Phenytoin - an old but underestimated drug to treat atrial arrhythmias?

Background

Phenytoin, a hydantoin derivative, is an anti-seizure drug, that is used clinically in the prevention of convulsions, the acute treatment of status epilepticus, as well as the modulation of neuropathic pain. It is also used in humans to terminate ventricular arrythmias due to its well-known ability to block Na⁺-channels, leading to it being classified as a type Ib antiarrhythmic drug. Furthermore, it may be utilized to cardiovert atrial fibrillation (AF) in horses. Given the wide range of medical uses, as well as recent studies indicating pleiotropic effects beyond Na⁺-channel inhibition in the heart, a systematic analysis of phenytoin’s ion channel inhibition profile is needed to define its most optimal use in cardiology practice.

Purpose

This study aims to analyze the effects of phenytoin on cardiac K⁺-channels that are involved in action potential formation. Members of the presently understudied K2P channel family will be a key focus area.

Methods

The effects of phenytoin on cardiac ionic currents were assessed using two-electrode voltage clamp measurements of K⁺- channels heterologously expressed in Xenopus laevis oocytes. Alanine mutagenesis scanning and in-silico docking simulations were performed to identify potential sites of interaction with phenytoin.

Results

At a screening concentration of 100 µM, phenytoin showed statistically significant inhibition of K2P17.1 (37% ±3.1%, p=0.0015, n=4). Inhibition of the K2P3.1 (23% ±1.9%, p=0.0666, n=7) channel came close to reaching significance. In contrast, the closely related alkaline-activated K2P16.1 channel (10% ±0.8%, p=0.7485, n=4), as well as the other 10 K⁺-channels tested (i.e. K2P2.1, K2P9.1, K2P13.1, K2P18, K_V2.1, K_V1.4, K_V1.5, K_V4.3, K_ir2.1, hERG), displayed only minor, non-significant inhibition. Due to its use in the cardioversion of AF in horses, we also tested the equine orthologue of the K2P3.1 channel, for which we observed significant inhibition (33% ±2.8%, p=0.0067, n=4).

Prior studies have shown that phenytoin blocks Na⁺-channels more potently at higher frequencies of stimulation. So further analysis into the frequency dependence of K2P3.1 and K2P17.1 channel inhibition followed. At stimulation rates of 1.0 Hz (K2P17.1 21% ±1.5%, p=0.0002, n=6; K2P3.1 39% ±2.7%, p=0.0001, n=4) and 1.5 Hz (K2P17.1 33% ±3.6%, p<0.0001, n=3; K2P3.1 41% ±3.7%, p=0,0001, n=3) vs. 0.1 Hz, we observed significantly faster and more robust inhibition of both channels.

To characterize the interaction between phenytoin and the most potently inhibited K⁺- channel, K2P 17.1, in-silico docking simulations were performed. Using the known crystal structure of K2P3.1 as a homology model, our studies suggest that phenytoin may bind to the inner entrance of the channel’s cavity. Alanine mutagenesis scanning revealed that the L154A, K261A, L262A, I263A, L264A mutants displayed a marked reduction in the current inhibition by 100 µM phenytoin, indicating a potential site of interaction. For comparison, we tested the clinically relevant, but topologically distant G88R mutant. The block by 100 µM phenytoin was unaffected, which is consistent with our proposed interaction site in the channel cavity.

Conclusion

The results suggest that phenytoin may exert its antiarrhythmic effects by inhibiting K⁺-channels in addition to Na⁺-channels. In particular, the relatively high affinity for the K2P3.1 and K2P17.1 channels suggest potential use of this drug in the treatment of AF in humans.