ABSTRACT
1Dept. of Pharmaco-Biology, Section of Pharmacology, University of Bari, Italy
2Dept of Medicinal Chemistry, University of Bari, Italy
The voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability. To date nine VGSCs isoforms have been identified –from Nav1.1 to Nav1.9- and have a distinct pattern of expression within the human organism. Nav1.4 and Nav1.5 are expressed in the skeletal muscle and in the heart, respectively, and are the target of clinical important drugs such as antimyotonics and antiarrhythmics, including mexiletine and tocainide. In this study, we evaluated the effects of newly-designed mexiletine and tocainide derivatives on sodium currents in HEK293 cells transfected with the hNav1.4 and hNav1.5 channels using patch-clamp experiments. Sodium currents were elicited by 25 ms-long depolarizing test pulses at -30 mV from the holding potential of -120 mV at two stimulation frequencies: 0.1 Hz to determine tonic block and 10 Hz to determine use-dependent block. The IC50 values were calculated by fitting the concentration-effect relationship with a first order binding function. Mexiletine and its derivative metahydroxymexiletine (MHM), a natural metabolite that accounts for 2% of oral mexiletine, produced similar effects on hNav1.4 channels (IC50 values of mexiletine: 246 ± 15 µM at 0.1 Hz and 24 ± 2 µM at 10 Hz; IC50 values of MHM: 259 ± 28 µM at 0.1 Hz and 41 ± 5 µM at 10 Hz). In contrast, the metabolite was two-fold more potent than its parent compound on hNav1.5 channels (IC50 values of mexiletine: 421 ± 69 µM at 0.1 Hz and 58 ± 13 µM at 10 Hz; IC50 values of MHM: 174 ± 6 µM at 0.1 Hz and 30 ± 3 µM at 10 Hz). Because of its reduced lipophilia, MHM may be less able than mexiletine to permeate the blood-brain barrier and so may be used in the treatment of peripheral hyperexcitability disorders without producing the limiting central nervous side effects. Regarding tocainide, we previously showed that a benzylated beta-proline derivative of tocainide, namely To10 or NeP1, was a very potent and use-dependent blocker of human sodium channels (Ghelardini et al., 2010). The newly synthesized To040 was obtained by the rupture of the beta-proline cycle of To10. In To042, the benzyl of To040 was substituted by a naphtyl. The effects of To040 on hNav1.4 channels were very similar to those of To10 (IC50 values of To10: 67 ± 5 µM at 0.1 Hz and 5 ± 0.3 µM at 10 Hz; IC50 values of To040: 79 ± 17 µM at 0.1 Hz and 4 ± 0.8 µM at 10 Hz), suggesting no change of constricting the amine group in a cycle. In contrast, To042 showed a greater effect than To10 on hNav1.4 channels (IC50 values of To042: 12 ± 1 µM at 0.1 Hz and 0.8 ± 0.1 µM at 10 Hz), indicating that increased hindrance and/or lipophilia on the N-terminal improve the binding of the drug to the channel. The tocainide derivatives were also tested on the F1586C hNav1.4 channel mutant, to verify whether they bind to the local anesthetic receptor. The IC50 values of To040 and To042 (To040 on Nav1.4 FC: 138 ± 9 µM at 0.1 Hz and 62 ± 4 µM at 10 Hz; To042 on Nav1.4 FC: 44 ± 4 µM at 0.1 Hz and 14 ± 2 µM at 10 Hz) demonstrate that Phe in position 1586 remain important for the binding of To040 and To042 to the channel. To042 shows a further improvement of sodium channel blockade compared to To10, which may be of interest in the treatment of hyperexcitability disorders, including myotonia and chronic pain (Ghelardini et al., 2010). Supported by the “Association Française contre les Myopathies”.
1. Ghelardini, Desaphy, Muraglia, Corbo, Matucci, Dipalma, Bertucci, Pistolozzi, Nesi, Norcini, Franchini, and Conte. Neuroscience. 2010 Aug 25;169(2):863-73