Gated Sodium Channel Isoform NaV1.7 - ACS Publications

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Perspective

Challenges and Opportunities for Therapeutics Targeting the Voltage-Gated Sodium Channel Isoform Na 1.7 V

John V. Mulcahy, Hassan Pajouhesh, Jacob Beckley, Anton Delwig, J. Du Bois, and John Hunter J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01906 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019

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Journal of Medicinal Chemistry

Challenges and Opportunities for Therapeutics Targeting the VoltageGated Sodium Channel Isoform NaV1.7 John V. Mulcahy*#, Hassan Pajouhesh#, Jacob T. Beckley##, Anton Delwig#, J. Du Bois‡, John C. Hunter# # SiteOne Therapeutics, 280 Utah Ave, Suite 250, South San Francisco, CA 94080 ## SiteOne Therapeutics, 351 Evergreen Drive, Suite B1, Bozeman, MT 59715 ‡ Stanford University, Lokey Chemistry and Biology, 337 Campus Drive, Stanford, CA 94305

Abstract Voltage-gated sodium ion channel subtype 1.7 (NaV1.7) is a high interest target for the discovery of non-opioid analgesics. Compelling evidence from human genetic data, particularly the finding that persons lacking functional NaV1.7 are insensitive to pain, has spurred considerable effort to develop selective inhibitors of this Na+ ion channel target as analgesic medicines. Recent clinical setbacks and disappointing performance of preclinical compounds in animal pain models, however, have led to skepticism around the potential of selective NaV1.7 inhibitors as human therapeutics. In this Perspective, we discuss the attributes and limitations of recently disclosed investigational drugs targeting NaV1.7, and review evidence that, by better understanding the requirements for selectivity

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and target engagement, the opportunity to deliver effective analgesic medicines targeting NaV1.7 endures.

A. Introduction Voltage-gated sodium ion channels (NaVs) are integral membrane proteins comprising a pore-forming -subunit and two accessory -subunits.1 These ~260 kD transmembrane proteins undergo conformational changes in response to membrane depolarization and open transiently to allow passage of Na+ ions down an electrochemical gradient into the cell. The fast depolarization events produced by NaV channel opening result in generator potentials, action potentials, and pacemaker potentials depending on cell localization and tissue distribution. Ten mammalian isoforms of the NaV -subunit have been identified (NaV1.1–1.9, Nax), which differ in primary sequence, tissue distribution and gating characteristics.2,3 A number of clinically prescribed small molecule therapeutics modulate NaV activity such as local anesthetics, anticonvulsants and antiarrhythmics.4 Most drugs exhibit limited NaV subtype-selectivity but display remarkably distinct pharmacology, a property that has been attributed to differences in drug affinities for closed, open and inactivated conformations of the channel (“state-dependence”). Drug efficacy may also reflect the influence of ligand


2

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binding on the distribution of protein conformations, for instance, by shifting the voltage dependence of activation or inactivation.5 Design of therapeutics targeting NaVs is complicated by the difficulty of understanding the biophysical and pharmacodynamic (PD) consequences of ligand binding to different conformations of the channel. The finding that certain NaV isoforms, particularly NaV1.7, 1.8 and 1.9, are expressed predominantly in unmyelinated and small diameter myelinated afferents that transmit nociceptive signals has led to widespread efforts to discover selective inhibitors of these particular subtypes.6 Selectivity is necessary for drug safety given the obligatory role of off-target NaV isoforms in central and peripheral neuronal conduction (NaV1.1–1.3, 1.6), skeletal muscle contraction (NaV1.4) and the cardiac action potential (NaV1.5).2 Interest in the discovery of selective inhibitors of NaV1.7 was further kindled by the findings that a rare genetic condition in which the channel is nonfunctional leads to insensitivity to pain, and that gain-of-function mutations to the same subtype are associated with episodic extreme pain syndromes such as erythromelalgia and paroxysmal extreme pain disorder.7– 9

Based on the biophysical properties of NaV1.7, localization in small diameter peripheral

afferents, human genetic findings and studies in transgenic mice, NaV1.7 has been vaunted as one the best validated targets for analgesic drug discovery.10,11 The availability of safe and effective NaV1.7 inhibitors would provide the prospect of reducing prescription of opioids and help alleviate the opioid crisis.12 Despite this excitement, clinical proof-of-concept has remained elusive with investigation of early lead compounds


3


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in acute and chronic pain trials resulting in limited analgesic benefit. Such clinical setbacks have led some to question the obligatory role of NaV1.7 in nociception and to suggest alternative hypotheses to explain the insensitivity to pain observed in humans with NaV1.7 loss-of-function mutations and rodent knockouts.13 In this Perspective, we highlight recent advances in the discovery of small molecule and peptide selective inhibitors of NaV1.7 and discuss a series of pressing questions regarding the physicochemical and pharmacological requirements for a safe and effective antagonist of this target. We review evidence that modest efficacy of early NaV1.7 inhibitors in preclinical and clinical studies stems from insufficient target engagement and discuss strategies to overcome the limitations of disclosed lead compounds in order to realize the therapeutic promise of analgesic medicines targeting NaV1.7.

B. Contribution of Sodium Channel Subtypes to Pain Signaling Multiple NaV isoforms including NaV1.3, NaV1.6, NaV1.7, NaV1.8 and NaV1.9 are involved in the transmission of pain signals by primary afferent neurons and have been proposed as targets for subtype-selective analgesics.14 NaV1.3 is not normally detected in adult primary afferents but is re-expressed and accumulates at the end of the transected fiber following nerve injury.15 This result led to interest in selective inhibitors of NaV1.3 as


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therapeutics for neuropathic pain. However, the finding that mechanical allodynia develops normally in NaV1.3 knockout mice calls into question whether selective inhibition of NaV1.3 would be sufficient for analgesia.16 NaV1.6 is expressed throughout both the central and peripheral nervous systems and concentrated at nodes of Ranvier in myelinated neurons where it is involved in the saltatory conduction of action potentials.14 Increased density of NaV1.6-positive nodes of Ranvier is observed in the spared nerve injury (SNI) model of neuropathic pain in which two of the three branches of the sciatic nerve are cut.17 Further, adult-onset knockout of NaV1.6 in primary sensory neurons attenuates development of mechanical allodynia, suggesting a role in peripheral sensitization.17 Despite these findings, achieving safe and effective analgesia by inhibition of NaV1.6 is likely to be challenging due to the broad expression of the isoform. Three sodium channel isoforms, NaV1.7, NaV1.8 and NaV1.9, are preferentially expressed in peripheral afferents and have garnered attention as targets for novel analgesics.14 NaV1.7 is expressed in different types of primary sensory neurons, including myelinated

A-

and

A-fibers,

and

unmyelinated

C-fibers.

Evidence

from

immunocytochemistry and electrophysiology recordings indicates that NaV1.7 expression is inversely correlated with neuronal size and conduction velocity, suggesting preferential expression in C- and A-fibers that convey nociceptive information.18 Staining with a NaV1.7-selective antibody has demonstrated that the channel is present throughout the length of the neuron, from the free nerve endings to the central terminals within the dorsal


5


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horn.19 Despite its association with the peripheral nervous system, NaV1.7 is also expressed in olfactory sensory neurons and discrete regions of the CNS including hypothalamic neurons.20,21 The gating properties of NaV1.7 are characterized by a voltage of half-maximal activation and fast activation kinetics similar to other tetrodotoxinsensitive (TTX-s) subtypes, but significantly slower closed-state inactivation (~150 ms for NaV1.7 compared to 20 ms for NaV1.6).22 The slow kinetics of closed-state inactivation allow the channel to remain in the closed state during subthreshold depolarizations and generate substantial ramp currents.23 The observations of localization at peripheral terminals, the ability to generate ramp currents and a low threshold for activation have led to a hypothesis that, in addition to contributing to axonal conduction, NaV1.7 serves as a threshold channel in nociceptors, amplifying small depolarizations caused by other channels to generate the action potential (Figure 1).11,14

Figure 1. Contribution of NaV1.7, NaV1.8 and NaV1.9 to the action potential in peripheral nociceptive neurons, adapted from Bennett et al.14 NaV1.7 and NaV1.9 function as threshold channels, amplifying small depolarizations driven by upstream ion channels.11,14,24 NaV1.7 also contributes to the rising phase of the action potential. NaV1.8 is activated at more depolarized potentials near 0 mV, contributes the majority of current to the rising phase of the action potential, and is capable of high frequency firing due to rapid recovery from inactivation.25–27


