Although the sodium channel blocker mexiletine is considered the first-line drug in myotonia, some patients experiment adverse effects, while others do not gain any benefit. Other antimyotonic drugs are thus needed to offer mexiletine alternatives. In the present study, we used a previously-validated rat model of myotonia congenita to compare six marketed sodium channel blockers to mexiletine. Myotonia was induced in the rat by injection of anthracen-9-carboxylic acid, a muscle chloride channel blocker. The drugs were given orally and myotonia was evaluated by measuring the time of righting reflex. The drugs were also tested on sodium currents recorded in a cell line transfected with the human skeletal muscle sodium channel hNav1.4 using patch-clamp technique. In vivo, carbamazepine and propafenone showed antimyotonic activity at doses similar to mexiletine (ED50 close to 5 mg/kg); flecainide and orphenadrine showed greater potency (ED50 near 1 mg/kg); lubeluzole and riluzole were the more potent (ED50 near 0.1 mg/kg). The antimyotonic activity of drugs in vivo was linearly correlated with their potency in blocking hNav1.4 channels in vitro. Deviation was observed for propafenone and carbamazepine, likely due to pharmacokinetics and multiple targets. The comparison of the antimyotonic dose calculated in rats with the current clinical dose in humans strongly suggests that all the tested drugs may be used safely for the treatment of human myotonia. Considering the limits of mexiletine tolerability and the occurrence of non-responders, this study proposes an arsenal of alternative drugs, which may prove useful to increase the quality of life of individuals suffering from non-dystrophic myotonia. Further clinical trials are warranted to confirm these results.

We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003), suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogs by adding one or two hydroxyl groups to the aryloxy moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration–response relationships were constructed using 25-ms-long depolarizing pulses at −30 mV applied from an holding potential of −120 mV at 0.1 Hz (tonic block) and 10 Hz (use-dependent block) stimulation frequencies. The half-maximum inhibitory concentrations (IC50) were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. In particular, replacement of Phe1586 and Tyr1593 by non-aromatic cysteine residues may help in the understanding of the role of π–π or π–cation interactions in mexiletine binding. Alteration of tonic block suggests that the aryloxy moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryloxy moiety may modify high-affinity binding of the drug amine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these results confirm our former hypothesis by showing that the presence of hydroxyl groups to the aryloxy moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with their receptor.

Orphenadrine is a drug acting on multiple targets, including muscarinic, histaminic, and NMDA receptors. It is used in the treatment of Parkinson's disease and in musculoskeletal disorders. It is also used as an analgesic, although its mechanism of action is still unknown. Both physiological and pharmacological results have demonstrated a critical role for voltage-gated sodium channels in many types of chronic pain syndromes. We tested the hypothesis that orphenadrine may block voltage-gated sodium channels. By using patch-clamp experiments, we evaluated the effects of the drug on whole-cell sodium currents in HEK293 cells expressing the skeletal muscle (Nav1.4), cardiac (Nav1.5) and neuronal (Nav1.1 and Nav1.7) subtypes of human sodium channels, as well as on whole-cell tetrodotoxin (TTX)-resistant sodium currents likely conducted by Nav1.8 and Nav1.9 channel subtypes in primary culture of rat DRG sensory neurons. The results indicate that orphenadrine inhibits sodium channels in a concentration-, voltage- and frequency-dependent manner. By using site-directed mutagenesis, we further show that orphenadrine binds to the same receptor as the local anesthetics. Orphenadrine affinities for resting and inactivated sodium channels were higher compared to those of known sodium channels blockers, such as mexiletine and flecainide. Low, clinically relevant orphenadrine concentration produces a significant block of Nav1.7, Nav1.8, and Nav1.9 channels, which are critical for experiencing pain sensations, indicating a role for sodium channel blockade in the clinical efficacy of orphenadrine as analgesic compound. On the other hand, block of Nav1.1 and Nav1.5 may contribute to the proconvulsive and proarrhythmic adverse reactions, especially observed during overdose.

