First Demonstration of a Functional Role for Central Nervous System Betaine/γ-Aminobutyric Acid Transporter (mGAT2) Based on Synergistic Anticonvulsant Action among Inhibitors of mGAT1 and mGAT2

Abstract

In a recent study, EF1502 [N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [d]isoxazol-3-ol], which is an N-substituted analog of the GAT1-selective GABA uptake inhibitor exo- 5,6,isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1-4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor, another GAT1-selective N-substituted analog of exo-THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 (K i of 4 and 5 M, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.Reduction of GABA-mediated inhibitory neurotransmission is associated with seizure activity and drugs that elevate synaptic GABA levels either by inhibition of GABA degradation or inhibition of high-affinity transport have been demonstrated to possess anticonvulsant activity (see Dalby, 2003;Sarup et al., 2003). For example, the GABA-transaminase inhibitor vigabatrin and the GABA-transport inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] are clinically effective antiepileptic drugs (for review and references, see Ben-Menachem, 2002;Kalviainen, 2002).Since the advent of cloning of several GABA transporters from different species including mouse, rat, and human, in-

Molecular Targets for Antiepileptic Drug Development

“…330 Studies with a novel GABA-transporter inhibitor, EF1502, that potently inhibits GAT-1 and GAT-2 (BGT-1)-but not GAT-3 and GAT-4 -indicate that inhibition of GAT-2 potentiates the anticonvulsant activity of GAT-1. 331,332 GAT-3 can be selectively inhibited with SNAP-5114, which enhances GABA-mediated synaptic inhibition in the neocortex, indicating that GAT-3 plays a role in this brain region. 333 Studies in resected tissue from patients with complex partial seizures and hippocampal sclerosis indicate that the expression of GAT-1 is decreased, but that of GAT-3 may be increased, possibly explaining prolonged IPSCs and increased excitability reported in electrophysiological studies with this tissue.…”

Astrocytic transporters in Alzheimer's disease

“…inhibitor, EF1502, has been used in preclinical studies and showed additional anticonvulsant effects when administered with tiagabine (177,178). While we await clinical development of astrocyte specific GATs inhibitors, examining astrocyte GABAergic transport may provide both insight into potential biomarker validation (astrocyte GABA content) and the efficacy of targeting astrocytic GABA transporters in combatting AD pathology.…”

Selective GABA Transporter Inhibitors Tiagabine and EF1502 Exhibit Mechanistic Differences in Their Ability to Modulate the Ataxia and Anticonvulsant Action of the Extrasynaptic GABAAReceptor Agonist Gaboxadol

“…Proof of concept that GAT inhibitors mediate anticonvulsant activity in rodents has been firmly established (Nielsen et al, 1991;White et al, 1993White et al, , 2002White et al, , 2005Madsen et al, 2009Madsen et al, , 2010. Furthermore, it has been demonstrated that an inhibitor of BGT1 having no affinity for GABA i.e., 4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) when administered in combination with inhibitors of GAT1 (i.e., tiagabine) and N- [4,5,6,, results in a synergistic anticonvulsant effect in audiogenic seizure (AGS)-susceptible Frings mice (White et al, 2005).…”

“…Furthermore, it has been demonstrated that an inhibitor of BGT1 having no affinity for GABA i.e., 4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) when administered in combination with inhibitors of GAT1 (i.e., tiagabine) and N- [4,5,6,, results in a synergistic anticonvulsant effect in audiogenic seizure (AGS)-susceptible Frings mice (White et al, 2005). Because EF1502 also inhibits GAT1 , additional experiments examining the combinatorial effect of the two GAT1 selective inhibitors tiagabine and LU-32-176 were performed.…”