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The functional role of NaV1.7 as a threshold channel and significant contributor to the upstroke of the action potential in nociceptors may explain the profound insensitivity to acute thermal and mechanical pain observed in NaV1.7 knockout, conditional knockout and inducible knockout mice.28–30 Whereas global NaV1.7 knockout mice exhibit intact intra-epidermal C-fibers, certain human patients with NaV1.7 loss-of-function are reported to lack intra-epidermal C-fiber nociceptors as measured anatomically by skin biopsy and functionally by microneurography recordings.28,31 This finding suggests an important anatomic difference between mice and humans, although the functional consequence of NaV1.7 loss-of-function, insensitivity to acute pain, is similar in both species. The role of NaV1.7 in chronic, and particularly neuropathic pain states is less clear, with evidence that certain types of pain may be independent of NaV1.7.32 One case report describes a woman with genetic NaV1.7 loss-of-function who developed certain symptoms of neuropathic pain including reduced mechanical withdrawal thresholds, tingling and numbness following a pelvic fracture that resulted in an epidural hematoma impinging on the L5 nerve root.33 In adult onset inducible NaV1.7 knockout mice, mechanical allodynia developed following SNI, but was attenuated compared to control animals.30 In contrast, acetone-induced cold allodynia did not develop in the inducible knockout model.30 In a conditional mouse knockout in which NaV1.7 is not present in dorsal root ganglia (DRG) or sympathetic neurons, neither mechanical nor cold allodynia developed in chronic constriction injury (CCI) or spinal nerve transection (SNT) models


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but both were observed following treatment with the chemotherapeutic oxaliplatin.32 Viewed together, studies involving human patients with NaV1.7 loss-of-function and rodent knockout models confirm the obligatory role of NaV1.7 in acute nociception, but suggest a more nuanced role in the development and maintenance of allodynia in nerve injury models. NaV1.8, similar to NaV1.7, has generated significant interest as a target for the identification of isoform-selective analgesics. Early reports found that NaV1.8 is preferentially expressed in small diameter primary afferents with the functional characteristics of nociceptors.34 More recent studies demonstrated that NaV1.8 is also present in a population of low threshold mechanoreceptors and hypothalamic neurons.35,36 In contrast to NaV1.7, which exhibits a voltage of half maximal activation near –30 mV in multiple expression systems, NaV1.8 is half-maximally activated at more depolarized membrane potentials near 0 mV.25,26 This difference in the threshold for activation, taken in the context of other biophysical properties of the isoforms, has led to a model for action potential generation in nociceptive sensory neurons in which NaV1.7 sets the threshold for firing and NaV1.8 contributes the majority of current to the upstroke the action potential, setting the amplitude (Figure 1).31 NaV1.8 is also likely to be the main NaV isoform involved in repetitive firing, made possible by fast recovery from inactivation.27 Isoform selective NaV1.8 inhibitors have been reported by Abbott, Pfizer, Vertex and others, and characterized in rodent pain models.37–39 Recently Vertex reported


8

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promising clinical results with a selective NaV1.8 inhibitor, VX-150, in three phase 2 studies in patients with osteoarthritis, small fiber neuropathy, and following bunionectomy.40 Collectively, the available evidence suggest that NaV1.8 functions in concert with NaV1.7 and plays a major role in establishing the excitability of nociceptive sensory neurons. Characterization of the role of NaV1.9 in pain signaling has proven challenging due to instability of current in native neurons and difficulty expressing the isoform in heterologous systems. Recently, interest has been piqued by the identification of genetically-defined human pain disorders involving NaV1.9 and certain technical challenges to studying its functional properties have been overcome.41,42 Subcellular localization at the peripheral and central terminals of small diameter primary afferents and biophysical evidence that NaV1.9 is active at voltages near the resting membrane potential suggest that it serves as a threshold channel, albeit with slower activation/inactivation kinetics than the TTX-s isoforms NaV1.3 and NaV1.7.24 Behavioral studies in rodent knockouts indicate that NaV1.9 has a subtle role in setting thresholds for acute thermal and mechanical pain, but a more profound effect on hyperalgesia following inflammation and cold allodynia following nerve injury.43,44 Like NaV1.7, mutations in NaV1.9 are associated with rare pain disorders in humans, with examples of both episodic extreme pain and insensitivity to pain.41,42 Paradoxically, one mutation in NaV1.9 found in patients with insensitivity to pain, L811P, results in a hyperpolarized voltage-dependence of activation, a property that would be expected to render neurons hyperexcitable.41


9


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Functional characterization of L811P and a related mutation, L1302F, expressed heterologously in rat DRG neurons demonstrated persistent current and depolarization of the resting membrane potential to voltages that inactivated other NaV isoforms, effectively silencing a subpopulation of neurons.45 These results imply an important functional difference between insensitivity to pain associated with NaV1.7 and NaV1.9 mutations in humans.

C. Sodium Channel Conformational Changes and Binding Sites The voltage-gated sodium channel -subunit is composed of four homologous domains, each containing six transmembrane helices (S1–S6), arranged symmetrically around the ion-conducting pore. Each of the four domains undergoes conformational changes in response to variation in the electrical potential across the cell membrane. Cationic “voltage sensors” partially traverse the membrane and drive conformational changes that affect Na+ ion conductance (Figure 2). This mechanism was suggested in prescient work by Hodgkin and Huxley, and confirmed by experimental measurement of gating currents.46,47 Recent X-ray and cryoelectron microscopy (cryo-EM) studies of bacterial NaV homologs (NaVAb, NaVMs, NaVRh), cockroach sodium channel (NaVPaS), electric eel channel (NaV1.4-1), and human channels (hNaV1.4-1, hNaV1.7-1-2) reveal the structure and trajectory of the voltage sensors in exquisite detail.48–55 At hyperpolarized membrane potentials the S4 helices, which contain arginine residues, are


10

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drawn inward – the closed, “resting” conformation of the channel. Depolarization of the membrane shifts the equilibrium toward alternate conformations in which one or more S4 helices move ~5–10Å toward extracellular space. Movement of the S4 helices is coupled to “activation” and “inactivation” gating mechanisms that regulate conduction of Na+ ions through the central pore. While significant questions remain regarding the coupling of specific voltage sensor movements with the opening and closing of different gating mechanisms, a compelling body of evidence indicates that activation of the domain I–III voltage sensors is associated with channel opening whereas activation of the domain

IV

voltage

sensor

results

in

movement

of

a

hydrophobic

isoleucine/phenylalanine/methionine (IFM) motif and fast inactivation.56–58 Channel closing, recovery from fast inactivation, and onset and recovery of slow inactivation are also voltage-dependent and likely coupled to changes in the position of voltage sensors, but the specific relationships are unclear. An incomplete understanding of the structure, dynamics and relative populations of the different NaV conformers and how small molecule ligands affect the conformational state of the channel present a substantial impediment to drug discovery programs.


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Figure 2. A) The NaV alpha subunit consists of four homologous domains arranged symmetrically around an ion-conducting pore. Each domain contains six transmembrane alpha helices, S1–S6. The voltage sensor (S4, green cylinder) partially traverses the membrane in response to voltage changes. B) Activation of domain I–III voltage sensors is coupled to NaV channel opening. Activation of domain IV is coupled to movement of the inactivation motif (IFM) and fast-inactivation.

At least seven discrete binding sites have been identified for ligands that modulate NaVs (Figure 3).59 At the voltage sensor domains (VSDs), peptide anemone, cone snail, scorpion and spider toxins bind to extracellular loops, interfering with NaV activation and inactivation.60 Cysteine rich peptides isolated from spider venoms that engage the S3–S4 loop of VSD II and trap the channel in the resting state have attracted particular attention for their selective pharmacology against NaV1.7 (Figure 3, binding site 2).61 Additionally, a class of small molecule sulfonamides that bind to a distinct site on VSD IV of NaV1.7 was disclosed in patent applications filed in 2009 (Figure 4, binding site 4).62,63 Whereas the spider toxins are potent against the resting state of the channel, the sulfonamide ligands preferentially bind the depolarized conformation of VSD IV.64,65 At the pore, cationic guanidinium compounds, saxitoxin (STX) and tetrodotoxin (TTX), associate with the extracellular vestibule and sterically obstruct Na+ ion conductance (Figure 3, binding site 1).66,67 Peptide µ-conotoxins isolated from the marine cone snail such as KIIIA bind to a proximal site.68 Subtype-selectivity has been profiled for a series of conotoxins across


12

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NaV1.1–NaV1.8.69 Classical local anesthetics, such as lidocaine, benzocaine and bupivacaine, bind to a discrete region of NaV that is accessible from the intracellular side of the channel or by diffusion through intramembrane fenestrations (Figure 3, binding site 3).70 Like the sulfonamides, local anesthetics preferentially bind to certain open or inactivated conformations of the channel.71 While both state-dependent, the preference of local anesthetics and sulfonamides for specific conformers of the channel are distinct, and may influence their pharmacology. Of the known NaV ligand binding sites, three have shown particular promise as targets for selective inhibition of NaV1.7: the sulfonamide binding site in VSD IV, the peptide toxin site at VSD II, and, recently, the extracellular pore.72,73

Figure 3. Approximate binding sites of compounds targeting NaV1.7 overlaid on cryo-EM structure of hNaV1.7-1-2 complex.55 (Left) Extracellular view of hNaV1.7. (Right) Cross section through hNaV1.7. Approximate ligand binding sites: (1) extracellular vestibule, (2) VSD II, (3) local anesthetic binding site, (4) VSD IV.

