Rationale:
GABA, the primary inhibitory neurotransmitter in the adult CNS, is catabolized by GABA-amino transferase (GABA-AT). Therefore, inactivation of GABA-AT can elevate GABA level within CNS and potentially reduce neuronal hyperexcitability associated with seizures and epilepsies. OV329 is a clinical stage compound that is a highly potent, GABA-AT inhibitor, a mechanism similar to that of vigabatrin (VGB), that holds the potential to be the best-in-class antiepileptic drug with this mechanism. It has been previously shown that a low (3 mg/kg), repeat dose of OV329 (q.d, 8 days) can lead to significant reductions of focal seizures in a mouse model of mesial-temporal lobe epilepsy. The t
max of OV329 is ~30 minutes following i.p or p.o dosing with an approximate half-life of 1.0 - 1.5 hours. Given this short exposure window, it is critical to understand how OV329 inhibits enzymatic activity of GABA-AT relative to its PK profile. The mechanistic insights gained here will potentially inform how to optimally dose OV329 in the clinic.
Methods:
A reliable biochemical assay to measure GABA-AT activity using tissue homogenates was established. For PD studies, mice (8-12 wks) were dosed with OV329, either p.o or i.p, and tissues were collected at various time points. For PK, plasma and tissue samples were analyzed by LC-MS/MS to detect OV329. To characterize the expression and cellular distribution, western blot and immunostaining assays were performed using an anti-GABA-AT antibody.
Results:
Compared to VGB (IC
50 ~126 µM), OV329 (IC
50 ~114 nM) was found to be a highly potent GABA-AT inhibitor as measured by GABA-AT enzyme activity using mouse brain lysates. A single, acute dose of OV329 (30 mg/kg) led to a significant reduction in GABA-AT activity in the brain (~30%) and the liver (~60%) within 4 hours of dosing and the reduction of GABA-AT activity persisted for almost a week. In comparison, GABA-AT activity regained to baseline within 3-days, after single dose (300 mg/kg) of vigabatrin. Repeated doses of OV329 (10 mg/kg/day x 3 days) resulted in an equivalent or greater GABA-AT inhibition in brain and elevated GABA levels compared to a single 30 mg/kg dose. Additionally, in a dose-response study, OV329 did not inhibit alanine or aspartate aminotransferases, major liver enzymes, within efficacious dose range, suggesting minimal, potential off target effect on other aminotransferases in contrast to VGB.
Conclusions:
This data demonstrates that OV329 inhibits the activity of GABA-AT in brain, and a significant level of GABA-AT inhibition with a concomitant change in GABA level can be achieved by repeat low, dose administration of OV329. Future studies are warranted to gain further insights on the inactivation mechanism of GABA-AT by OV329. Finally, low and multiple doses of OV329 may be a viable strategy for treating patients, which also may help avoid potential adverse effects without compromising efficacy.
Funding:
Ovid Therapeutics