Rationale:
OV329 is a highly potent, clinical stage GABA-amino transferase (GABA-AT) inhibitor, that holds the potential to be the best-in-class antiepileptic drug with a mechanism of action similar to that of vigabatrin. GABA, the primary inhibitory neurotransmitter in the adult CNS, is catabolized by GABA-AT. Inactivation of GABA-AT is a viable option to enhance GABA levels, which may help reduce neuronal hyperexcitability associated with seizures and epilepsies. Therefore, it is critical to broaden our understanding of the functional implications of GABA-AT inhibition and the significance of elevated GABA, at a cellular level. We aim to understand how GABA-AT inhibition by OV329 modulates phasic and tonic GABAergic transmission in hippocampal dentate gyrus, a brain region intimately associated with temporal lobe epilepsy. Mechanistic insights gained here will potentially inform the appropriate indication and how to use OV329 in the clinic.
Methods:
We examined the effect of OV329 on both forms of ionotropic GABAergic inhibition using whole-cell patch clamp electrophysiology in the Dentate Gyrus Granule Cell (DGGC) layer of six to eight week-old male C57Bl6/J mice using acute slices and a repeated dose
in vivo paradigm. The acute effects of OV329 were tested on hippocampal brain slices after a one hour incubation with either PBS (vehicle), 0.25mM or 10mM OV329. The cumulative effects of an
in vivo repeated dosing paradigm were tested following six days of IP doses of either vehicle or 5 mg/kg OV329 injections. Electrophysiological recordings were carried out in DGCCs and spontaneous inhibitory postsynaptic currents (sIPSCs) were captured prior to the application of picrotoxin to block GABA
A receptors and, measure the resulting shift in the holding current caused by the block of extrasynaptic GABA
A receptors. GABA-AT activity was measured from tissue homogenates using a biochemical assay.
Results:
Acute inhibition of GABA-AT resulted in a significant increase in the frequency of sIPSCs at both doses, while higher concentration (10mM) of OV329 significantly increased the amplitude of sIPSCs. Tonic current was significantly larger after 10mM OV329 treatment. GABA-AT activity was significantly reduced (~50%) with 10 mM treatment compared to vehicle, while the extent of GABA-AT inhibition was less (~15%) with 0.25mM OV329.
In vivo, six days of daily 5 mg/kg OV329 treatment, did not alter the amplitudes of sIPSCs while the frequencies of sIPSCs and tonic current were significantly increased.Conclusions:
Results from this study suggest inhibition of GABA-AT is a powerful tool by which both phasic and tonic inhibition can be modulated. Further, these data suggest that the effects on phasic and tonic inhibition are dependent on the extent of GABA-AT inhibition. Finally, repeated administration of OV329 led to robust DGGC tonic currents
in vivo, likely due to increased extracellular GABA levels.
Funding:
Ovid Therapeutics and NIH/NINDS (NS108378, NS111064, NS111338)