D. Classes of Selective NaV1.7 Inhibitors Sulfonamides binding to VSD IV


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A series of aryl sulfonamides that bind to VSD IV of NaV1.7 first appeared in patents filed by Pfizer and Icagen in 2009 (Figure 4).62,63 Whereas other compounds characterized as NaV1.7 inhibitors such as XEN-402 (1) and CNV-1014802 (2) exhibit limited isoform selectivity, early sulfonamides were reported as highly selective for NaV1.7 over a subset of off-target isoforms including NaV1.3, 1.4, 1.5 and 1.8; selectivity over NaV1.1, NaV1.2 and NaV1.6 is generally more modest.74 A development candidate PF-05089771 (3) was advanced to the clinic and studied in healthy volunteers with evoked pain endpoints, and in subjects with postoperative dental pain, osteoarthritis of the knee, diabetic peripheral neuropathy and hereditary erythromelalgia.75–77 Results disclosed from these studies have been disappointing (Table 1). Despite an IC50 of 11 nM against hNaV1.7 measured with a voltage protocol that favors the inactivated state and maximum plasma exposures > 10,000 ng/mL, a study in healthy volunteers that measured evoked pain and a study in patients with peripheral diabetic neuropathy did not meet efficacy endpoints.75,76 Observations from two studies, one with 235 subjects with postoperative dental pain, and the other with five subjects with inherited erythromelalgia were more promising, showing marginally significant reductions in pain scores at one or more time points.76,77 As of Oct 27, 2015 PF-05089771 (3) was no longer listed in Pfizer’s drug development pipeline.78


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Figure 4. Potency and selectivity of NaV1.7 inhibitors advanced to clinical development. 64,73,79–81 Table 1. Clinical Studies with PF-05089771

Indication Evoked pain in healthy volunteers75 Diabetic peripheral neuropathy76 Postoperative dental pain76

Subjects 25

141

Endpoint

Result

PF-05089771 (300 mg), PF05089771 + pregabalin, pregabalin, ibuprofen, placebo PF-05089771 (150 mg), PF05089711 + pregabalin, pregabalin, placebo

Thermal, UV, pressure, electrical and cold pain thresholds

Not significantly different than placebo

Daily pain numeric rating (NRS)

Trend toward lower NRS, not statistically significant

235

PF-05089771 (150–1600 mg), ibuprofen, placebo

Total pain relief, 0–6 hours (TOTPAR[6])

5

PF-05089771 (1600 mg), placebo, crossover design

Average pain score post dose (PI-NRS)

80

PF-05089771, placebo

Safety, tolerability, pharmacokinetics

Inherited erythromelalgia77 Osteoarthritis of the knee

Arms

Small statisticallysignificant effect at 150 mg dose Lower pain score at 4–5 h and 8–9 h post dose (P < 0.1) Not disclosed

Additional characterization of PF-05089771 (3) and related compounds has been disclosed,64,82

and

groups

from

Amgen,

Bristol-Myers

Squibb,

Chromocell,

Genentech/Xenon, Merck and other companies have pursued related chemical series (Figure 5).83–87 In several cases, high unbound plasma exposure (i.e. drug concentration corrected for plasma protein binding) relative to in vitro potency against NaV1.7 is


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required to achieve a pharmacodynamic effect in animal pain models.82,83,88,89 Sulfonamide 5 was studied in a mouse formalin pain model.82 Using an intravenous infusion to maintain unbound plasma concentrations > 60x the mouse NaV1.7 IC50 (< 0.1 nM), no evidence of analgesia was observed. First-generation sulfonamide inhibitors are largely sequestered by plasma proteins (≥ 99% plasma protein bound). Standard equilibrium dialysis methods of determining plasma protein binding are known to underestimate binding in such instances, possibly explaining the need for high unbound exposures.90 Merck has explored multiple series of aryl sulfonamides bearing basic amine functional groups with an emphasis on improving ligand efficiency and lipophilic ligand efficiency.87,88 Compound 6 exhibits an unbound fraction (fu) of 0.065 in mouse and was efficacious by subcutaneous (SC) administration in a formalin model at an unbound concentration of 1.5 µM, ~50x the mNaV1.7 IC50; however oral bioavailability was low (F = 2%, 10 mpk in rat).88 A related compound, 7, with improved oral availability (F = 50%) was effective at an unbound concentration ~100x the mNaV1.7 IC50. These exposure multiples are consistent with other reports of the pharmacokinetic/pharmacodynamic (PK/PD) relationship of sulfonamide inhibitors despite the greater unbound fraction and reduced lipophilicity. A more striking result from the team at Merck was achieved with compound 8, which is effective in a mouse formalin model at low multiples of 3.5x the mNaV1.7 IC50.87 This result is a significant improvement from the profile of earlier sulfonamides. Interpretation of these data are complicated,


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however, by the absence of potency measurements against off-target isoforms such as NaV1.2 and NaV1.6, inhibition of which could contribute to efficacy but also produce undesirable central nervous system, sensory or motor deficits.91,92


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Figure 5. NaV1.7 inhibitors targeting VSD IV evaluated in rodent pain behavior models. “+” indicates effective in PD model. “–” indicates not effective. *Kd of GX-585 against mouse TTX-resistant current = 22 µM **Kd of GX-201 against mouse TTX-resistant current = 13 µM. 30,82–84,86–89,93

An additional hypothesis for why high exposure multiples are required for analgesia with many NaV1.7-selective sulfonamides proposes that free drug concentration in plasma is not representative of drug concentration at the site of action.84 A team at Bristol Myers Squibb identified a series of sulfonamide derivatives bearing amine functional groups, which display good selectivity over NaV1.5, reminiscent of compounds disclosed by Merck.84 Selectivity over other off-target NaV isoforms was not reported. Quantification of drug concentrations in dissected mouse DRG one hour after oral (po) dosing showed that total DRG concentrations correlated with apparent permeability (Papp) and ranged from 0.23–6.3x free plasma concentration across a series of four compounds. Two compounds, 9 and 10, were identified with high permeability in a PAMPA assay (Papp pH 7.4 > 20 x 10-6 cm/s), total DRG/free plasma ratios > 5 and similar potency against mNaV1.7. Compound 10 reduced pain behaviors in the mouse formalin assay at total DRG concentrations corresponding to 30x the mNaV1.7 IC50 but 9, at a similar exposure multiple, was ineffective. A measurement of the unbound drug concentrations in DRG would improve interpretation of these findings. Unbound DRG concentrations were measured for a series of indole-acylsulfonamides including 11, however the results remain confounding. 11 was effective in the mouse formalin, complete Freund’s adjuvant (CFA), and CCI pain models at doses that achieve unbound DRG exposures of ~3x the mNaV1.7 IC50 (35 nM) and ~9x the mNaV1.7 DRG IC50 (11 nM), whereas a closely related compound


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that achieved similar unbound DRG exposures was not. One explanation is that measurement of unbound drug concentration in DRG tissue is technically challenging, and may not accurately represent DRG exposure. Alternatively, factors other than poor distribution to DRG neuronal tissue may be responsible for the high exposures required for analgesia with many but not all sulfonamides. Genentech elucidated the X-ray structure of a NaV1.7/NaVAb chimera with a bound sulfonamide, confirming the association of this ligand with the S4 helix of VSD IV.94 In a collaboration with Xenon Pharmaceuticals, two candidates GDC-0276 (4) and GDC-0310 were advanced for clinical development.81 As of October 2018, development of both compounds has been discontinued for reasons that have not been disclosed. Recent publications have revealed specific in vivo target engagement and PK/PD models that presumably supported these development programs.86 By expressing the I848T hNaV1.7 gain-of-function mutation in mice, an aconitine-induced pain behavior model was established as a preclinical tool to assess NaV1.7 target engagement (IEM model).86 Whereas competitor compounds PF-05089771 (3) and MRL-5 (similar to 8) require free plasma concentrations > 30x NaV1.7 IC50 to reduce flinching in this model, a number of Genentech/Xenon acyl sulfonamide NaV1.7 inhibitors such as 12, 13 and 14 are active in the target engagement assay at concentrations ≤ 5x the NaV1.7 IC50. 30,86,89 Compound 12 is highly potent (IC50 = 0.6 nM) and closely resembles the development candidate GDC0276. A hypothesis has been put forward that improved target engagement at low


