Inhibition of frog skeletal muscle sodium channels by newly synthesized chiral derivatives of mexiletine and tocainide

Abstract

To search for potent use-dependent blockers of skeletal muscle sodium channels as potential antimyotonic agents, the actions of newly synthesized chiral analogs of mexiletine and tocainide were tested in vitro on sodium currents of single fibers of frog semitendinosus muscle by vaseline-gap voltage clamp method. The effect of each drug on the maximal peak Na+ transient (I(Na) max) was evaluated as both tonic and use-dependent block by using infrequent depolarizing stimulation and trains of pulses at 2-10 Hz frequency, respectively. The mexiletine analog 3-(2,6-dimethylphenoxy)-2-methylpropanamine (Me2), having an increased distance between the phenyl and the amino groups, was less potent than mexiletine in producing a tonic block but produced a remarkable use-dependent block. In fact, the half-maximal concentration (IC50) for tonic block of S(-)-Me2 was 108 microM vs. 54.5 microM of R(-)-mexiletine, but the IC50 was 6.2 times lowered by the 10 Hz stimulation with respect to the 2.4 fold decrease observed with mexiletine. The R(-)-mexiletine and the S(-)-Me2 were about twofold more potent than the corresponding enantiomers in producing a tonic block, but the stereoselectivity attenuated during use-dependent blockade. The more lipophilic 2-(4-chloro-2-methylphenoxy)-1-phenylethylamine (Me1), presently available as raceme, produced a potent and irreversible tonic block of the sodium currents with an IC50 of 29 microM, but had a less pronounced use-dependent inhibition, with a 1.9 fold decrease of the IC50 at 10 Hz. The R(-) isomer of 2',6'-valinoxylidide (To1), a tocainide derivative with an increased hindrance on the chiral carbon atom, was twofold (IC50 = 209 microM) and tenfold (IC50 = 27.4 microM) more potent than R(-)-tocainide in tonic and use-dependent block, respectively. Tocainide was almost devoid of stereoselectivity, whereas the eudismic ratio of To1 [(IC50 S(+)-To1/IC50 R(-)-To1] was 1.7. As for mexiletine and Me2, the stereoselectivity of To1 was the weaker the higher the frequency of stimulation. The cyclic pyrrolo-imidazolonic tocainide analog To2 produced a small tonic block at 500 microM, and 1 min stimulation at 10 Hz was needed to show up a 50% block of I(Na) max. All the compounds produced a left-shift of the steady-state inactivation curve correlated positively with the extent of use-dependent inhibition, with the exception of the cyclic To2 that acted as an open-channel blocker. The highly use-dependent blockers Me2 and To1 might be promising drugs to solve high frequency discharges of action potentials typical of myotonic muscles. Concomitantly the high potency of Me1 and the open-channel block exerted by To2 can represent important features to get selective blockers for skeletal muscle sodium channels.

Optimal Requirements for High Affinity and Use-Dependent Block of Skeletal Muscle Sodium Channel by N-Benzyl Analogs of Tocainide-Like Compounds

“…The width of the gaps of the central pools (A and B) had been previously set to 70 to 100 m and 200 m, respectively. Four KCl/agar bridges electrodes connected the recording chamber to the voltage clamp amplifier based on methods described by Hille and Campbell (1976) and detailed elsewhere (Hille and Campbell, 1976;De Luca et al, 1997a. For recordings, the solution in pool A was replaced with the external solution; after about 10 min of equilibration, the recordings were performed at 10°C.…”

“…Each sequence consisted of a conditioning pulse to Ϫ140 mV for 500 ms (to have most of the sodium channels in the "activatable" state), a prepulse of variable potential of 1000-ms duration, and the 10-ms test pulse to Ϫ20 mV; after a pause of 1 s, the sequence was cyclically repeated 18 to 20 times, with the prepulse potential value increased each time by 5-mV steps (De Luca et al, 1997a.…”

“…The estimates of S.E.M. of normalized I Na values have been obtained as described previously (De Luca et al, 1997a. Molar concentrations of the drugs tested that produce a 50% block of I Na (IC 50 ) in the various experimental conditions were determined by using a nonlinear least-squares fit of the concentrationresponse curves to the following logistic equation:…”

“…The parts of LA molecule with recognized pharmacophore roles are the ionizable amino group and the aromatic ring at the opposite extremity, which are proposed to establish cation-and -interactions, respectively, with the two hydrophobic domains cited above (Dougherty, 1996;Ragsdale et al, 1996). In addition, a site in D1-S6 segment may account for the stereoselective action of chiral compounds (De Luca et al, 1997aNau et al, 1999), probably as a consequence of a third point interaction driven by the stereogenic center, which is the carbon atom near the pharmacophore amino group. An increase in the lipophilicity and/or hindrance at this level (i.e., with an aromatic ring) enhances the potency of stereoselective compounds for blocking Na ϩ currents in skeletal muscle by up to 10-fold (De Luca et al, 1997aDesaphy et al, 2001).…”

“…In addition, a site in D1-S6 segment may account for the stereoselective action of chiral compounds (De Luca et al, 1997aNau et al, 1999), probably as a consequence of a third point interaction driven by the stereogenic center, which is the carbon atom near the pharmacophore amino group. An increase in the lipophilicity and/or hindrance at this level (i.e., with an aromatic ring) enhances the potency of stereoselective compounds for blocking Na ϩ currents in skeletal muscle by up to 10-fold (De Luca et al, 1997aDesaphy et al, 2001). We proposed that the lipophilic group at this level could establish with the hydrophobic pockets in D4-S6 a -interaction stronger than the -cation interaction made by the amino group (Wright et al, 1998;De Luca et al, 2000.…”