Development of Riluzole Analogs with Improved Use-Dependent Inhibition of Skeletal Muscle Sodium Channels.

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

Safinamide's potential in treating nondystrophic myotonias: Inhibition of skeletal muscle voltage-gated sodium channels and skeletal muscle hyperexcitability in vitro and in vivo.

The antiarrhythmic sodium-channel blocker mexiletine is used to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or suboptimal response. Safinamide is an add-on therapy to levodopa for Parkinson's disease. In addition to MAOB inhibition, safinamide inhibits neuronal sodium channels, conferring anticonvulsant activity in models of epilepsy. Here, we investigated the effects of safinamide on skeletal muscle hNav1.4 sodium channels and in models of myotonia, in-vitro and in-vivo. Using patch-clamp, we showed that safinamide reversibly inhibited sodium currents in HEK293T cells transfected with hNav1.4. At the holding potential (hp) of -120 mV, the half-maximum inhibitory concentrations (IC50) were 160 and 33 µM at stimulation frequencies of 0.1 and 10 Hz, respectively. The calculated affinity constants of safinamide were dependent on channel state: 420 µM for closed channels and 9 µM for fast-inactivated channels. The p.F1586C mutation in hNav1.4 greatly impaired safinamide inhibition, suggesting that the drug binds to the local anesthetic receptor site in the channel pore. In a condition mimicking myotonia, i.e. hp. of -90 mV and 50-Hz stimulation, safinamide inhibited INa with an IC50 of 6 µM, being two-fold more potent than mexiletine. Using the two-intracellular microelectrodes current-clamp method, action potential firing was recorded in vitro in rat skeletal muscle fibers in presence of the chloride channel blocker, 9-anthracene carboxylic acid (9-AC), to increase excitability. Safinamide counteracted muscle fiber hyperexcitability with an IC50 of 13 µM. In vivo, oral safinamide was tested in the rat model of myotonia. In this model, intraperitoneal injection of 9-AC greatly increased the time of righting reflex (TRR) due to development of muscle stiffness. Safinamide counteracted 9-AC induced TRR increase with an ED50 of 1.2 mg/kg, which is 7 times lower than that previously determined for mexiletine. In conclusion, safinamide is a potent voltage and frequency dependent blocker of skeletal muscle sodium channels. Accordingly, the drug was able to counteract abnormal muscle hyperexcitability induced by 9-AC, both in vitro and in vivo. Thus, this study suggests that safinamide may have potential in treating myotonia and warrants further preclinical and human studies to fully evaluate this possibility.

Pharmacogenetics of myotonic hNav1.4 sodium channel variants situated near the fast inactivation gate.

Sodium channel myotonia and paramyotonia congenita are caused by gain-of-function mutations in the skeletal muscle voltage-gated sodium channel hNav1.4. The first-line drug is the sodium channel blocker mexiletine; however, some patients show side effects or limited responses. We previously showed that two hNav1.4 mutations, p.G1306E and p.P1158L, reduce mexiletine potency in vitro, whereas another sodium channel blocker, flecainide, is less sensitive to mutation-induced gating defects. This observation was successfully translated to p.G1306E and p.P1158L carriers. Thus, the aim of this study was to perform a pharmacological characterization of myotonic Nav1.4 mutations clustered near the fast inactivation gate of the channel. We chose seven mutations (p.V1293I, p.N1297S, p.N1297K, p.F1298C, p.G1306E, p.I1310N, and p.T1313M) from the database of Italian and French networks for muscle channelopathies. Recombinant hNav1.4 mutants were expressed in HEK293T cells for functional and pharmacological characterization using the patch-clamp technique. All the studied mutations impair the kinetics and/or voltage dependence of fast inactivation, which is likely the main mechanism responsible for myotonia. The severity of myotonia is well-correlated to the enhancement of window currents generated by the intersection of the activation and fast inactivation voltage dependence. Five of the six mutants displaying a significant positive shift of fast inactivation voltage dependence reduced mexiletine inhibition in an experimental condition mimicking myotonia. In contrast, none of the mutations impairs flecainide block nor does p.T1313M impair propafenone block, indicating that class Ic antiarrhythmics may constitute a valuable alternative. Our study suggests that mutation-driven therapy would be beneficial to myotonic patients, greatly improving their quality of life.