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exposure multiples compared to competitor compounds is driven by slow ligand-protein dissociation kinetics (koff < 10–4 sec-1).86 Because the kinetics of association are also slow, equilibrium IC50 values for 13 and 14 are difficult to measure.30 The equilibrium dissociation constant (Kd), determined by the kinetics of inhibition (koff/kon) is used as an alternative to IC50 for these compounds in PK/PD assessments. In wild type mice, 13 and 14 were effective in the formalin, CFA, streptozocin (STZ) diabetic neuropathy and SNI models of inflammatory and chronic pain at unbound plasma concentrations 2–12x NaV1.7 Kd. The half maximal effective concentration (EC50) of both compounds in the STZ model decreased by 3–10 fold following 12 days of dosing for reasons that are not understood but may involve a reversal of sensitization driven by suppression of electrical activity. Higher unbound plasma exposures of 13 and 14, 10–30x mNaV1.7 Kd (Figure 6), were required to affect the threshold for response to noxious thermal stimulus (hot plate, tail immersion), noxious mechanical stimulus (Randall-Selitto assay), and thermal stimulus after inflammation (CFA-Hargreaves).30 The availability of an inducible NaV1.7 knockout model makes possible direct comparison of the pharmacodynamic effect of drug treatment to the theoretical maximum, the phenotype of the NaV1.7 knockout. While the effects of compounds 13 and 14 reached statistical significance in acute thermal and mechanical pain behavior assays, animals did not routinely reach the cut-off, as was the case with the knockout. This finding suggests that a larger effect size is possible at higher exposures or with superior target engagement.


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Figure 6. PK/PD relationship of GX-585 (13) and GX-201 (14) in mouse models of thermal, mechanical, chemical and inflammatory pain. “+” indicates effective in PD model. “–” indicates not effective. Ave. Cp = average total plasma concentration.30

One interpretation of the finding that higher unbound plasma concentrations of 13 and 14 are required to demonstrate a PD effect in acute thermal and mechanical pain models than in inflammatory or neuropathic pain models is that the steady-state dynamics between channel conformers in uninjured tissue favors the resting state for which the sulfonamide inhibitors have low affinity. In chronic or inflammatory pain states, depolarization of the resting membrane potential may shift the population of channel conformers toward the inactivated state, improving target engagement in a variable manner that is dependent on the specific pain condition. If this explanation is correct, state-independent inhibitors of NaV1.7 should exhibit a different PK/PD relationship than state-dependent inhibitors. The former may display more consistent pharmacology across a range of pain states because target engagement is not affected by differences in the distribution of NaV1.7 between channel conformers. For certain types of pain, statedependent inhibition would have the advantage of addressing pathological pain while leaving normal, protective pain reflexes intact. For other types of pain, state-independent inhibition may be necessary to achieve a robust analgesic effect at well-tolerated exposures. While studies with animal PD models provide useful insight into these


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questions, ultimately the comparative effects of state-dependent and state-independent NaV1.7 inhibitors will need to be explored in human patients to be properly understood. Amgen has reported a pair of atropisomeric quinolone sulfonamides having differential potencies against hNaV1.7.83 The more potent enantiomer, AMG8379 (15), exhibits an IC50 < 10 nM against hNaV1.7 and > 100x selectivity over NaV1.1–1.6. This compound was effective in mouse UVB burn-induced hyperalgesia (UVB), capsaicininduced nociceptive behavior and histamine-induced itch models at doses of 30–100 mg/kg po with terminal free plasma concentrations in the range of 5–23x mNaV1.7 IC50. The absence of locomotor effects in an open field assay at any dose tested and the lack of efficacy with the less potent atropisomer, AMG8380 (16), support the view that the anti-nociceptive effects are the result of on-target inhibition of mNaV1.7. Collectively, the available data generated with aryl and acyl sulfonamide inhibitors of NaV1.7 indicate that substantial improvements in NaV subtype selectivity and target engagement have been made since the discovery of first-generation investigational drug candidates such as PF05089771. Whether these improvements are sufficient to achieve robust analgesia in human pain indications with an acceptable margin of safety remains to be demonstrated.

Cystine knot peptides binding to VSD II


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A number of structurally-related, cysteine-rich peptides that bind to the extracellular face of VSD II have been isolated from venomous spiders.61 These cystine knot compounds, named for their three-dimensional loop structures arising from multiple disulfide bridges, preferentially bind to the resting state of the channel and trap the channel in a closed conformation. Certain peptides exhibit selectivity for hNaV1.7 over other human isoforms.95 However, interpretation of in vivo PD results is often complicated by off-target activity against other families of ion channels (e.g. CaV channels).96 GpTx-1, JzTx-V, Huwentoxin IV (HwTx-IV), ProTx-II (17) and Pn3a (18) have been the subject of investigation as therapeutics targeting NaV1.7 and are discussed as representative examples of this class (Figure 7). An initial report characterized ProTx-II as 100 to 500-fold selective for NaV1.7 (IC50 = 0.3 nM) over other hNaV subtypes (NaV1.2–1.6, 1.8 IC50s = 26–146 nM). 97 This study also found that ProTx-II was not analgesic in a rat CFA model of acute hyperalgesia by intravenous (iv) or intrathecal (it) administration at doses of 0.1 and 0.01 mg/kg, respectively.97 Higher doses were not tolerated and the absence of efficacy was attributed to limited permeability through the peripheral nerve sheath. Subsequent reports have indicated that the potency of ProTx-II against at least one subunit, NaV1.2, is affected by the presence of an accessory 4 subunit and that NaV subtype-selectivity is more modest than originally reported.98,99 Intrathecal administration of 2 µg of toxin in rats produced elevated thermal withdrawal latencies in the Hargreaves test and a significant reduction


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in phase I and phase II flinching in the formalin test.99 The therapeutic window was narrow; a dose of 0.8 µg had no effect and doses > 2 µg resulted in motor deficits, possibly due to inhibition of NaV1.1 or 1.6. In the absence of published pharmacokinetics (PK) data, it is difficult to understand what level of exposure relative to on- and off-target NaV IC50s was achieved by these dosing regimens.

Figure 7. Selective cystine knot toxins that bind to VSD II in NaV1.7. “+” indicates effective in PD model. “– ” indicates not effective. r = rat. m = mouse.99–101

µ-TRTX-Pn3a (Pn3a, 18), isolated from a tarantula venom, is also highly potent against hNaV1.7 (IC50 = 0.9 nM) with selectivity ranging from 40 to 1000-fold over all other NaV isoforms.100 Pn3a dose-dependently reverses pain behaviors induced by the NaV1.7 activator OD1 at 1 and 3 mg/kg ip, however 3 mg/kg ip produced no analgesic effect in response to inflammatory challenges induced by formalin, carrageenan, or CFA. Interestingly, Pn3a reduces mechanical hypersensitivity in a mouse model of post-surgical pain, an effect that was reversed by co-administration of the opioid antagonist naloxone.102 Pn3a acts on NaV1.7 by reducing peak current and shifting the voltagedependence of activation to more depolarized membrane potentials; due to this


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mechanism, NaV1.7 current is not fully abolished at all membrane potentials.100 The PK profile of Pn3a has not been reported. In the absence of additional data, these findings suggest that the lack of analgesic efficacy in certain rodent pain models may be due to insufficient inhibition of NaV1.7. The limited analgesic efficacy and narrow therapeutic window observed in studies with natural cystine knot peptides motivated the design of synthetic peptides with improved NaV subtype selectivity. Amgen and Janssen have advanced synthetic peptides that target VSD II with improved selectivity for NaV1.7 over other hNaV isoforms.95,99,101,103 Using a multi-attribute positional scan approach, Amgen has identified NaV1.7-selective analogues of the tarantula venom peptides GpTx-1 and JzTx-V. A single amino acid variation in GpTX-1, Ile10Glu, confers > 500-fold selectivity over two other peripheral subtypes, NaV1.4 and NaV1.5.101 Similarly, an analogue of JzTx-V AMG6120 (20), was identified that exhibits > 100-fold selectivity over NaV1.4–1.6 and NaV1.8.103 20 elicited an anti-pruritic effect in a mouse histamine-induced itch model following sc administration but required high plasma exposures, ~100x mNaV1.7 IC50. Interestingly, similar exposures were not effective in a capsaicin induced nociceptive behavior model. Following a different approach, Janssen prepared a library of over 1500 analogues of ProTx-II, from which JNJ63953918 (21) was identified and found to exhibit 100 to > 1000 fold selectivity over NaV1.1, 1.2, 1.4, 1.5 and 1.6 in a manual patch clamp assay.99 Like ProTx-II, 21 is believed to bind the closed, resting state of NaV1.7. Similar potency is


