Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1, a toxin from the scorpion Odonthobuthus doriae

Voltage-gated sodium channels are essential for the propagation of action potentials in nociceptive neurons. Nav1.7 is found in peripheral sensory and sympathetic neurons and involved in short-term and inflammatory pain. Nav1.8 and Nav1.3 are major players in nociception and neuropathic pain, respectively. In our effort to identify isoform-specific and high-affinity ligands for these channels, we investigated the effects of OD1, a scorpion toxin isolated from the venom of the scorpion Odonthobuthus doriae. Nav1.3, Nav1.7, and Nav1.8 channels were coexpressed with beta1-subunits in Xenopus laevis oocytes. Na+ currents were recorded with the two-electrode voltage-clamp technique. OD1 modulates Nav1.7 at low nanomolar concentrations: 1) fast inactivation is dramatically impaired, with an EC50 value of 4.5 nM; 2) OD1 substantially increases the peak current at all voltages; and 3) OD1 induces a substantial persistent current. Nav1.8 was not affected by concentrations up to 2 microM, whereas Nav1.3 was sensitive only to concentrations higher than 100 nM. OD1 impairs the inactivation process of Nav1.3 with an EC50 value of 1127 nM. Finally, the effects of OD1 were compared with a classic alpha-toxin, AahII from Androctonus australis Hector and a classic alpha-like toxin, BmK M1 from Buthus martensii Karsch. At a concentration of 50 nM, both toxins affected Nav1.7. Nav1.3 was sensitive to AahII but not to BmK M1, whereas Nav1.8 was affected by neither toxin. In conclusion, the present study shows that the scorpion toxin OD1 is a potent modulator of Nav1.7, with a unique selectivity pattern.     

State- and use-dependent block of muscle Nav1.4 and neuronal Nav1.7 voltage-gated Na+ channel isoforms by ranolazine

Ranolazine is an antianginal agent that targets a number of ion channels in the heart, including cardiac voltage-gated Na(+) channels. However, ranolazine block of muscle and neuronal Na(+) channel isoforms has not been examined. We compared the state- and use-dependent ranolazine block of Na(+) currents carried by muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.7 isoforms expressed in human embryonic kidney 293T cells. Resting and inactivated block of Na(+) channels by ranolazine were generally weak, with a 50% inhibitory concentration (IC(50)) >/= 60 microM. Use-dependent block of Na(+) channel isoforms by ranolazine during repetitive pulses (+50 mV/10 ms at 5 Hz) was strong at 100 microM, up to 77% peak current reduction for Nav1.4, 67% for Nav1.5, and 83% for Nav1.7. In addition, we found conspicuous time-dependent block of inactivation-deficient Nav1.4, Nav1.5, and Nav1.7 Na(+) currents by ranolazine with estimated IC(50) values of 2.4, 6.2, and 1.7 microM, respectively. On- and off-rates of ranolazine were 8.2 microM(-1) s(-1) and 22 s(-1), respectively, for Nav1.4 open channels and 7.1 microM(-1) s(-1) and 14 s(-1), respectively, for Nav1.7 counterparts. A F1579K mutation at the local anesthetic receptor of inactivation-deficient Nav1.4 Na(+) channels reduced the potency of ranolazine approximately 17-fold. We conclude that: 1) both muscle and neuronal Na(+) channels are as sensitive to ranolazine block as their cardiac counterparts; 2) at its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na(+) channels; and 3) ranolazine block of open Na(+) channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.     

Animal Toxins Can Alter the Function of Nav1.8 and Nav1.9

Human voltage-activated sodium (Nav) channels are adept at rapidly transmitting electrical signals across long distances in various excitable tissues. As such, they are amongst the most widely targeted ion channels by drugs and animal toxins. Of the nine isoforms, Nav1.8 and Nav1.9 are preferentially expressed in DRG neurons where they are thought to play an important role in pain signaling. Although the functional properties of Nav1.8 have been relatively well characterized, difficulties with expressing Nav1.9 in established heterologous systems limit our understanding of the gating properties and toxin pharmacology of this particular isoform. This review summarizes our current knowledge of the role of Nav1.8 and Nav1.9 in pain perception and elaborates on the approaches used to identify molecules capable of influencing their function.     