Effects of Benzothiazolamines on Voltage-Gated Sodium Channels.

Benzothiazole is a versatile fused heterocycle that aroused much interest in drug discovery as anticonvulsant, neuroprotective, analgesic, anti-inflammatory, antimicrobial, and anticancer. Two benzothiazolamines, riluzole and lubeluzole, are known blockers of voltage-gated sodium (Nav) channels. Riluzole is clinically used as a neuroprotectant in amyotrophic lateral sclerosis. Inhibition of Nav channels by riluzole is voltage-dependent due to preferential binding to inactivated sodium channels. Yet the drug exerts little use-dependent block, probably because it lacks protonable amine. One important property is riluzole ability to inhibit persistent Na+ currents, which likely contributes to its neuroprotective activity. Lubeluzole showed promising neuroprotective effects in animal stroke models, but failed to show benefits in acute ischemic stroke in humans. One important concern is its propensity to prolong the cardiac QT interval, due to hERG K+ channel block. Lubeluzole very potently inhibits Nav channels in a voltage- and use-dependent manner, due to its great preferential affinity for inactivated channels and the presence of a protonable amine group. Patch-clamp experiments suggest that the binding sites of both drugs overlap the local anesthetic receptor within the ion-conducting pathway. Riluzole and lubeluzole displayed very potent antimyotonic activity in a rat model of myotonia, a pathological skeletal muscle condition characterized by high-frequency runs of action potentials. Such results well support the repurposing of riluzole as an antimyotonic drug, allowing the launch of a pilot study in myotonic patients. Riluzole, lubeluzole, and new Nav channel blockers built on the benzothiazolamine scaffold will certainly continue to be investigated for possible clinical applications.

Coexistence of CLCN1 and SCN4A mutations in one family suffering from myotonia.

Non-dystrophic myotonias are characterized by clinical overlap making it challenging to establish genotype-phenotype correlations. We report clinical and electrophysiological findings in a girl and her father concomitantly harbouring single heterozygous mutations in SCN4A and CLCN1 genes. Functional characterization of N1297S hNav1.4 mutant was performed by patch clamp. The patients displayed a mild phenotype, mostly resembling a sodium channel myotonia. The CLCN1 c.501C>G (p.F167L) mutation has been already described in recessive pedigrees, whereas the SCN4A c.3890A>G (p.N1297S) variation is novel. Patch clamp experiments showed impairment of fast and slow inactivation of the mutated Nav1.4 sodium channel. The present findings suggest that analysis of both SCN4A and CLCN1 genes should be considered in myotonic patients with atypical clinical and neurophysiological features.

Increased sodium channel use-dependent inhibition by a new potent analogue of tocainide greatly enhances in vivo antimyotonic activity.

Although the sodium channel blocker, mexiletine, is the first choice drug in myotonia, some myotonic patients remain unsatisfied due to contraindications, lack of tolerability, or incomplete response. More therapeutic options are thus needed for myotonic patients, which require clinical trials based on solid preclinical data. In previous structure-activity relationship studies, we identified two newly-synthesized derivatives of tocainide, To040 and To042, with greatly enhanced potency and use-dependent behavior in inhibiting sodium currents in frog skeletal muscle fibers. The current study was performed to verify their potential as antimyotonic agents. Patch-clamp experiments show that both compounds, especially To042, are greatly more potent and use-dependent blockers of human skeletal muscle hNav1.4 channels compared to tocainide and mexiletine. Reduced effects on F1586C hNav1.4 mutant suggest that the compounds bind to the local anesthetic receptor, but that the increased hindrance and lipophilia of the N-substituent may further strengthen drug-receptor interaction and use-dependence. Compared to mexiletine, To042 was 120 times more potent to block hNav1.4 channels in a myotonia-like cellular condition and 100 times more potent to improve muscle stiffness in vivo in a previously-validated rat model of myotonia. To explore toxicological profile, To042 was tested on hERG potassium currents, motor coordination using rotarod, and C2C12 cell line for cytotoxicity. All these experiments suggest a satisfactory therapeutic index for To042. This study shows that, owing to a huge use-dependent block of sodium channels, To042 is a promising candidate drug for myotonia and possibly other membrane excitability disorders, warranting further preclinical and human studies.