ABSTRACT
Title
Toward the development of dually acting sodium channel blockers and anti-oxidant compounds with wider therapeutic application in neuromuscular disorders: effects of newly synthesized pyrroline derivatives of mexiletine.
Authors
M. De Bellis1, V. Sblendorio1, A. Carocci2, MM. Cavalluzzi2, G. Lentini2, C. Franchini2, A. De Luca1and D. Conte Camerino1,
1. Unit of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari Aldo Moro and 2. Department of Medicinal Chemistry, Faculty of Pharmacy University of Bari Aldo Moro, Italy.
1. Unit of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari Aldo Moro and 2. Department of Medicinal Chemistry, Faculty of Pharmacy University of Bari Aldo Moro, Italy.
Abstract
The classical anesthetic-like (LA) drugs mexiletine (Mex) and tocainide (Toc), formerly used for treating rare myotonic syndromes, have been withdrawn from the market, thus further reducing the few therapeutic options for these diseases. A large screening of newly synthesized analogues helped us to clarify the drug structural requirements for ameliorating the therapeutic profile, in terms of potency and use-dependent block of skeletal muscle sodium currents. In particular, the presence of a lipophilic moiety (phenyl, benzyl group) on either the chiral center or the amino group nearby enhances potency up to 30-fold (De Luca et al., Mol. Pharmacol. 2000; 2003).At the light of these results, we decided to introduce further structural changes in the pharmacophore amino terminal group of Mex and in particular we focused on the effects of the introduction of a pyrroline ring. This group may change the physicochemical properties (logP and pKa values) improving both the hydrophobic interaction with the binding site and the use-dependent behaviour as well as conferring new pharmacological activities. In fact, the pyirroline ring has an antioxidant activity and previous studies have shown that this group confers to Mex and to others antiarrhytmic drugs protective effects against ischemia-reperfusion myocardial injury in vivo (Twomey et al., 1997; Ravi et al., 2000). Then, the combination of antioxidant and membrane stabilizing mechanisms may enlarge the therapeutic potential to other muscle disorders, such as muscular dystrophies.
Then we tested two newly synthesized analogs of Mex: VM11, in which the pyrroline ring was introduced on the amino group of Mex and CI16, in which the same structural change has been introduced in the isopropyl analogue of Mex. The pyrroline derivatives were tested on sodium currents (INa) of single frog skeletal muscle fibers by means of voltage-clamp recordings. Steps to -20 mV from the holding potential of -100 mV at different stimulation frequencies (0.3 up to 10Hz), were applied in order to evaluate tonic (TB) and use-dependent blocks (UDB) by drugs. VM11 and CI16 were 3 and 6 fold more potent than Mex in producing a TB of INa with IC50values of 23.4 ± 0.9 μM and 12.6 ± 0.2 μM, respectively. In particular, CI16 showed the highest use-dependent behaviour with ratio (TB IC50/10Hz-UDB IC50) of 21 vs. 11.1 and 3.2 of VM11 and Mex, respectively. The IC50value of CI16 for UDB was 0.6 ± 0.1 μM, up to 40 fold more potent than Mex.
To monitor the anti-oxidant profile of the new derivatives, we started to evaluate the potential protection of Mex and of the two pyrroline derivatives against the oxidative cytotoxic effect of H2O2on C2C12 cells. Cell viability has been determined by means of a standard colorimetric test. Preliminary experiments show that Mex had a cytoprotective effect only at the concentration of 1 mM, while CI16 produced a significant, although modest, reduction of H2O2induced cytotoxicity at concentrations close to the IC50value for blocking Nav1.4. These results support our working hypothesis; however the modest antioxidant effect observed suggest that this latter could be related to the in vivo production of a nitroxide metabolite. The verification of this hypothesis will provide the basis for in vivo/ex vivo studies on proper animal models of muscular dystrophy.
Supported by the “Association Française contre les Myopathies” (AFM).
Twomey et al. (1997). Free Radic Biol Med 22(5):909-16.
Ravi et al. (2000). JPET 292: 838-845.
De Luca et al. (2000). Mol. Pharmacol. 57: 268-277.
De Luca et al. (2003). Mol. Pharmacol. 64: 932-945.
Then we tested two newly synthesized analogs of Mex: VM11, in which the pyrroline ring was introduced on the amino group of Mex and CI16, in which the same structural change has been introduced in the isopropyl analogue of Mex. The pyrroline derivatives were tested on sodium currents (INa) of single frog skeletal muscle fibers by means of voltage-clamp recordings. Steps to -20 mV from the holding potential of -100 mV at different stimulation frequencies (0.3 up to 10Hz), were applied in order to evaluate tonic (TB) and use-dependent blocks (UDB) by drugs. VM11 and CI16 were 3 and 6 fold more potent than Mex in producing a TB of INa with IC50values of 23.4 ± 0.9 μM and 12.6 ± 0.2 μM, respectively. In particular, CI16 showed the highest use-dependent behaviour with ratio (TB IC50/10Hz-UDB IC50) of 21 vs. 11.1 and 3.2 of VM11 and Mex, respectively. The IC50value of CI16 for UDB was 0.6 ± 0.1 μM, up to 40 fold more potent than Mex.
To monitor the anti-oxidant profile of the new derivatives, we started to evaluate the potential protection of Mex and of the two pyrroline derivatives against the oxidative cytotoxic effect of H2O2on C2C12 cells. Cell viability has been determined by means of a standard colorimetric test. Preliminary experiments show that Mex had a cytoprotective effect only at the concentration of 1 mM, while CI16 produced a significant, although modest, reduction of H2O2induced cytotoxicity at concentrations close to the IC50value for blocking Nav1.4. These results support our working hypothesis; however the modest antioxidant effect observed suggest that this latter could be related to the in vivo production of a nitroxide metabolite. The verification of this hypothesis will provide the basis for in vivo/ex vivo studies on proper animal models of muscular dystrophy.
Supported by the “Association Française contre les Myopathies” (AFM).
Twomey et al. (1997). Free Radic Biol Med 22(5):909-16.
Ravi et al. (2000). JPET 292: 838-845.
De Luca et al. (2000). Mol. Pharmacol. 57: 268-277.
De Luca et al. (2003). Mol. Pharmacol. 64: 932-945.