Altered gating and local anesthetic block mediated by residues in the I-S6 and II-S6 transmembrane segments of voltage-dependent Na+ channels

The cytoplasmic side of the voltage-dependent Na+ channel pore is putatively formed by the S6 segments of domains I to IV. The role of amino acid residues of I-S6 and II-S6 in channel gating and local anesthetic (LA) block was investigated using the cysteine scanning mutagenesis of the rat skeletal muscle Na+ channel (Nav1.4). G428C uniquely reduced sensitivity to rested state or first-pulse block by lidocaine without alterations in the voltage dependence or kinetics of gating that would otherwise account for the increase in the IC50 for block. Mutations in I-S6 (N434C and I436C) and in II-S6 (L785C and V787C) increased sensitivity to first-pulse block by lidocaine. Enhanced inactivation accounted for the increased sensitivity of N434C to lidocaine and hastening of inactivation of I436C in the absence of drug could account for higher affinity first-pulse block. Mutations in I-S6 (I424C, I425C, and F430C) and in II-S6 (I782C and V786C) reduced the use-dependent lidocaine block. The reduction in use-dependent block of F430C was consistent with alterations in inactivation gating; the other mutants did not exhibit gating changes that could explain the reduced sensitivity to lidocaine. Therefore, several amino acids (I424, I425, G428, I782, and V786), in addition to those previously identified (Yarov-Yarovoy et al., 2002), alter the sensitivity of Nav1.4 to lidocaine, independent of mutation-induced changes in gating. The magnitude of the change in the IC50 values, the isoform, and LA dependence of the changes in affinity suggest that the determinants of binding in I-S6 and II-S6 are subsidiary to those in IV-S6.     

State-dependent mibefradil block of Na+ channels

Mibefradil is a T-type Ca2+ channel antagonist with reported cross-reactivity with other classes of ion channels, including K+, Cl-, and Na+ channels. Using whole-cell voltage clamp, we examined mibefradil block of four Na+ channel isoforms expressed in human embryonic kidney cells: Nav1.5 (cardiac), Nav1.4 (skeletal muscle), Nav1.2 (brain), and Nav1.7 (peripheral nerve). Mibefradil blocked Nav1.5 in a use/frequency-dependent manner, indicating preferential binding to states visited during depolarization. Mibefradil blocked currents of all Na+ channel isoforms with similar affinity and a dependence on holding potential, and drug off-rate was slowed at depolarized potentials (k(off) was 0.024/s at -130 mV and 0.007/s at -100 mV for Nav1.5). We further probed the interaction of mibefradil with inactivated Nav1.5 channels. Neither the degree nor the time course of block was dependent on the stimulus duration, which dramatically changed the residency time of channels in the fast-inactivated state. In addition, inhibiting the binding of the fast inactivation lid (Nav1.5 ICM + MTSET) did not alter mibefradil block, confirming that the drug does not preferentially interact with the fast-inactivated state. We also tested whether mibefradil interacted with slow-inactivated state(s). When selectively applied to channels after inducing slow inactivation with a 60-s pulse to -10 mV, mibefradil (1 microM) produced 45% fractional block in Nav1.5 and greater block (88%) in an isoform (Nav1.4) that slow-inactivates more completely. Our results suggest that mibefradil blocks Na+ channels in a state-dependent manner that does not depend on fast inactivation but probably involves interaction with one or more slow-inactivated state(s).     

Effect of 8-bromo-cAMP on the tetrodotoxin-resistant sodium (Nav 1.8) current in small-diameter nodose ganglion neurons

We examined whether 8-bromo-cAMP (8-Br-cAMP)-induced modification of tetrodotoxin-resistant (TTX-R) sodium current in neonatal rat nodose ganglion neurons is mediated by the activation of protein kinase A (PKA) and/or protein kinase C (PKC). In 8-Br-cAMP applications ranging from 0.001 to 1.0mM, 8-Br-cAMP at 0.1mM showed a maximal increase in the peak TTX-R Na(+) (Nav1.8) current and produced a hyperpolarizing shift in the conductance-voltage (G-V) curve. The PKC inhibitor bisindolylmaleimide Ro-31-8425 (Ro-31-8425, 0.5microM) decreased the peak Nav 1.8 current. The Ro-31-8425-induced modulation of the G(V)(1/2) baseline (a percent change in G at baseline V1/2) was not affected by additional 8-Br-cAMP application (0.1mM). The maximal increase in Nav 1.8 currents was seen at 0.1microM after the application of a PKC activator, phorbol 12-myristate 13-acetate (PMA) and forskolin. The PMA-induced increase in Nav 1.8 currents was not significantly affected by additional 0.1mM 8-Br-cAMP application. Intracellular application of a PKA inhibitor, protein kinase inhibitor (PKI, 0.01mM), inhibited the baseline Nav 1.8 current, significantly attenuated the 8-Br-cAMP-and PMA-induced increase in the peak Nav 1.8 current, and caused a significant increase in the slope factor of the inactivation curve. The PKI application at a higher concentration (0.5mM) greatly inhibited the PMA (0.1microM)-induced increase in the peak Nav 1.8 current amplitude and further enhanced the Ro-31-8425-induced decrease in the current. These results suggest that the 8-Br-cAMP-induced increase in Nav 1.8 currents may be mediated by activation of both PKA and PKC.     