The sodium channel blocker mexiletine is considered the first-line drug in myotonic syndromes, a group of muscle disorders characterized by membrane over-excitability. We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, block voltage-gated sodium channels in a manner reminiscent to mexiletine, whereas salbutamol and nadolol do not. We now developed a pharmacological rat model of myotonia congenita to perform in vivo preclinical test of antimyotonic drugs. Myotonia was induced by i.p. injection of 30 mg/kg of anthracene-9-carboxylic acid (9-AC), a muscle chloride channel blocker, and evaluated by measuring the time of righting reflex (TRR). The TRR was prolonged from <0.5 s in control conditions to a maximum of ∼4 s, thirty minutes after 9-AC injection, then gradually recovered in a few hours. Oral administration of mexiletine twenty minutes after 9-AC injection significantly hampered the TRR prolongation, with an half-maximum efficient dose (ED50) of 12 mg/kg. Both propranolol and clenbuterol produced a dose-dependent antimyotonic effect similar to mexiletine, with ED50 values close to 20 mg/kg. Antimyotonic effects of 40 mg/kg mexiletine and propranolol lasted for 2 h. We also demonstrated, using patch-clamp methods, that both propranolol enantiomers exerted a similar block of skeletal muscle hNav1.4 channels expressed in HEK293 cells. The two enantiomers (15 mg/kg) also showed a similar antimyotonic activity in vivo in the myotonic rat. Among the drugs tested, the R(+)-enantiomer of propranolol may merit further investigation in humans, because it exerts antimyotonic effect in the rat model, while lacking of significant activity on the β-adrenergic pathway. This study provides a new and useful in vivo preclinical model of myotonia congenita in order to individuate the most promising antimyotonic drugs to be tested in humans.

Background and purpose: Pilsicainide, an anti-arrhythmic drug used in Japan, is described as a pure sodium channel blocker. We examined the mechanisms by which it is able to block open channels, because these properties may be especially useful to reduce hyperexcitability in pathologies characterized by abnormal sodium channel opening. Experimental approach: The effects of pilsicainide on various heterologously expressed human sodium channel subtypes and mutants were investigated using the patch clamp technique. Key results: Pilsicainide exhibited tonic and use-dependent effects comparable to those of mexiletine and flecainide on hNav1.4 channels. These use-dependent effects were abolished in the mutations F1586C and Y1593C within segment 6 of domain IV, suggesting that the interaction of pilsicainide with these residues is critical for its local anaesthetic action. Its affinity constants for closed channels (KR) and channels inactivated from the closed state (KI) were high, suggesting that its use-dependent block (UDB) requires the channel to be open for it to reach a high-affinity blocking site. Accordingly, basic pH, which slightly increased the proportion of neutral drug, dramatically decreased KR and KI values. Effects of pilsicainide were similar on skeletal muscle hNav1.4, brain hNav1.1 and heart hNav1.5 channels. The myotonic R1448C and G1306E hNav1.4 mutants were more and less sensitive to pilsicainide, respectively, due to mutation-induced gating modifications. Conclusions and implications:Although therapeutic concentrations of pilsicainide may have little effect on resting and closed-state inactivated channels, it induces a strong UDB due to channel opening, rendering the drug ideally suited for inhibition of high-frequency action potential firing. Abbreviations: h and (1 -h), proportions of closed and inactivated channels at a particular potential; ICTRL, peak current amplitude measured in the same cell before drug application; IDRUG, peak current amplitude measured in the presence of drug; INa, whole-cell sodium current; KAPP, apparent affinity constant at a given potential; KI, affinity for inactivated channels; KR, affinity for closed channels; LA, local anaesthetic; S, slope factor; TB, tonic block; UDB, use-dependent block; V, conditioning pulse potential; V1/2, half-maximum inactivation potential British Journal of Pharmacology IntroductionDue to their ability to act as sodium channel blockers, local anaesthetic (LA)-like drugs can be used to treat a large spectrum of disorders including cardiac arrhythmias, epilepsy, myotonia and neuropathic pain (Conte Camerino et al., 2007). Many of these drugs present a tertiary amine associated with a hydrophobic aromatic tail through an amide, ester or ether link. The two ends of the drug may interact with channel pore-lining amino acids through hydrophobic or p-cation interactions (Ragsdale et al., 1994;Wright et al., 1998;Nau et al., 1999;Wang et al., 2000;Yarov-Yarovoy et al., 2001;O'Leary and Chahine, 2002;McNulty et al., 2007;Ahern et al., 2008;Sunam...