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

Abstract

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.

Dual Action of Mexiletine and Its Pyrroline Derivatives as Skeletal Muscle Sodium Channel Blockers and Anti-oxidant Compounds: Toward Novel Therapeutic Potential

“…Another main aim of our study was to investigate about a potential anti-oxidant effect of the tetramethyl-pyrroline-derivatives, by means of assessing their cyto-protective action on a model of H 2 O 2 -induced oxidative stress cytotoxicity. However, data from our and other laboratories underlined a direct potential cytotoxicity of local anesthetic-like drugs on various cell types (Hofmann et al, 2013 ; Sung et al, 2014 ; De Bellis et al, 2017a ). Then we first evaluated the ability of the lead compound Mex, to modulate by itself C 2 C 12 cell viability with respect to H 2 O 2 .…”

“…We then assessed the activity of the new Mex and Me5 derivatives on Na V 1.4 channel (De Bellis et al, 2017a ). This was of importance considering the presence of the pyrroline moiety on the main pharmacophore group (amino-terminal group).…”

“…Surprisingly, the new analog CI16 is the most use-dependent mexiletine-like sodium channel blocker described so far, with a ratio IC 50 TB/IC 50 10-Hz UDB of 21 (Table 2 ). The use-dependent behavior is a complex dynamic process involving the kinetics of drug binding to and unbinding from the channel in relation to both state-dependent drug affinity and physicochemical properties (De Bellis et al, 2017a , b ). The recovery from inactivation of drug-bound channels was determined.…”

“…The acquired traces were analyzed offline using Clampfit software (pClamp6; Axon Instruments). This method was shown to be predictable of drug efficacy on mammalian sodium channel and myotonic conditions (De Luca et al, 2004 ; De Bellis et al, 2017a ).…”

Digging into Lipid Membrane Permeation for Cardiac Ion Channel Blocker d-Sotalol with All-Atom Simulations

“…Even more importantly, similar computational approaches can be used as one of the steps to design drugs, which have similar membrane binding affinities and bind around mutated protein residues that result in altered channel function. Such an approach focusing on a desired drug lipophilicity and spatial arrangement of crucial functional groups was used, for instance, to design selective sodium channel blockers (Muraglia et al, 2007 ; De Luca et al, 2012 ; De Bellis et al, 2017 ). Such a structure-function based approach was shown to improve drug safety profile through mitigation of off-target effects, including hERG block (De Bellis et al, 2017 ).…”