Isolation and structure-activity of mu-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels

Mu-conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new mu-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other mu-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for mu-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of mu-conotoxins.     

A comparative study of the action of tolperisone on seven different voltage dependent sodium channel isoforms

The specific, acute interaction of tolperisone, an agent used as a muscle relaxant and for the treatment of chronic pain conditions, with the Na(v1.2), Na(v1.3), Na(v1.4), Na(v1.5), Na(v1.6), Na(v1.7), and Na(v1.8) isoforms of voltage dependent sodium channels was investigated and compared to that of lidocaine. Voltage dependent sodium channels were expressed in the Xenopus laevis oocyte expression system and sodium currents were recorded with the two electrode voltage clamp technique. Cumulative dose response relations revealed marked differences in IC(50) values between the two drugs on identical isoforms, as well as between isoforms. A detailed kinetic analysis uncovered that tolperisone as well as lidocaine exhibited their blocking action not only via state dependent association/dissociation with voltage dependent sodium channels, but a considerable fraction of inhibition is tonic, i.e. permanent and basic in nature. Voltage dependent activation was affected to a minor extent only. A shift in steady-state inactivation to more negative potentials could be observed for most drug/isoform combinations. The contribution of this shift to overall block was, however, small at drug concentrations resulting in considerable overall block. Recovery from inactivation was affected notably by both drugs. Lidocaine application led to a pronounced prolongation of the time constant of the fast recovery process for the Na(v1.3), Na(v1.5), and Na(v1.7) isoforms, indicating common structural properties in the local anesthetic receptor site of these three proteins. Interestingly, this characteristic drug action was not observed for tolperisone.     

Molecular diversity of structure and function of the voltage-gated Na+ channels

A variety of different isoforms of voltage-sensitive Na+ channels have now been identified. The recent three-dimensional analysis of Na+ channels has unveiled a unique and unexpected structure of the Na+ channel protein. Na+ channels can be classified into two categories on the basis of their amino acid sequence, Nav1 isoforms currently comprising nine highly homologous clones and Nax that possesses structure diverging from Nav1, especially in several critical functional motifs. Although the functional role of Nav1 isoforms is primarily to form an action potential upstroke in excitable cells, recent biophysical studies indicate that some of the Nav1 isoforms can also influence subthreshold electrical activity through persistent or resurgent Na+ currents. Nav1.8 and Nav1.9 contain an amino acid sequence common to tetrodotoxin resistant Na+ channels and are localized in peripheral nociceptors. Recent patch-clamp experiments on dorsal root ganglion neurons from Nav1.8-knock-out mice unveiled an additional tetrodotoxin-resistant Na+ current. The demonstration of its dependence on Nav1.9 provides evidence for a specialized role of Nav1.9, together with Nav1.8, in pain sensation. Although Nax has not been successfully expressed in an exogenous system, recent investigations using relevant native tissues combined with gene-targeting have disclosed their unique "concentration"-sensitive but not voltage-sensitive roles. In this context, these emerging views of novel functions mediated by different types of Na+ channels are reviewed, to give a perspective for future research on the expanding family of Na+ channel clones.     

