In human dorsal root ganglia (DRG), and trigeminal (TG) neurons, the various voltage-gated sodium channel (Nav) isoforms play critical roles in the firing of action potentials, which drive electrical impulses that encode somatosensations including, itch, and pain. The SCN11A gene encodes the tetrodotoxin (TTX)-resistant voltage-gated sodium channel Nav1.9, characterized by unique gating properties. Unlike other isoforms, the Nav1.9 channel activates and inactivates slowly and has a hyperpolarized voltage-dependence of activation and depolarized voltage-dependence of inactivation. This leads to a large window current that has been suggested to function as a regulator of the resting membrane potential of neurons. Mutations in Nav1.9 channels lead to congenital insensitivity to pain (gain-of-function) or familial episodic pain syndrome (loss-of-function) suggesting the channel is a critical mediator of pain. Despite its relevance in pain pathophysiology, most existing data relies on rodent models or heterologous expression systems, leaving the specific pharmacology and biophysical behavior of these channels in human primary neurons largely unknown. In this study, we pharmacologically isolated and characterized native Nav1.9 channel currents in human DRG and TG neurons to compare their biophysical profiles. Our findings reveal significant kinetic and voltage-dependent differences between the two populations. Specifically, Nav1.9 channels in TG neurons exhibit a right-shifted steady-state inactivation curve, a larger window current, and faster activation kinetics compared to those in DRG neurons. In addition, conditions that simulate inflammatory states in-vivo greatly potentiates the Nav1.9 currents consistent with similar observations in rodent models. By detailing these distinct biophysical properties, this research offers crucial insights into Nav1.9 channel function relevant for drug discovery efforts aimed at developing analgesics for both acute and chronic pain.

The voltage-gated sodium channel Nav1.8 (SCN10A) has strong genetic and pharmacological validation as a potential target for treating acute and chronic pain. While several different chemotypes have been advanced as selective inhibitors, a quinoxaline carboxamide core structure was identified as a particularly attractive core structure due to very high sodium channel subtype selectivity. However, poor solubility and overall ADME properties need to be improved. Scaffold hopping to a central trifluoromethyl pyridine followed by optimization of distal substituents resulted in improved overall properties. Several advanced lead compounds have been identified with excellent potency, selectivity, solubility, and pharmacokinetics. Preliminary mechanism of action studies suggest that this class of compounds are voltage and state independent inhibitors that bind to a novel site on the Nav1.8 channel.

The gene KCNT1 encodes the sodium-activated potassium channel KNa1.1 (Slack, Slo2.2). Variants in the KCNT1 gene induce a gain-of-function (GoF) phenotype in ionic currents and cause a spectrum of intractable neurological disorders in infants and children, including epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Effective treatment options for KCNT1-related disease are absent, and novel therapies are urgently required. We describe the development of a novel class of oxadiazole KNa1.1 inhibitors, leading to the discovery of compound 31 that reduced seizures and interictal spikes in a mouse model of KCNT1 GoF.

The Nav1.9 voltage dependent sodium channel has long been a desirable target for potential pain therapeutics owing to the highly restricted expression in peripheral sensory neurons. More recently human genetic evidence has been produced which directly links Nav1.9 to human pain disorders, increasing the motivation to develop pharmacological screening methodologies. The primary barrier to assay development has been removed with the generation of robust recombinant Nav1.9 cell lines which recapitulate the unique biophysical properties of the channel. We have previously reported the generation and characterization of human, mouse and rat Nav1.9 channels stably expressed in human HEK-293 cells which exhibit the slowly activating and inactivating inward sodium channel currents that are characteristic of native Nav1.9 (Lin et al, 2016). Nanion's SyncroPatch 384PE high throughput electrophysiology (HTEP) system allows for high fidelity assessment and detailed biophysical characterization of NaV channels with up to 384 simultaneous recordings. To accelerate the pharmacological characterization of NaV1.9 we have sought to develop HTEP assays on the Syncropatch platform. Here we report that SyncroPatch recordings of recombinant Nav1.9 channels can recapitulate the pharmacological properties of Nav1.9. Our results show stable recordings of human and rat orthologs of Nav1.9. Recordings in the absence and presence of known Nav1.9 inhibitors (lidocaine, tetracaine, TC-N 1752) were robust and stable, allowing for accurate measurements of compound IC50s. Additionally, biophysical properties of voltage-dependent activation and inactivation properties were characterized. Collectively, these results show that Nav1.9 channels can studied on high throughput electrophysiological systems, facilitating the search for novel pain therapeutics.

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective NaV1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective NaV1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.

In this article, we describe the discovery of an aryl ether series of potent and selective Nav1.3 inhibitors. Based on structural analogy to a similar series of compounds we have previously shown bind to the domain IV voltage sensor region of Nav channels, we propose this series binds in the same location. We describe the development of this series from a published starting point, highlighting key selectivity and potency data, and several studies designed to validate Nav1.3 as a target for pain.

