel. The estimated IC for inhibition of ectopic activity in the SNL model occurred at similar unbound concentrations as clinical effective concentrations in humans. Conclusions: Inhibition of ectopic activity in the SNL model could be useful in predicting clinical effective concentrations for novel sodium channel blockers. In addition, in vitro potency could be used for screening, characterization and selection of compounds, thereby reducing the need for in vivo testing. [ABSTRACT FROM AUTHOR]

Chronic pain often represents a severe, debilitating condition. Up to 10% of the worldwide population are affected, and many patients are poorly responsive to current treatment strategies. Nociceptors detect noxious conditions to produce the sensation of pain, and this signal is conveyed to the CNS by means of action potentials. The fast upstroke of action potentials is mediated by voltage-gated sodium channels, of which nine pore-forming α-subunits (Nav1.1–1.9) have been identified. Heterogeneous functional properties and distinct expression patterns denote specialized functions of each subunit. The Nav1.7 and Nav1.8 subunits have emerged as key molecules involved in peripheral pain processing and in the development of an increased pain sensitivity associated with inflammation and tissue injury. Several mutations in the SCN9A gene encoding for Nav1.7 have been identified as important cellular substrates for different heritable pain syndromes. This review aims to cover recent progress on our understanding of how biophysical properties of mutant Nav1.7 translate into an aberrant electrogenesis of nociceptors. We also recapitulate the role of Nav1.8 for peripheral pain processing and of additional sodium channelopathies which have been linked to disorders with pain as a significant component. [ABSTRACT FROM AUTHOR]

Familial primary erythromelalgia is a rare autosomal dominant disease characterized by redness and painful episodes of the feet and hands, which is often triggered by heat or exercise. In this report, a Taiwanese family with the characteristic features of erythromelalgia is described. Genetic linkage studies established that the disease locus maps to human chromosome 2. Sequence analysis indicated that the disease segregates with a novel mutation in the alpha subunit of the voltage-gated sodium channel (SCN9A or Nav1.7). The change observed is predicted to cause the substitution of a highly conserved isoluecine 136 for a valine within the first segment of the transmembrane domain (D1S1). Using immuno-histochemistry to stain a skin biopsy specimen from the affected region, we demonstrate that there is a significant reduction in the number of small fibers. [ABSTRACT FROM AUTHOR]

Neuroendocrine differentiation has been associated with prostate cancer (CaP). Brn-3a (short isoform) and Brn-3c, transcriptional controllers of neuronal differentiation, were readily detectable in human CaP both in vitro and in vivo. Brn-3a expression, but not Brn-3c, was significantly upregulated in >50% of tumours. Furthermore, overexpression of this transcription factor in vitro (i) potentiated CaP cell growth and (ii) regulated the expression of a neuronal gene, the Nav1.7 sodium channel, concomitantly upregulated in human CaP, in an isoform-specific manner. It is concluded that targeting Brn-3a could be a useful strategy for controlling the expression of multiple genes that promote CaP.Prostate Cancer and Prostatic Diseases (2006) 9, 83–91. doi:10.1038/sj.pcan.4500837; published online 8 November 2005 [ABSTRACT FROM AUTHOR]

Functional expression of voltage-gated sodium channel α-subunits (VGSCαs), specifically Nav1.7, is associated with strong metastatic potential in prostate cancer (CaP) in vitro. Furthermore, VGSC activity in vitro directly potentiates processes integral to metastasis. To investigate VGSCα expression in CaP in vivo, immunohistochemistry and real-time PCR were performed on human prostate biopsies (n>20). VGSCα immunostaining was evident in prostatic tissues and markedly stronger in CaP vs non-CaP patients. Importantly, RT-PCRs identified Nav1.7 as the VGSCα most strikingly upregulated (∼20-fold) in CaP, and the resultant receiver-operating characteristics curve demonstrated high diagnostic efficacy for the disease. It is concluded that VGSCα expression increases significantly in CaP in vivo and that Nav1.7 is a potential functional diagnostic marker.Prostate Cancer and Prostatic Diseases (2005) 8, 266–273. doi:10.1038/sj.pcan.4500796; published online 9 August 2005 [ABSTRACT FROM AUTHOR]