Inhibition of cardiac sodium currents by toluene exposure

Toluene is an industrial solvent widely used as a drug of abuse, which can produce sudden sniffing death due to cardiac arrhythmias. In this paper, we tested the hypothesis that toluene inhibits cardiac sodium channels in Xenopus laevis oocytes transfected with Nav1.5 cDNA and in isolated rat ventricular myocytes. In oocytes, toluene inhibited sodium currents (INa+) in a concentration-dependent manner, with an IC50 of 274 microm (confidence limits: 141-407 microm). The inhibition was complete, voltage-independent, and slowly reversible. Toluene had no effect on: (i). the shape of the I-V curves; (ii). the reversal potential of Na+; and (iii). the steady-state inactivation. The slow recovery time constant from inactivation of INa+ decreased with toluene exposure, while the fast recovery time constant remained unchanged. Block of INa+ by toluene was use- and frequency-dependent. In rat cardiac myocytes, 300 microm toluene inhibited the sodium current (INa+) by 62%; this inhibition was voltage independent. These results suggest that toluene binds to cardiac Na+ channels in the open state and unbinds either when channels move between inactivated states or from an inactivated to a closed state. The use- and frequency-dependent block of INa+ by toluene might be responsible, at least in part, for its arrhythmogenic effect.     

A phenylalanine residue at segment D3-S6 in Nav1.4 voltage-gated Na(+) channels is critical for pyrethroid action

Mammalian voltage-gated Na(+) channels were less sensitive to pyrethroids than their insect counterparts by 2 to 3 orders of magnitude. Deltamethrin at 10 microM elicited weak gating changes in rat skeletal muscle alpha-subunit Na(+) channels (Nav1.4) after > 30 min of perfusion. About 10% of the peak current was maintained during the 8-ms, +50-mV pulse and, upon repolarization to -140 mV, the amplitude of the slow tail current corresponded to less than 3% of total Na(+) channels modified by deltamethrin. A background mutation, Nav1.4-I687M (within D2:S4-S5 cytoplasmic linker), enhanced the deltamethrin-induced maintained current by approximately 2.5-fold, whereas Nav1.4-I687T, a homologous superkdr mutation, reduced it by approximately 2-fold. Repetitive pulses at 2 Hz further augmented the effects of deltamethrin on Nav1.4-I687M mutant channels so that approximately 75% of peak currents were maintained. A second mutation, Nav1.4-I687M/F1278I at the middle of D3-S6, rendered the channel greatly resistant to deltamethrin. This double mutant channel remained sensitive to batrachotoxin (BTX), even though nearby residues S1276 and L1280 were critical for BTX action. We hypothesize that the deltamethrin receptor and the BTX receptor are situated at the middle but opposite surface of the D3-S6 alpha-helical structure. Another mutant, Nav1.4-I687M/N784K, exhibited a partial deltamethrin-resistant phenotype but was completely resistant to BTX. Consistent with the BTX-resistant phenotype of N784K and the known adjacent kdr mutation at position L785F, deltamethrin and BTX were probably situated next to each other upon binding at D2-S6. Evidently, distinct residues from multiple S6 segments were critical for deltamethrin and BTX actions.     

Lidocaine block of neonatal Nav1.3 is differentially modulated by co-expression of beta1 and beta3 subunits

The effects of lidocaine on neonatal Na(v)1.3 (Na(v)1.3n) expressed alone and in combination with beta1 and beta3 subunits in Xenopus oocytes were examined. Lidocaine reversibly inhibited the peak Na(v)1.3n current, shifted the steady-state inactivation curve to hyperpolarized potentials and delayed recovery from inactivation. These effects were attenuated by the co-expression of the beta subunits, with greater attenuating effects being observed in oocytes co-expressing beta1 compared to those co-expressing beta3. Use-dependent block by lidocaine was assessed at 1 Hz train frequency for 60 pulses. Lidocaine caused similar use-dependent block of current amplitude at pulse 60 for Na(v)1.3n and Na(v)1.3n+beta3. In oocytes co-expressing beta1, these use-dependent actions were reduced. In conclusion, the effects of lidocaine on Na(v)1.3n are differentially modulated by beta1 and beta3 subunits. Since these subunits exhibit a complementary distribution, this finding may have importance in our understanding of lidocaine action.     