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observed in electrophysiological assays that favor the resting state (holding potential = – 120 mV) and an approximately 1:1 ratio of the resting/inactivated states (V1/2 inactivation). Species variability, which is often a confounding factor in interpreting behavioral results, was found to be minimal, with similar IC50s measured against human and rat peripheral isoforms NaV1.5, NaV1.6 and NaV1.7. Central and peripheral administration of 21 was studied in rat models of chemical and thermal pain; 0.3–7.5 µg JNJ63953918 (21) it dosedependently reduced flinching in phase 1 and phase 2 of the rat formalin model and increased withdrawal latencies in the tail flick and hot plate assays. Peri-sciatic (PS) administration of 0.8–2.5 mg increased the thermal withdrawal latency of the ipsilateral paw in a rat Hargreaves test with no evidence of discoordination or motor effects, indicating that the peptide is also effective by local peripheral administration. Interestingly, rats receiving the highest 2.5 mg PS dose exhibited increased thermal withdrawal latencies at both the ipsilateral and contralateral paws with no evidence of gross motor deficits. These findings support the obligatory role of NaV1.7 in the peripheral and spinal transmission of pain and suggest that inhibition in either compartment may be analgesic. Absent PK measurements with 17 or 21, it is difficult to understand the level of exposure required for analgesia by this mechanism. The finding that peripheral exposures of ~100x mNaV1.7 are required for a pharmacodynamic effect with 20 suggests that at least a subset of cystine knot peptides suffer from similar challenges with target engagement as first-generation aryl sulfonamides (vide supra).


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Guanidinium compounds binding to the extracellular vestibule TTX (22), STX (23) and the gonyautoxins are naturally occurring sodium channel inhibitors that bind to the extracellular vestibule and occlude the ion-conducting pore (Figure 8). These agents show little state-dependence, exhibiting < 3-fold variation in potency between electrophysiology protocols that favor the resting, open and inactivated conformations of the channel.104 While compounds that block the outer pore of the sodium channel are often dismissed in the pursuit of selectivity given the highly conserved nature of this region in all NaV subtypes, small sequence differences between isoforms exist and markedly affect ligand potency. A single amino acid variation in the domain I pore loop is responsible for the insensitivity of NaV1.5, 1.8, and 1.9 to the natural product TTX.105,106 TTX has been investigated in a number of rodent models of acute and chronic pain, and was effective at doses of 1–6 µg/kg sc in the formalin, acetic acid induced writhing, and sciatic nerve ligation (SNL) models.107 The finding that, despite relatively low potency against NaV1.5 compared to TTX-s NaV isoforms, TTX exhibits a median lethal dose of 12.5 µg/kg sc in mice is evidence that selectivity over non-cardiac subtypes (e.g., NaV1.4, NaV1.6) is necessary to achieve a high margin of safety with state-independent inhibitors.108 A formulation of TTX is currently in development at Wex Pharmaceuticals for the treatment of chemotherapy-induced peripheral neuropathy and cancer-related pain,


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and has been evaluated in multiple clinical studies at a doses up to 30 µg administered IM or sc.109

Figure 8. Natural and synthetic guanidinium compounds that bind to the extracellular pore. 105,107,110–112

STX (23) and certain gonyautoxins are ≥ 100-fold less potent against hNaV1.7 in comparison to other TTX-s hNaV isoforms.113 This difference in potency is primate specific; STX is essentially equipotent against other TTX-s NaV isoforms expressed in other species such as mouse, rat and dog. Mutagenesis experiments have demonstrated that a two amino acid variation in the pore loop of domain III is responsible for the difference in potency between hNaV1.7 and other TTX-s isoforms.111 In principal, this sequence variation could be leveraged to design selective, state-independent inhibitors of hNaV1.7. Modification of the C13-substituent in compound 23 to an acetate (24) improves potency against hNaV1.7 and selectivity over hNaV1.5.111 SiteOne Therapeutics has capitalized on these findings and disclosed a series of compounds that exhibit > 1000x selectivity for NaV1.7 over NaV1.4 and NaV1.6 (e.g., compound 25, Figure 8). Selectivity over other


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isoforms and characterization in pain behavior models is not reported.112 Future work with this class of compounds will inform our understanding of whether highly selective, stateindependent inhibitors of NaV1.7 more fully recapitulate the insensitivity to pain observed in humans with genetic NaV1.7 loss-of-function in comparison to state-dependent inhibitors with low activity against the (closed) resting state of the channel.

E. Discussion The limited clinical efficacy of certain potent and selective inhibitors of NaV1.7 raises a series of questions regarding the pharmacology of NaV1.7: 1. Is acute inhibition of NaV1.7 sufficient to produce robust analgesia? 2. If so, what level of NaV1.7 target occupancy is required for meaningful analgesia? 3. Do factors such as tissue barriers, underestimated protein binding or statedependence limit the efficacy of existing chemical series targeting NaV1.7? 4. What level of selectivity is required to avoid off-target effects mediated by other NaV isoforms? 5. Aside from analgesia, what other on-target effects may be expected from truly selective NaV1.7 inhibitors? In the discussion that follows, we review data and hypotheses surrounding each of these questions.


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Analgesia by acute inhibition Genetic and pharmacological evidence from humans and rodents supports the obligatory role of NaV1.7 in the transmission of nociceptive signals. NaV1.7 is the most abundant TTX-s sodium channel in small diameter human and mouse DRG neurons, where it accounts for 75–100% of TTX-s current.64 Inhibitors of NaV1.7 increase the action potential rheobase and elicit action potential failure in human DRG neurons at concentrations ~10x IC50.64 Unfortunately, disappointing outcomes from early clinical studies have generated skepticism that pharmacological inhibition of NaV1.7 is sufficient to produce robust analgesia. These studies were conducted with the nonselective statedependent NaV inhibitors XEN402 (1) and CNV1014802 (2), and with PF-05089771 (3), an isoform selective, state-dependent inhibitor that targets the inactivated state of the channel and shows very high plasma protein binding, both properties that may increase the plasma exposure required for efficacy. The failure of clinical drug candidates has been ascribed to the expression of putative gene products other than NaV1.7 that are required for loss of pain sensation.13 The observation that an endogenous opioid peptide, met-enkephalin, is upregulated 2–3 fold in conditional NaV1.7 knockout mice has been noted by multiple laboratories and supports the above hypothesis.13,114 However, the finding that an opioid antagonist, naloxone, partially reverses insensitivity to pain in NaV1.7 knockout mice is controversial.


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Minett et al. found that naloxone (2 mg/kg ip) reversed insensitivity to thermal (Hargreaves) and mechanical (Randall-Selitto) noxious stimuli in a sensory-neuron specific NaV1.7 KO.13 In contrast, researchers at Genentech found that naloxone did not affect thermal or mechanical nociceptive deficits in an adult onset conditional NaV1.7 knockout and a team from Merck found that naloxone did not affect analgesia mediated by a preclinical NaV1.7 inhibitor.114,115 Moreover, studies with the highly selective gating modifier peptide JNJ63953918 (21) indicate that true NaV1.7 inhibitors are effective in pain models in morphine-tolerant animals and within minutes of administration, faster than can be explained by changes in gene expression and are effective in morphinetolerant animals.99 While there may be an additive effect of increased expression of endogenous opioids, current evidence supports the conclusion that acute inhibition of NaV1.7 is sufficient to produce analgesia.

