Jennifer R. Deuis

An animal model of oxaliplatin-induced cold allodynia reveals a crucial role for Nav1.6 in peripheral pain pathways.

Summary Cold allodynia elicited by local intraplantar injection of the chemotherapeutic agent oxaliplatin is mediated through Nav1.6‐expressing peripheral sensory fibres. Activation of Nav1.6 alone elicits only mechanical allodynia and spontaneous pain, but when combined with inhibition of Kv channels, profound cold allodynia develops. ABSTRACT Cold allodynia, pain in response to cooling, occurs during or within hours of oxaliplatin infusion and is thought to arise from a direct effect of oxaliplatin on peripheral sensory neurons. To characterize the pathophysiological mechanisms underlying acute oxaliplatin‐induced cold allodynia, we established a new intraplantar oxaliplatin mouse model that rapidly developed long‐lasting cold allodynia mediated entirely through tetrodotoxin‐sensitive Nav pathways. Using selective inhibitors and knockout animals, we found that Nav1.6 was the key isoform involved, while thermosensitive transient receptor potential channels were not involved. Consistent with a crucial role for delayed‐rectifier potassium channels in excitability in response to cold, intraplantar administration of the K+‐channel blocker 4‐aminopyridine mimicked oxaliplatin‐induced cold allodynia and was also inhibited by Nav1.6 blockers. Intraplantar injection of the Nav1.6 activator Cn2 elicited spontaneous pain, mechanical allodynia, and enhanced 4‐aminopyridine‐induced cold allodynia. These findings provide behavioural evidence for a crucial role of Nav1.6 in multiple peripheral pain pathways including cold allodynia.

Pharmacological characterisation of the highly NaV1.7 selective spider venom peptide Pn3a

Human genetic studies have implicated the voltage-gated sodium channel NaV1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits NaV1.7 (IC50 0.9 nM) with at least 40–1000-fold selectivity over all other NaV subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by NaV1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective NaV1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective NaV1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted NaV1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

NaV1.7 as a pain target - From gene to pharmacology.

Na(V)1.7, a subtype of the voltage-gated sodium channel family that is highly expressed in peripheral sensory neurons, remains one of the most promising targets for the treatment of pain. However, despite compelling genetic evidence supporting a key role for Na(V)1.7 in regulating excitability of peripheral sensory neurons, the development of truly subtype-selective inhibitors has been challenging. Here, we discuss complexities surrounding targeting Na(V)1.7 pharmacologically for treatment of pain and explore future opportunities for development of effective analgesic Na(V)1.7 inhibitors

Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain.

Loss-of-function mutations of NaV1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of NaV1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of NaV1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of NaV1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with NaV1.7 inhibitors and significantly reduced in NaV1.7−/− mice. To validate the use of the model for profiling NaV1.7 inhibitors, we determined the NaV selectivity and tested the efficacy of the reported NaV1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited NaV1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited NaV1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited NaV channels and was only effective in the OD1 model when delivered systemically. Our novel model of NaV1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of NaV1.7 inhibitors.

Analgesic treatment of ciguatoxin-induced cold allodynia

Summary Intracutaneous administration of ciguatoxin in humans replicates the sensory disturbances associated with ciguatera. Lamotrigine and flupirtine were efficacious for the treatment of ciguatoxin‐induced cold allodynia. Abstract Ciguatera, the most common form of nonbacterial ichthyosarcotoxism, is caused by consumption of fish that have bioaccumulated the polyether sodium channel activator ciguatoxin. The neurological symptoms of ciguatera include distressing, often persistent sensory disturbances such as paraesthesias and the pathognomonic symptom of cold allodynia. We show that intracutaneous administration of ciguatoxin in humans elicits a pronounced axon‐reflex flare and replicates cold allodynia. To identify compounds able to inhibit ciguatoxin‐induced Nav responses, we developed a novel in vitro ciguatoxin assay using the human neuroblastoma cell line SH‐SY5Y. Pharmacological characterisation of this assay demonstrated a major contribution of Nav1.2 and Nav1.3, but not Nav1.7, to ciguatoxin‐induced Ca2+ responses. Clinically available Nav inhibitors, as well as the Kv7 agonist flupirtine, inhibited tetrodotoxin‐sensitive ciguatoxin‐evoked responses. To establish their in vivo efficacy, we used a novel animal model of ciguatoxin‐induced cold allodynia. However, differences in the efficacy of these compounds to reverse ciguatoxin‐induced cold allodynia did not correlate with their potency to inhibit ciguatoxin‐induced responses in SH‐SY5Y cells or at heterologously expressed Nav1.3, Nav1.6, Nav1.7, or Nav1.8, indicating cold allodynia might be more complex than simple activation of Nav channels. These findings highlight the need for suitable animal models to guide the empiric choice of analgesics, and suggest that lamotrigine and flupirtine could be potentially useful for the treatment of ciguatera.

Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens.