Nav1.8 voltage gated sodium channels are expressed predominantly in peripheral nociceptors, playing a key role in action potential propagation in these neurons. Nav1.8 knockout and antisense studies indicate that the channel has a major function in nociceptive processing in preclinical models of pain and histological samples suggest that Nav1.8 in expressed on primary afferent terminals innervating ‘painful’ human tissues. Small molecule modulators selective for Nav1.8 from several chemical series have been disclosed including PF-1247324 (IC50: 199 nM against human Nav1.8) and A-803467 (IC50: 9 nM). Here we describe the in vitro biophysical properties of these compounds and identify amino acid residues within the pore domain that are important for compound-channel interaction. While the compounds are broadly similar in terms of selectivity, they can be differentiated by their unique use- and state-dependence of inhibition. PF-1247324 exhibits significant positive use- and state-dependence of block similar to that observed for local anesthetics (LA). In contrast, A-803467 shows negative use and state dependence (partial loss of block) that depends on the magnitude and duration of membrane depolarization. These different biophysical profiles of the two chemotypes appear to result from distinct interactions with the channel. Alanine mutations were constructed at two sites important for LA binding (F1710A and Y1717A). In addition, unique residues present in the S6 segments of Domains II and III of Nav1.8 but not TTX-sensitive Nav channels were mutated. We found that the affinity of PF-1247324 was significantly reduced by mutations at the LA binding site as well as Nav1.8 specific residues, whereas A-803467 affinity was reduced most dramatically by mutation of one unique site. The data suggest PF-1247324 and A-803467 interact with distinct yet partially overlapping regions of the pore domain of Nav1.8.

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7’s role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.

Voltage-gated sodium (Nav) channel inhibitors are used clinically as analgesics and local anesthetics. However, the absence of Nav channel isoform selectivity of current treatment options can result in adverse cardiac and central nervous system side effects, limiting their therapeutic utility. Human hereditary gain- or loss-of-pain disorders have demonstrated an essential role of Nav1.7 sodium channels in the sensation of pain, thus making this channel an attractive target for new pain therapies. We previously identified a novel, state-dependent human Nav1.7 selective inhibitor (PF-05089771, IC50 = 11 nM) that interacts with the voltage-sensor domain (VSD) of domain IV. We further characterized the state-dependent interaction of PF-05089771 by systematically varying the voltage, frequency, and duration of conditioning prepulses to provide access to closed, open, and fast- or slow-inactivated states. The current study demonstrates that PF-05089771 exhibits a slow onset of block that is depolarization and concentration dependent, with a similarly slow recovery from block. Furthermore, the onset of block by PF-05089771 develops with similar rates using protocols that bias channels into predominantly fast- or slow-inactivated states, suggesting that channel inhibition is less dependent on the availability of a particular inactivated state than the relative time that the channel is depolarized. Taken together, the inhibitory profile of PF-05089771 suggests that a conformational change in the domain IV VSD after depolarization is necessary and sufficient to reveal a high-affinity binding site with which PF-05089771 interacts, stabilizing the channel in a nonconducting conformation from which recovery is slow.

Voltage-gated sodium channel (Nav) inhibitors are used clinically as analgesics and local anesthetics. However, the absence of Nav channel isoform selectivity of current treatment options can result in adverse cardiac and CNS side effects, limiting their therapeutic utility. Human hereditary gain- or loss-of-pain disorders have demonstrated an essential role of Nav1.7 sodium channels in the sensation of pain, thus making this channel an attractive target for new pain therapies. We have identified a novel, human Nav1.7 selective inhibitor (PF-05089771, IC50 = 11 nM) that preferentially interacts with, and stabilizes, inactivated conformation(s) of the channel via an interaction with the voltage-sensor domain (VSD) of Domain 4. The current study demonstrates that PF-05089771 exhibits concentration-dependent slowly developing inhibition (tau = 209 sec and 33 sec, at 100 nM and 1 μM, respectively), and a similarly slow recovery from block upon washout (tau ∼7 min). PF-05089771 exhibits minimal use-dependent inhibition until concentrations exceed 10-fold the IC50, which is consistent with the observed slow onset of block and/or a low affinity for resting or fast-inactivated channel conformations. To evaluate this further, we employed whole cell patch clamp protocols to separate channels into predominantly fast- or slow-inactivated Nav populations. Inhibition by PF-05089771 develops with similar rates using protocols that biases for either fast- or slow-inactivated states, suggesting that preference for a particular inactivated state (fast, intermediate or slow) appears less critical than the relative time that the channel is in an inactivated state during compound exposure. The inhibition profile of PF-05089771 suggests that a conformational change in the Domain 4 VSD couples to multiple downstream inactivated states and immobilizing the voltage-sensor via a small molecule interaction with this site may lock the channel into long term inactivation from which recovery is slow.