NaV1.7 target occupancy Genetic loss-of-function of NaV1.7 results in essentially complete elimination of Na+ currents mediated by this subtype. In comparison, pharmacological inhibition, especially at concentrations that minimize effects on off-target isoforms, is often incomplete. At this time, the level of NaV1.7 inhibition needed to reduce sensitivity to noxious stimuli remains an outstanding question. Some insight is obtained from ex vivo dose-response studies that determine the relationship between drug concentration and


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neuronal excitability. TTX is a useful tool compound for performing such measurements because it exhibits similar potency against human, rat, and mouse NaV1.7, very limited state- or use-dependence, and excellent selectivity over the two other principal NaV isoforms expressed in nociceptive afferent neurons, NaV1.8 and NaV1.9. In a study that used live cell imaging of rat DRG cultures to measure calcium release triggered by electrical field stimulation, 100 nM TTX (22) had a maximal effect, reducing the Ca2+ response by ~90%.116 Accordingly, concentrations of TTX ~10x the IC50 against rNaV1.7, which inhibit ~90% of NaV1.7 current, are sufficient to almost completely silence firing of cultured DRGs. Additional insight into the level of NaV1.7 inhibition required to affect pain behavior is gleaned from recent work from Genentech with an adult onset inducible knockout mouse.30 Following treatment with tamoxifen, NaV1.7 protein decreases exponentially with a half-life of ~0.7 weeks. Significant deficits in pain sensitivity are apparent two weeks after treatment (i.e., roughly 10–15% protein remaining) and continue to develop up to 6–8 weeks. These results are consistent with the view that inhibition of 80–90% of NaV1.7 is necessary for robust pharmacodynamic effects on pain behavior.

Factors that may limit efficacy One explanation for the limited efficacy of investigational drugs targeting NaV1.7 posits that certain factors conspire to prevent target engagement of ostensibly potent


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and selective chemotypes. Understanding the nature of these factors is an active area of research in NaV1.7 drug discovery programs. Outstanding questions include the importance of central vs peripheral exposure, the level of inhibition required for analgesia (vide supra), the relative availability of the resting vs the inactivated channel conformation, and barriers that may limit drug access or distribution to the target. Expression of NaV1.7 extends from the peripheral terminals of DRG neurons to the central terminals within the dorsal horn.19 The presence of NaV1.7 in the spinal cord raises the question of whether inhibition of the peripheral population is sufficient for analgesia, or whether distribution within the CNS is required. Preclinical results with aryl and acyl sulfonamides that have low brain/plasma ratios suggest that peripheral inhibition of NaV1.7 is sufficient for analgesia.83 Some caution is necessary in interpreting these data; given the high exposures required, one cannot rule out that efficacy in pain models could be a result of inhibition of multiple peripheral isoforms. The finding that the synthetic peptide JNJ63953918 (21) is analgesic by it or PS administration supports the position that inhibition of either the central or peripheral subpopulation of NaV1.7 is effective. The high selectivity of this compound and absence of gross motor deficits in treated animals increases confidence that the effects are mediated by inhibition of NaV1.7. Many NaV1.7-selective inhibitors including small molecules from the sulfonamide series and at least one cystine knot peptide (20) require unbound plasma concentrations ≥ 20–100x IC50 for efficacy in rodent PD models. The high exposures needed for efficacy


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may be partially explained if in fact 80–90% NaV1.7 occupancy is required for meaningful analgesia. In addition, factors such as very high protein binding, tissue barriers including the peripheral nerve sheath or blood brain barrier, rapid protein-ligand dissociation kinetics,86 state-dependent activity, and reduced potency in the presence of accessory subunits may adversely influence target engagement in vivo, giving rise to an apparent PK/PD disconnect. Accurate assessment of plasma protein binding can be difficult for lipophilic compounds, particularly as the bound fraction approaches ≥ 99%. Alternatively, the concentration of unbound drug in plasma may not correlate well with levels of exposure in neuronal tissue.84 Finally, the state-dependent nature of most inhibitors that target NaV1.7 (as measured against cells in culture) may complicate predictions of target engagement in live organisms, as the in vivo distribution of NaV1.7 conformers is not well understood and likely varies between tissues and pain states. The sulfonamide class of inhibitors binds to a site on VSD IV that is exposed upon membrane depolarization. In contrast, cystine knot peptides preferentially bind to the resting conformation of VSD II; guanidinium pore blockers exhibit little if any conformational preference. Understanding which of these factors limit efficacy of certain sulfonamide inhibitors and related investigational drugs is critical for advancing new small molecules that target NaV1.7.

Selectivity


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Achieving selectivity over off-target NaV isoforms is a formidable challenge in the development of NaV1.7 inhibitors. Due to the obligatory role of NaVs in the generation and propagation of action potentials, inhibition of off-target isoforms is a profound safety liability. An open question is what level of selectivity over different off-target isoforms, as measured by a functional in vitro assay such as whole-cell electrophysiology, is necessary for a satisfactory therapeutic window in vivo. The requirement for selectivity is compound specific, as differences in tissue distribution, state- and frequency dependent binding will augment or erode the margin of safety in ways that are difficult to predict from in vitro measurements. The gating modifier toxin JNJ63953918 (21) exhibits > 100x selectivity for hNaV1.7 over hNaV1.1, 1.2 and 1.4–1.6, as measured by whole-cell electrophysiology against recombinant NaV isoforms.99 A more modest 7–16x therapeutic window between the lowest analgesic dose and the onset of muscle weakness was observed in rats following intrathecal administration. This margin of safety is consistent with the view that analgesia requires > 80% block of NaV1.7 and that sensory-motor effects may occur at lower levels of off-target NaV inhibition. Amgen has reported that AMG8379 (15), a state-dependent sulfonamide NaV1.7 inhibitor that exhibits 100–1000x selectivity over other NaV isoforms, is effective in mouse histamine-induced itch and capsaicin-induced nociceptive behavior models at doses of 30 and 100 mg/kg po. The high dose did not cause a decrease in locomotor activity, as measured in an open field assay by automated beam breaks. The


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finding that a less potent atropisomer AMG8380 (16) is not effective in either behavioral model supports the conclusion that the anti-itch and anti-nociceptive effects of 15 are mediated by NaV1.7. Other reports describing the efficacy of sulfonamides do not discuss the influence of these compounds on locomotion. In addition, absent knowledge of the potency of sulfonamides towards off-target NaVs (preferably with appropriate rodent isoforms), it is not possible to conclude with certainty that analgesia is mediated by NaV1.7 block or by inhibition of more than one NaV isoform. In light of evidence that a high level of NaV1.7 inhibition is required for analgesia by acute dosing and that ostensibly potent and selective NaV1.7 inhibitors exhibit a narrow window between analgesia and adverse pharmacological effects, a high level of selectivity for NaV1.7, perhaps > 100x, may be necessary to attain the former. This level of selectivity may be achieved with compounds that display differential potency against NaV isoforms, functional selectivity driven by differences in NaV isoform tissue distribution, action potential frequency, and statedependent binding, or a combination of the two. If mild adverse physiological effects such as paresthesias or CNS side effects precede gross motor deficits, selectivity levels greatly exceeding 100x may be required to achieve a robust margin of safety in humans.

Additional on-target effects While pharmaceutical interest in NaV1.7 has primarily focused on the role of this subtype in pain sensation, recent work has demonstrated that NaV1.7 also mediates


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olfaction, cough, regulation of glucose sensing, and body weight.21,117–119 NaV1.7 is highly expressed in olfactory sensory neurons and appears to have a non-redundant role in the transmission of action potentials along a section of olfactory sensory neurons within the olfactory glomerulus.20 Mice and humans lacking functional NaV1.7 exhibit a loss of smell (anosmia), and NaV1.7 inhibitors have been shown to impact odor recognition. 30,117 In vagal sensory neurons that arise from the trachea, larynx and the lungs, NaV1.7 is the predominant NaV isoform, thus raising the possibility that NaV1.7 inhibitors could affect the cough reflex or urge to cough. Bilateral treatment of guinea pig nodose ganglia with adeno-associated virus containing NaV1.7 shRNA nearly abolished the cough response to a chemical irritant (0.1 M citric acid).118 NaV1.7 is also expressed in pancreatic -cells where it is involved in glucose sensing and insulin secretion. NaV1.7 knockout mice exhibit > 3fold higher insulin content in insulin-producing  cells than wild type controls.119 In mouse hypothalamic neurons expression of NaV1.7 prolongs excitatory postsynaptic potentials and is involved in integration of synaptic input. NaV1.7 knockout in Agrp+ and Pomc+ subtypes of neurons, respectively, resulted in a decrease and increase in bodyweight.21 These observations suggest additional indications for which selective inhibitors of NaV1.7 may prove useful and draw attention to potential on-target side effects of analgesics targeting NaV1.7 that will need to be monitored during development.