A mutation in the local anaesthetic binding site abolishes toluene effects in sodium channels

Toluene is a solvent of abuse that inhibits cardiac sodium channels in a manner that resembles the action of local anaesthetics. The purpose of this work was to analyze toluene effects on skeletal muscle sodium channels with and without beta1 subunit (Nav1.4+beta1 and Nav1.4-beta1, respectively) expressed in Xenopus laevis oocytes and to compare them with those produced in the F1579A mutant channel lacking a local anaesthetic binding site. Toluene inhibited Nav1.4 sodium currents (IC50=2.7 mM in Nav1.4+beta1 and 2.2 mM in Nav1.4-beta1 in a concentration dependent way. Toluene (3 mM) blocked sodium currents in Nav1.4 channels proportionally throughout the entire current-voltage relationship producing inactivation at more negative potentials. Minimal inhibition was produced by 3 mM toluene in F1579A mutant channels. Recovery from inactivation was slower both in Nav1.4 and F1579A channels in the presence of 3 mM toluene. The solvent blocked sodium currents in a use-dependent and frequency-dependent manner in Nav1.4 channels. A single mutation in the local anaesthetic binding site of Nav1.4 channels almost abolished toluene effects. These results suggest that this site is important for toluene action.     

Discovery and biological evaluation of 5-aryl-2-furfuramides, potent and selective blockers of the Nav1.8 sodium channel with efficacy in models of neuropathic and inflammatory pain

Nav1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons and has been implicated in the pathophysiology of inflammatory and neuropathic pain. Recent studies using an Nav1.8 antisense oligonucleotide in an animal model of chronic pain indicated that selective blockade of Nav1.8 was analgesic and could provide effective analgesia with a reduction in the adverse events associated with nonselective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 5-substituted 2-furfuramides, which are potent, voltage-dependent blockers (IC50 < 10 nM) of the human Nav1.8 channel. Selected derivatives, such as 7 and 27, also blocked TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons with comparable potency and displayed >100-fold selectivity versus human sodium (Nav1.2, Nav1.5, Nav1.7) and human ether-a-go-go (hERG) channels. Following systemic administration, compounds 7 and 27 dose-dependently reduced neuropathic and inflammatory pain in experimental rodent models.     

Inhibition of Nav1.7 channel by a novel blocker QLS-81 for alleviation of neuropathic pain

Voltage-gated sodium channel Nav1.7 robustly expressed in peripheral nociceptive neurons has been considered as a therapeutic target for chronic pain, but there is no selective Nav1.7 inhibitor available for therapy of chronic pain. Ralfinamide has shown anti-nociceptive activity in animal models of inflammatory and neuropathic pain and is currently under phase III clinical trial for neuropathic pain. Based on ralfinamide, a novel small molecule (S)-2-((3-(4-((2-fluorobenzyl) oxy) phenyl) propyl) amino) propanamide (QLS-81) was synthesized. Here, we report the electrophysiological and pharmacodynamic characterization of QLS-81 as a Nav1.7 channel inhibitor with promising anti-nociceptive activity. In whole-cell recordings of HEK293 cells stably expressing Nav1.7, QLS-81 (IC₅₀ at 3.5 ± 1.5 μM) was ten-fold more potent than its parent compound ralfinamide (37.1 ± 2.9 μM) in inhibiting Nav1.7 current. QLS-81 inhibition on Nav1.7 current was use-dependent. Application of QLS-81 (10 μM) caused a hyperpolarizing shift of the fast and slow inactivation of Nav1.7 channel about 7.9 mV and 26.6 mV, respectively, and also slowed down the channel fast and slow inactivation recovery. In dissociated mouse DRG neurons, QLS-81 (10 μM) inhibited native Nav current and suppressed depolarizing current pulse-elicited neuronal firing. Administration of QLS-81 (2, 5, 10 mg· kg⁻¹· d⁻¹, i.p.) in mice for 10 days dose-dependently alleviated spinal nerve injury-induced neuropathic pain and formalin-induced inflammatory pain. In addition, QLS-81 (10 μM) did not significantly affect ECG in guinea pig heart ex vivo; and administration of QLS-81 (10, 20 mg/kg, i.p.) in mice had no significant effect on spontaneous locomotor activity. Taken together, our results demonstrate that QLS-81, as a novel Nav1.7 inhibitor, is efficacious on chronic pain in mice, and it may hold developmental potential for pain therapy.     