BACKGROUND AND PURPOSE:NaV 1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF-01247324, a new generation, selective, orally bioavailable Nav 1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACH:The inhibition of Nav 1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain. KEY RESULTS:PF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50 : 331 nM) and in recombinantly expressed h Nav 1.8 channels (IC50 : 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav 1.5 channels (IC50 : ∼10 μM) and 65-100-fold selectivity over TTX-sensitive (TTX-S) channels (IC50 : ∼10-18 μM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC50 448 nM, and the block of both human recombinant Nav 1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models. CONCLUSIONS AND IMPLICATIONS:Using PF-01247324, we have confirmed a role for Nav 1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav 1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.

Voltage-gated sodium channels (Navs) are an important family of transmembrane ion channel proteins and Nav drug discovery is an exciting field. Pharmaceutical investment in Navs for pain therapeutics has expanded exponentially due to genetic data such as SCN10A mutations and an improved ability to establish an effective screen sequence for example IonWorks Barracuda®, Synchropatch® and Qube®. Moreover, emerging clinical data (AZD-3161, XEN402, CNV1014802, PF-05089771, PF-04531083) combined with recent breakthroughs in Nav structural biology pave the way for a future of fruitful prospective Nav drug discovery.

Significance Voltage-gated sodium (Na v ) channels contribute to physiological and pathophysiological electrical signaling in nerve and muscle cells. Because Na v channel isoforms exhibit tissue-specific expression, subtype selective modulation of this channel family provides important drug development opportunities. However, most available Na v channel modulators are unable to distinguish between Na v channel subtypes, which limits their therapeutic utility because of cardiac or nervous system toxicity. This study describes a new class of subtype selective Na v channel inhibitors that interact with a region of the channel that controls voltage sensitivity. This interaction site may enable development of selective therapeutic interventions with reduced potential for toxicity.

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Nav1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Nav1.8 channel blockers. A-887826 exemplifies this series and potently (IC50=11nM) blocked recombinant human Nav1.8 channels. A-887826 was ∼3 fold less potent to block Nav1.2, ∼10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na+) currents and was >30 fold less potent to block NaV1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na+) currents (IC50=8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund’s adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by ∼4mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Nav1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Nav1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Nav1.8 sodium channels in pathological pain states.

Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.

A series of aryl-substituted nicotinamide derivatives with selective inhibitory activity against the Na(v)1.8 sodium channel is reported. Replacement of the furan nucleus and homologation of the anilide linker in subtype-selective blocker A-803467 (1) provided potent, selective derivatives with improved aqueous solubility and oral bioavailability. Representative compounds from this series displayed efficacy in rat models of inflammatory and neuropathic pain.

BACKGROUND: A non-opioid receptor-mediated inhibition of sodium channels in dorsal root ganglia (DRGs) by kappa-opioid receptor agonists (kappa-ORAs) has been reported to contribute to the antinociceptive actions in animals and humans. In this study, we examined structurally diverse kappa-ORAs for their abilities to inhibit tetrodotoxin-resistant (TTX-r) sodium channels in adult rat DRGs.METHODS: Whole-cell recordings of TTX-r sodium currents were performed on cultured adult rat DRGs. Structurally diverse kappa-ORAs were studied for their abilities to inhibit TTX-r sodium channels.RESULTS: The racemic kappa-ORA, (+/-)U50,488, inhibited TTX-r sodium currents in a voltage-dependent manner, yielding IC50, values of 49 and 8 mu M, at prepulse potentials of -100 and -40 mV, respectively. Furthermore, we found that both the kappa-ORA U50,488 active enantiomer 1S,2S U50,488 and the inactive enantiomer 1R,2R U50,488 were equally potent inhibitors of TTX-r sodium currents. Structurally related kappa-ORAs, such as BRL 52537 and ICI 199,441 also inhibited TTX-r sodium currents. However, sodium channel inhibition and kappa-opioid receptor agonism have a distinct structure-activity relationship because another kappa-ORA (ICI 204,488) was inactive versus TTX-r sodium channels. We further investigated the sodium channel block of this class of compounds by studying (+/-)U50,488. (+/-)U50,488 was found to preferentially interact with the slow inactivated state of TTX-r sodium channels and to retard recovery from inactivation.CONCLUSION: Our results suggest that TTX-r sodium channels can be inhibited by many kappa-ORAs via an opioid receptor-independent mechanism. Although the potency for sodium channel inhibition is typically much less than apparent affinity for opioid receptors, sodium channel block may still contribute to the antinociceptive effects of this class of compounds. (Anesth Analg 2009;109:632-40)