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (NaV) channels, we screened spider venoms for inhibitors of human NaV1.7 (hNaV1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel NaV1.7 inhibitor, μ-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNaV1.7 > hNaV1.6 > hNaV1.2 > hNaV1.1 > hNaV1.3 channels in fluorescent assays. NaV1.7 inhibition was diminished (IC50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 μM. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNaV1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNaV1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNaV1.7 inhibitors for treatment of chronic pain.

Transcriptomic and behavioural characterisation of a mouse model of burn pain identify the cholecystokinin 2 receptor as an analgesic target

Burn injury is a cause of significant mortality and morbidity worldwide and is frequently associated with severe and long-lasting pain that remains difficult to manage throughout recovery. We characterised a mouse model of burn-induced pain using pharmacological and transcriptomic approaches. Mechanical allodynia elicited by burn injury was partially reversed by meloxicam (5 mg/kg), gabapentin (100 mg/kg) and oxycodone (3 and 10 mg/kg), while thermal allodynia and gait abnormalities were only significantly improved by amitriptyline (3 mg/kg) and oxycodone (10 mg/kg). The need for relatively high opioid doses to elicit analgesia suggested a degree of opioid resistance, similar to that shown clinically in burn patients. We thus assessed the gene expression changes in dorsal root ganglion neurons and pathophysiological mechanisms underpinning burn injury-induced pain using a transcriptomic approach. Burn injury was associated with significantly increased expression of genes associated with axon guidance, neuropeptide signalling, behavioural defence response and extracellular signalling, confirming a mixed neuropathic and inflammatory aetiology. Notably, among the pain-related genes that were upregulated post-injury was the cholecystokinin 2 receptor (Cckbr), a G protein-coupled receptor known as a pain target involved in reducing opioid effectiveness. Indeed, the clinically used cholecystokinin receptor antagonist proglumide (30 mg/kg) was effective at reversing mechanical allodynia, with additional analgesia evident in combination with low-dose oxycodone (1 mg/kg), including significant reversal of thermal allodynia. These findings highlight the complex pathophysiological mechanisms underpinning burn injury-induced pain and suggest that cholecystokinin-2 receptor antagonists may be useful clinically as adjuvants to decrease opioid requirements and improve analgesic management.

Analgesic effects of clinically used compounds in novel mouse models of polyneuropathy induced by oxaliplatin and cisplatin.

BACKGROUND Peripheral neuropathy is the major dose-limiting side effect of cisplatin and oxaliplatin, and there are currently no effective treatments available. The aim of this study was to assess the pharmacological mechanisms underlying chemotherapy-induced neuropathy in novel animal models based on intraplantar administration of cisplatin and oxaliplatin and to systematically evaluate the analgesic efficacy of a range of therapeutics. METHODS Neuropathy was induced by a single intraplantar injection of cisplatin or oxaliplatin in C57BL/6J mice and assessed by quantification of mechanical and thermal allodynia. The pharmacological basis of cisplatin-induced neuropathy was characterized using a range of selective pharmacological inhibitors. The analgesic effects of phenytoin, amitriptyline, oxcarbazepine, mexiletine, topiramate, retigabine, gabapentin, fentanyl, and Ca(2+/)Mg(2+) were assessed 24 hours after induction of neuropathy. RESULTS Intraplantar administration of cisplatin led to the development of mechanical allodynia, mediated through Nav1.6-expressing sensory neurons. Unlike intraplantar injection of oxaliplatin, cold allodynia was not observed with cisplatin, consistent with clinical observations. Surprisingly, only fentanyl was effective at alleviating cisplatin-induced mechanical allodynia despite a lack of efficacy in oxaliplatin-induced cold allodynia. Conversely, lamotrigine, phenytoin, retigabine, and gabapentin were effective at reversing oxaliplatin-induced cold allodynia but had no effect on cisplatin-induced mechanical allodynia. Oxcarbazepine, amitriptyline, mexiletine, and topiramate lacked efficacy in both models of acute chemotherapy-induced neuropathy. CONCLUSION This study established a novel animal model of cisplatin-induced mechanical allodynia consistent with the A-fiber neuropathy seen clinically. Systematic assessment of a range of therapeutics identified several candidates that warrant further clinical investigation.

Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.

The pharmacology of voltage-gated sodium channel activators

ABSTRACT Toxins and venom components that target voltage‐gated sodium (NaV) channels have evolved numerous times due to the importance of this class of ion channels in the normal physiological function of peripheral and central neurons as well as cardiac and skeletal muscle. NaV channel activators in particular have been isolated from the venom of spiders, wasps, snakes, scorpions, cone snails and sea anemone and are also produced by plants, bacteria and algae. These compounds have provided key insight into the molecular structure, function and pathophysiological roles of NaV channels and are important tools due to their at times exquisite subtype‐selectivity. We review the pharmacology of NaV channel activators with particular emphasis on mammalian isoforms and discuss putative applications for these compounds. This article is part of the Special Issue entitled ‘Venom‐derived Peptides as Pharmacological Tools.’ HIGHLIGHTSVoltage‐gated sodium channel activator toxins occur in many venoms.Sodium channel activators are important tool compounds and drug leads.These compounds modulate voltage‐gated sodium channels with high selectivity.