Use-dependent blockade of sodium channels by local anaesthetics and antiarrhythmic drugs. Effects of chloramine-T and calcium ions

Voltage clamp studies were carried out of the effects of chloramine-T(CT) and external Ca++ on the blocking interactions of local anaesthetics (LAs) and antiarrhythmic drugs (lidocaine, tetracaine, N-propyl ajmaline, compound KC 3791) with Na+ channels in frog Ranvier nodes. The results obtained provided direct evidence for the notion that: LAs interact preferentially with inactivated Na+ channels and stabilize their inactivated conformation ("drug-induced slow inactivation": SI); and SI underlies the cumulative inhibition of INa during repetitive membrane stimulation. Normal inactivation is not indispensable, but plays an auxiliary role in the mechanism of cumulative inhibition of INa by drugs interacting with open Na+ channels. This block results mainly from accumulation of the channels in the resting blocked state (due to the inability of charged drugs to leave the channel via a "hydrophobic pathway"). The contribution of the blockade-inactivated state to this type of block may depend on some properties of the drug and the holding membrane potential. The problem of the location of the binding site responsible for LA-induced SI requires further investigation in view of the fact that in the myocardium, along with LA, the lipid-insoluble tetrodotoxin (TTX) induces a pronounced SI.     

Analysis of human Nav1.8 expressed in SH-SY5Y neuroblastoma cells

The tetrodotoxin-resistant voltage-gated sodium channel alpha-subunit Nav1.8 is expressed in nociceptors and has been implicated in chronic pain. Difficulties of heterologous expression have so far precluded analysis of the pharmacological properties of human Nav1.8. To address this we have introduced human Nav1.8 in neuroblastoma SH-SY5Y cells. Voltage-clamp analysis showed that human Nav1.8 generated an inward tetrodotoxin-resistant sodium current with an activating threshold around -50 mV, half maximal activation at -11+/-3 mV and a reversal potential of 67+/-4 mV. These properties closely match those of the endogenous rat tetrodotoxin-resistant sodium current in dorsal root ganglia suggesting that the expressed human channel is in a near physiological conformation. Human Nav1.8 was resistant to tetrodotoxin and activated by the pyrethroid toxin deltamethrin. Both voltage-activated and deltamethrin-activated human Nav1.8 were inhibited by the sodium channel blockers BIII 890 CL, NW-1029, and mexiletine. Inhibition of Nav1.8 by these compounds may underlie their known analgesic effects in animal models.     

Ambroxol, a Nav1.8-preferring Na(+) channel blocker, effectively suppresses pain symptoms in animal models of chronic, neuropathic and inflammatory pain

Neuropathic pain affects many patients, and treatment today is far from being perfect. Nav1.8 Na⁺ channels, which are expressed by small fibre sensory neurons, are promising targets for novel analgesics. Na⁺ channel blockers used today, however, show only limited selectivity for this channel subtype, and can cause dose-limiting side effects. Recently, the secretolytic ambroxol was found to preferentially inhibit Nav1.8 channels. We used this compound as a tool to investigate whether a Nav1.8-preferring blocker can suppress symptoms of chronic, neuropathic and inflammatory pain in animal models. The drug was tested in the formalin paw model, two models of mononeuropathy, and a model of monoarthritis in rats. Ambroxol's effects were compared with those of gabapentin. Ambroxol at a dose of 1 g/kg had to be administered to rats to achieve the plasma levels that are reached in clinical use (for the treatment of infant and acute respiratory distress syndrome). Ambroxol (1 g/kg) was only weakly effective in models for acute pain, but effectively reduced pain symptoms in all other models; in some cases it completely reversed pain behaviour. In most cases the effects were more pronounced than those of gabapentin (at 100 mg/kg). These data show that a Nav1.8-preferring Na⁺ channel blocker can effectively suppress pain symptoms in a variety of models for chronic, neuropathic and inflammatory pain at plasma levels, which can be achieved in the clinic.     

Identification of a potent, state-dependent inhibitor of Nav1.7 with oral efficacy in the formalin model of persistent pain

Clinical human genetic studies have recently identified the tetrodotoxin (TTX) sensitive neuronal voltage gated sodium channel Nav1.7 (SCN9A) as a critical mediator of pain sensitization. Herein, we report structure-activity relationships for a novel series of 2,4-diaminotriazines that inhibit hNav1.7. Optimization efforts culminated in compound 52, which demonstrated pharmacokinetic properties appropriate for in vivo testing in rats. The binding site of compound 52 on Nav1.7 was determined to be distinct from that of local anesthetics. Compound 52 inhibited tetrodotoxin-sensitive sodium channels recorded from rat sensory neurons and exhibited modest selectivity against the hERG potassium channel and against cloned and native tetrodotoxin-resistant sodium channels. Upon oral administration to rats, compound 52 produced dose- and exposure-dependent efficacy in the formalin model of pain.