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.

Keywords: Membrane hyperexcitability; Mexiletine; Myotonia; Sodium channel; Tocainide derivative; Use-dependence.

Graphical abstract

Fig. 1

Chemical structures of tocainide and explorative compounds. The compound To10 is a benzyl-N-substituted β-proline derivative of tocainide (known also as NeP1, Ghelardini et al., 2010). In To040, the β-proline cycle was eliminated. In To042, the benzyl moiety was substituted for by a naphthalene group (Muraglia et al., 2014).

Fig. 2

Concentration-dependent effects of tocainide derivatives on hNav1.4 channels. (A) Representative traces of wild-type hNav1.4 sodium currents recorded in transfected HEK293 cells at steady-state before (CTRL) and during application of 30 μM To040 or To042 at 0.1 Hz and 10 Hz frequency stimulations. (B) Concentration-response relationships of To040 and To042 on WT hNav1.4 currents at 0.1 and 10 Hz. Each experimental point is the mean ± S.E.M. of at least 3 cells. The relationships were fit to the first-order binding equation (1) reported in the text, and fit parameter values are reported in Table 1 (C) bargraph shows IC₅₀ values calculated as in B for To042, To040, To10, mexiletine (Mex), and tocainide (Toc).

Fig. 3

Concentration-dependent effects of tocainide derivatives on F1586C hNav1.4 channel mutant. (A). Representative traces of F1586C hNav1.4 sodium currents recorded in transfected HEK293 cells at steady-state before (CTRL) and during application of 30 μM To040 or To042 at 0.1 Hz and 10 Hz frequency stimulations. (B) Concentration-response relationships of To040 and To042 on WT hNav1.4 currents at 0.1 and 10 Hz. Each experimental point is the mean ± S.E.M. of at least 3 cells. The relationships were fit to the first-order binding equation (1) reported in the text, and fit parameter values are reported in Table 1 (C) bargraph shows IC₅₀ values calculated as in B for To042, To040, and mexiletine (Mex).

Fig. 4

Effects of tocainide derivatives on hNav1.4 channels in a myotonia-like condition. (A) Representative traces of wild-type hNav1.4 sodium currents recorded in transfected HEK293 cells at steady-state before (CTRL) and during application of 0.1 μM To042. The cell was depolarized to −30 mV for 5 ms (as an action potential duration) from a holding potential of −90 mV (close to the resting membrane potential of skeletal muscle fibers) at 0.1 and 50 Hz frequency stimulations (as during a myotonic run). In absence of drug, a use-dependent reduction of sodium current developed to reach a steady-state, UDB-C. In presence of drug, the tonic block (TB) exerted by To042 was measured at 0.1 Hz stimulation, while the use-dependent sodium current reduction (UDB-D) was measured at 50 Hz. (B) Time course of hNav1.4 sodium current reduction at 50 Hz in absence (UDB-C) and presence (UDB-D) of 0.1 μM To042. The quantity of block directly attributable to the drug (TB + UDB) was calculated by subtracting UDB-C to UDB-D and adding TB. (C) Concentration-dependent inhibition of hNav1.4 channels by To042 as calculated in B. (D) Concentration-response relationships for To042 and mexiletine determined as in C. Each point is the mean ± S.E.M. from at least 3 cells. The relationships were fit to the first-order binding equation (2) reported in the text and fit parameter values are reported in Table 2.

Fig. 5

In vivo effects of tocainide derivatives in a rat model of myotonia. (A) Experimental protocol. Myotonia was induced in the rat by intraperitoneal injection of 30 mg/kg 9-anthracene carboxylic acid (9-AC) at time zero. The drug was administrated by os 20 min after 9-AC injection. The time of righting reflex (TRR) was measured at various before and after 9AC injection, as a measure of muscle stiffness. (B) Time course of the antimyotonic activity of To042 at different doses. The TRR was normalized with respect to that measured at 10 min time point. Experimental points are the means ± S.E.M. from at least 3 rats. (C) Dose-response relationship of To042, compared to the one of mexiletine, calculated as in B at 30 min time point. Each experimental point is the mean ± S.E.M. from at least 3 rats. The relationships were fit to the first-order binding equation (3) reported in the text, and fit parameter values are reported in Table 2.

Fig. 6

Comparative effects on To042 and mexiletine on hERG potassium channel. (A) Representative traces of hERG potassium currents recorded in transfected tsA201 cells at steady-state before (CTRL) and during application of 100 μM mexiletine (left panel) or 10 μM To042 (right panel). The cells were held at −70 mV and depolarized for 5 s to potentials ranging from −70 to +70 mV, applied in 10 mV increments. Tail currents were recorded at −50 mV. (B) Current-voltage relationships were drawn from steady-state current amplitudes measured at the end of the 5 s-long test pulse, before and during application of various mexiletine (left panel) and To042 concentrations (right panel). Each experimental relationship is the mean ± S.E.M. of at least 3 cells. (C) Concentration-response relationships drawn using tail current amplitudes measured at −50 mV after test depolarization at −10 mV. Each data point is the mean ± S.E.M. from at least 3 cells. The relationships were fit to the first-order binding equation (1) and fit parameter values are reported in the text.

Fig. 7

Effects of To042 on rat motor coordination measured as the latency to fall from the rotarod. Each data point is the mean ± S.E.M. from 6 rats.

Fig. 8

Citotoxic effects of tocainide and To042 on murine C2C12 cells, expressed as percentage of cell survival after 6 h of incubation with the drug. Each bar represents the mean ± SEM from 4 to 6 experiments. Statistical analysis was performed using unpaired Student's t-test and effect was considered significant for p < 0.05.