Conclusion


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Despite the limited clinical efficacy of first- and second-generation NaV1.7 inhibitors, this channel subtype remains a compelling target for analgesic drug development. Human and rodent genetic validation, and electrophysiological studies in DRG neurons support the view that NaV1.7 has an obligatory role in setting the threshold for action potential generation in nociceptive neurons. The poor showing of early clinical candidates may have resulted from limited selectivity over off-target NaV isoforms, preferential drug binding to the inactivated state, and/or poor drug properties such as very high plasma protein binding and high unbound clearance resulting in a level of target engagement that was insufficient to produce robust analgesia. Results from behavioral experiments conducted with compounds that are > 100x selective over other NaV isoforms such as the gating modifier toxin JNJ63953918 (21) and the sulfonamide AMG8379 (15) indicate that analgesia is possible by either central or peripheral inhibition of NaV1.7, but that relatively high levels of target engagement, perhaps 80–95%, are required to achieve an acute pharmacodynamic effect.83,99 Recent studies with the acyl sulfonamides GX-585 (13) and GX-201 (14) suggest that prolonged exposure to these inhibitors through repeat dosing may reverse sensitization in chronic pain models at exposures that do not affect sensitivity to acute thermal and mechanical pain. 30,86 Going forward, efforts that focus on identifying lead compounds with excellent selectivity (e.g. > 100x) over off-target NaV isoforms and evidence of target engagement at low exposure multiples hold promise of yielding safe and effective medicines.


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Relevant Funding Sources Authors Mulcahy, Pajouhesh, Beckley and Delwig received funding from National Institutes of Health grant NS081887 and USAMRMC contract W81XWH-17-1-0672.


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Ancillary Information Supporting Information – none Corresponding Author – John V. Mulcahy, [email protected] Abbreviations CFA – complete Freund’s adjuvant Cryo-EM – cryoelectron microscopy DRG – dorsal root ganglia fu – unbound fraction IEM – inherited erythromelalgia mouse model IFM – isoleucine/phenylalanine/methionine motif Kd – equilibrium dissociation constant NaV – voltage-gated Na+ ion channel NRS – numeric rating scale Papp – apparent permeability PS – peri-sciatic SNI – spared nerve injury SNL – sciatic nerve ligation SNT – spinal nerve transection STX – saxitoxin STZ – streptozocin


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TTX – tetrodotoxin TTX-s – tetrodotoxin-sensitive UVB – UVB burn-induced hyperalgesia VSD – voltage sensor domain Biographies John V. Mulcahy – John Mulcahy is a cofounder and Vice President of Research at SiteOne Therapeutics. He received his PhD from Stanford University, working in the fields of natural product total synthesis and synthetic methodology, and completed a postdoctoral fellowship at the Stanford University School of Medicine in the Department of Anesthesiology.

Hassan Pajouhesh Ph.D. is Director of Chemistry at SiteOne Therapeutics. He has a broad background in organic synthesis and medicinal chemistry and over 20 years of specific training and expertise in the ion channel research area. He has directed successful lead optimization efforts for multiple indications, resulting in clinical candidates and Investigational New Drug (IND) applications. He is author or coauthor of more than 22 peer-reviewed publications and a co-inventor on more than 30 issued or pending U.S. patents. Dr. Pajouhesh completed his Ph.D. in the field of organic chemistry at the University of Missouri-Kansas City. He received


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postdoctoral training at Southern Methodist University where his research contributed to the total synthesis of (+-)-Asparenomycin C.

Jacob T. Beckley is a Principal Scientist at SiteOne Therapeutics and leads the preclinical efficacy testing program. He received his PhD in Neuroscience from the Medical University of South Carolina under the direction of Dr. John J. Woodward. He then completed postdoctoral training in Dr. Dorit Ron’s laboratory at the University of California, San Francisco. His research is focused on assessing the electrophysiological and anti-nociceptive properties of selective sodium channel blockers.

Anton Delwig PhD is a Principal Scientist at SiteOne. He has 12 years of experience in molecular biology, neurophysiology and behavioral studies. Dr. Delwig received his PhD in neurobiology from the University of Vermont and completed his post-doctoral training at UC Berkeley and UCSF. He is a co-Principal Investigator on two small business grants awarded to SiteOne by The National Institutes of Health.

Justin Du Bois joined the Department of Chemistry at Stanford University in 1999 and is currently the Henry Dreyfus Professor. His research interests span chemical design and synthesis, chemical biology, and neuroscience. He is a cofounder and executive board member of SiteOne Therapeutics.


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Dr. John Hunter is the Chief Scientific Officer of SiteOne Therapeutics based in South San Francisco, CA. Previously, Dr. Hunter held leadership roles within Merck Research Laboratories, Schering-Plough, Roche Bioscience and the Parke-Davis Neuroscience Research Center. While at Roche he served as Consulting Professor of Anesthesiology at Stanford University School of Medicine. Dr. Hunter received his Bachelor of Science degree with honors and Ph.D. in Pharmacology from the University of Glasgow. MRC Post-Doctoral Fellowships in Neuroscience then followed at the University of Cambridge. His principal areas of research have been neurodegenerative disease and pain. He has authored more than 100 original scientific articles, as well as several reviews and/or book chapters and participated as either editor or reviewer for several journals.

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Figure 7 – original Figure 8 – original Table 1 – original Table of contents graphic – structure of GpTx-1 from ACS publication, reference 101

Table of Contents Graphic H2N

O

O O O S N N H F

HO HN HO N

NH

O O

Me

NH NH2

GDC-0276

GpTx-1

STX– OAc

Mode of Action

Inactivated State Inhibitor

Resting State Inhibitor

State-Independent Inhibitor

NaV1.7 Binding Site

Domain IV Voltage Sensor

Domain II Voltage Sensor

Extracellular Pore


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Table of Contents Graphic

H 2N

O

O O O S N N H F

HO HN HO N

NH

O O

Me

NH NH2

GDC-0276

GpTx-1

STX–OAc

Mode of Action

Inactivated State Inhibitor

Resting State Inhibitor

State-Independent Inhibitor

NaV1.7 Binding Site

Domain IV Voltage Sensor

Domain II Voltage Sensor

Extracellular Pore


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

• •

NaV1.8

Main contributor to rising phase Fast re-priming enables high frequency firing

NaV1.7

• • • •

Amplifies sub-threshold depolarizations Contributes to rising phase Fast kinetics of activation, inactivation Slow closed-state inactivation

• • •

Amplifies sub-threshold depolarizations Ultra-slow kinetics of activation, inactivation Large window current may contribute to resting membrane potential

NaV1.9


Page 70 of 78

Page 71 of 78 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


A. + + S1 S2 S3 S4 S5 + +

NH2+

B.

+ + S1 S2 S3 S4 S5 + +

S6

D1

D1

S6

+ + S1 S2 S3 S4 S5 + +

D2

S6

D3

+ + S1 S2 S3 S4 S5 + +

D4

D2 D3

D4

IFM

IFM

Resting (closed)

Open


Fast Inactivated

S6

CO2-



Page 72 of 78

Page 73 of 78 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


O

Cl

O

O

O

CF3

O

O

N

H 2N

Cl

O H N

O

F

O O N S N H F

S

O

H 2N

O O O S N N H F

N NH

1 funapide TV-45070/XEN-402 Teva/Xenon hNaV Isoform NaV1.7 NaV1.1 NaV1.2 NaV1.3 NaV1.4 NaV1.5 NaV1.6 NaV1.8

IC50 (µM) selectivity 0.054 – – 11x 0.60 – – – – 1.5x 0.084 3x 0.17 89x 4.8

2 vixotrigine BIIB074/CNV-1014802 Biogen/Convergence IC50 (µM) 32 22 13 23 10 84 15 6.0

selectivity < 1x < 1x < 1x < 1x 2.5x < 1x < 1x

3 PF-05089771 Pfizer

4 GDC-0276 Genentech/ Xenon

IC50 (µM) selectivity 0.011 77x 0.85 10x 0.11 1000x 11 900x 10 2000x 25 14x 0.16 > 900x >10

IC50 (µM) selectivity 0.0004 27x 0.011 50x 0.020 – – – – 120x 0.049 1200x 0.480 – –



O O N N S S N H

F3C O

H N

Me

CN H N HN

H 2N

F O O S N H

Me

N

Me Me Cl MeN N H

IC50 (µM) selectivity 0.027 0.030 – – – – –

Cl N H

IC50 (µM) selectivity 0.039 0.011 – – – – – – – – – 850x – – – – – exposure model formalin (+) 100x mIC50

