Mechanistic studies of novel sodium channel blockers

Phenytoin or diphenylhydantoin (DPH) is a clinically useful sodium channel blocker with efficacy against seizure disorders and neuropathic pain, and is a class 1b anti-arrhythmic. We have developed novel hydantoin and alpha-hydroxyphenylamide compounds designed using a pharmacophore for DPH and evaluated them for sodium channel blockade. While [3H]batrachotoxinin A 20-alpha-benzoate (BTX) displacement studies were completed and identified potential sodium channel blockers, little was known about the affects these compounds would have on sodium conductance and channel gating. Initially we examined these novel compounds on the neuronal sodium channel rNav1.2 expressed in Xenopus laevis oocytes. These results suggested that the novel analogues of DPH were more potent neuronal sodium channel blockers. We then examined a similar set of alpha-hydroxyphenylamide analogues of DPH with varying phenyl ring substitutions and examined their ability to inhibit hNav1.5 sodium channels expressed in Chinese Hamster Ovary (CHO) cells using the patch clamp recording technique. We found that phenyl ring substitutions with electron withdrawing properties resulted in compounds with greater activity. In comparison to DPH, the novel chloro-substituted alpha-hydroxyphenylamide compounds produced as much as a 20 fold greater tonic and frequency-dependent blockade of hNav1.5 sodium channels. In addition, we have found that the chloro-substitution position is a determinant for the state dependent blocking properties of the compound and these differences in affinity are due to a direct interaction with the receptor and not three dimensional differences between the molecules structures. Local anesthetics such as bupivacaine are potent sodium channel blockers and share several molecular determinants of binding with DPH. To this end we designed several compounds based on overlapping the phenyl ring of bupivacaine with that of DPH and examined their ability to block hNav1.5 sodium channels expressed in CHO cells. While all novel compounds significantly blocked sodium currents more effectively than DPH, only those containing a rather minimal overlap structure were more potent than bupivacaine. In summary we have demonstrated that analogues of DPH and bupivacaine are more potent sodium channel blockers and exhibit unique effects on channel gating, thus they may be useful for the treatment of epilepsy and other disorders.