Me N

O O N S S N H F

N

10 Bristol-Myers Squibb fu: 0.20 (mouse) IC50 (µM) selectivity 0.011 ACS Paragon Plus 0.051 – – – – –

F Me

Me

Cl

N

N H

7 Merck fu: 0.145 (mouse)

IC50 (µM) selectivity 0.021 0.030 – – – – – – – – – 1380x 29 – – – – exposure model formalin (+) 50x mIC50

O O N S S N H F

9 Bristol-Myers Squibb fu: 0.23 (mouse)

O

6 Merck fu: 0.065 (mouse)

5 PF-06456384 fu: 0.00281 (mouse)

Me Me

O

O

N

N

N O

NaV Isoform IC50 (µM) selectivity hNaV1.7 0.00001 mNaV1.7 < 0.0001 rNaV1.7 0.075 hNaV1.1 > 30,000x 0.314 hNaV1.2 300x 0.003 hNaV1.3 > 100,000x 6.4 hNaV1.4 > 100,000x 1.5 hNaV1.5 > 100,000x 2.6 hNaV1.6 580x 0.0058 hNaV1.8 > 100,000x 26 exposure Mouse PK/PD model formalin (–) 60x mIC50

NaV Isoform hNaV1.7 mNaV1.7 rNaV1.7 hNaV1.1 hNaV1.2

O O N N S S N H

Page 74 of 78

8 Merck fu: 0.18 (mouse) IC50 (µM) 0.012 0.013 – – – – – > 30 – – model formalin (+)

O O O S N Me H Me O

N

O O N S S N H F

selectivity

– – – – > 2500x – – exposure 3.5x mIC50

O O O S N H F

Me Cl Me

11 Bristol-Myers Squibb fu: 0.002 (mouse) IC50 (µM) selectivity

0.008 Environment 0.035 – – 16

– 2000

12 Genentech/Xenon fu: 0.001 (mouse) IC50 (µM) selectivity 0.0006 0.0022 0.0037 0.006 10x 0.002 3x

Page 75 of 78 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

hNaV1.1 hNaV1.2 hNaV1.3 hNaV1.4 hNaV1.5 hNaV1.6 hNaV1.8

– – – – > 30 – –

– – – – > 1000x – –

– – – – – Journal of –Medicinal – – 460x 5.1 – – – –

exposure Mouse PK/PD model formalin (–) 9x mIC50 (plasma) 50x mIC50 (DRG)

model exposure formalin (+) 5x mIC50 (plasma) 29x mIC50 (DRG)

O O O S N Me H F

O F Cl

Cl

N CH3

N N

O O O S N H

Mouse PK/PD

Kd (µM) 0.022 0.014 – 0.14 0.14 0.13 0.047 0.46 0.62 –*

0.006 0.002 – – 0.050 0.038 –

– 2000 3700 600 2300 – –

model exposure formalin (+) 3.4x mIC50 (plasma) 2.7x mIC50 (DRG) 3.7x mIC50 (plasma) CFA (+) O O S N H O MeO

model exposure IEM (+) 5.2x hIC50 formalin (+) 22x mIC50

O O S N H

O N

N

N

10x 3x – – 83x 63x –

O

O N

N OMe

F

F

F

Cl

Cl Me

13 GX-585 Genentech/Xenon fu: 0.007 NaV Isoform hNaV1.7 mNaV1.7 rNaV1.7 hNaV1.1 hNaV1.2 hNaV1.3 hNaV1.4 hNaV1.5 hNaV1.6 hNaV1.8

– 16 > 30 Chemistry 4.8 18.5 – –

selectivity

6x 6x 6x 2x 21x 28x –

exposure (EC50) model 2.3x hKd IEM (+) formalin (+) 3.3x mKd CFA, cold (+) 6.2x mKd 5.7x mKd SNI (+) 5x mKd (day 1) STZ (+)

Cl

CF3

14 GX-201 Genentech/Xenon fu: 0.002 Kd (µM) 0.0035 0.0021 – 0.14 0.064 0.18 0.028 0.37 0.21 –**

selectivity

40x 18x 51x 8x 100x 60x –

exposure (EC50) model 0.6x hKd IEM (+) formalin (+) 5.5x mKd CFA, cold (+) 7.5x mKd 11.9x mKd STZ (+)

F

15 AMG-8379 Amgen fu: 0.0017 (mouse)

16 AMG-8380 Amgen fu: 0.014 (mouse)

IC50 (µM) selectivity 0.0032 0.017 8.6 > 4300x > 14 690x 2.2 > 4300x > 14 > 4300x > 14 > 4300x > 14 410x 1.3 – –

IC50 (µM) selectivity 0.39 0.68 > 42 > 36x > 14 > 77x > 30 > 36x > 14 > 36x > 14 > 36x > 14 > 26x > 10 – –

exposure model histamine (+) 5.3x mIC50 23x mIC50 UVB (+) capsaicin (+) 21x mIC50


exposure model histamine (–) 4x mIC50 7x mIC50 UVB (–)


14 GX-201

13 GX-585 Mouse Model hot plate (+) tail immersion (+) Randall-Selitto (+) CFA, Hargreaves (+) capsaicin (+) odor (+)

Dose po 60 mg/kg 60 mg/kg 60 mg/kg 60 mg/kg 30 mg/kg 60 mg/kg

Ave. Cp 49.3 µM 49.3 µM 49.3 µM 67.9 µM 31.1 µM

Exposure 25x mIC50 25x mIC50 25x mIC50 34x mIC50 16x mIC50

Dose po 30 mg/kg 30 mg/kg 30 mg/kg 30 mg/kg 30 mg/kg 30 mg/kg

Ave. Cp 10.5 µM 10.5 µM

Exposure 10x mIC50 10x mIC50

16.1 µM

15x mIC50


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17 ProTx-II NaV Isoform hNaV1.7 mNaV1.7 rNaV1.7 hNaV1.1 hNaV1.2 hNaV1.3 hNaV1.4 hNaV1.5 hNaV1.6 hNaV1.8 Rodent PD

IC50 (µM) selectivity 0.0008 – – 20x 0.016 100x 0.079 30x 0.025 100x 0.079 500x 0.40 40x 0.032 – model (r) CFA (–) CFA (–) formalin (+) Hargreaves (+)

dose 0.1 mg/kg iv 0.01 mg/kg it 2 µg it 2 µg it

18 µ-TRTX-Pn3a IC50 (µM) selectivity 0.0009 0.0044 0.0015 41x 0.037 130x 0.12 230x 0.21 160x 0.14 890x 0.80 144x 0.13 > 10000x 50

19 [Ile10Glu]GpTx-1 Amgen IC50 (µM) selectivity 0.0021 – – – – – – – – 590x 1.3 > 4000 > 10 – – – –

20 AMG6120 Amgen IC50 (µM) selectivity 0.0008 0.0054 – – – – – – – – 130x 0.104 > 8000x 6.64 750x 0.604 > 1200x > 1.0 model (m) exposure histamine (+) 105x mIC50 capsaicin (–) 105x mIC50


21 JNJ63955918 Janssen IC50 (µM) selectivity 0.010 – – 1000x 10 160x 1.6 – – 530x 5.3 > 1000x > 10 100x 1.0 – – model (r) formalin (+) tail flick (+) hot plate (+) Hargreaves (+)

dose 1–5 µg it 0.3–7.5 µg it 0.3–7.5 µg it 1.4 mg PS


H 2N

OH HO HO HN

O

O

OH

OH NHOH

NH2

22 tetrodotoxin (TTX) hNaV Isoform NaV1.7 NaV1.1 NaV1.2 NaV1.3 NaV1.4 NaV1.5 NaV1.6 NaV1.8 NaV1.9 Rat PD

IC50 (µM) selectivity 0.036 < 1x 0.0041 < 1x 0.014 < 1x 0.0053 < 1x 0.0076 27x 1.0 800x > 30 > 800x > 30

HO HN HO N

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H 2N NH

O

13

O

NH2

NH NH2

O O

Me

NH

O Me Me

HO HN HO N N H

NH2

23 saxitoxin (STX) IC50 (µM) 0.408 0.0023 0.0010 0.013 0.019 0.213 0.0011 > 10 > 10

HO HN HO N

H 2N NH

selectivity < 1x < 1x < 1x < 1x < 1x < 1x > 20x > 20x

NH NH NH2

24

25 SiteOne

IC50 (µM) selectivity 0.019 – – – – – – – – 240 4.6 – – – – – –

IC50 (µM) selectivity 0.044 – – – – – – 1700x 75 – – 660x 29 – – – –

model dose formalin (+) 6 µg/kg sc writhing (+) 1 µg/kg sc 1–3 µg/kg sc SNL (+)


O N O

Gated Sodium Channel Isoform NaV1.7 - ACS Publications

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