Abstracts

Rationale: Benzodiazepines are GABA_A receptor positive allosteric modulators (GABA PAMs) used as first-line treatment for multiple seizure disorders. As seizures progress, synaptic (or benzodiazepine-sensitive) GABA_A receptors are actively internalized and become functionally inactive. Conversely, extra-synaptic GABA_A receptors are refractory to this internalization, thereby providing an attractive therapeutic target for treatment of acute and chronic benzodiazepine-resistant seizures. Allopregnanolone, a potent GABA PAM neuroactive steroid at both synaptic and extra-synaptic GABA_A receptors, has pre-clinical anti-convulsant efficacy, but limited oral bioavailability. In order to provide a GABA PAM neuroactive steroid with drug properties suitable for acute and chronic epilepsy, we have generated novel neuroactive steroid derivatives of allopregnanolone with equi-potency at synaptic and extra-synaptic receptors, as well as molecules selective for extra-synaptic receptors, with improved pharmacokinetic properties suitable for oral administration. Methods: In vitro electrophysiological recordings were made using whole-cell patch recordings from Ltk-1 and CHO cells expressing either 1 2 2 or 4 3 GABA_A receptors, respectively. Protection against PTZ-induced (120 mg/kg, s.c.) seizures was assessed in CD-1 mice 30 min after acute systemic administration of compounds (1, 3, and 10 mg/kg IP, or 1, 10, 30 mg/kg PO). Valproic acid (400 mg/kg, IP) served as a positive control in the model and provided robust seizure protection. Latency to seizure, seizure frequency, and lethality was monitored for 30 min following PTZ administration. Results: Allopregnanolone produced similar potency in the concentration dependent enhancement of GABA currents at 1 2 2 (EC50 = 180 nM; Emax=475%) and 4 3 (EC50 =80 nM; Emax=418%) receptors. SGE-001, a representative synthetic GABA PAM neuroactive steroid (NAS) also showed equi-potent activity at 1 2 2 (EC50 = 120 nM; Emax=652%) and 4 3 (EC50 = 190 nM; Emax=474%) GABAA receptors. SGE-002 is a representative synthetic GABA PAM NAS with a high 4 (EC50 = 250 nM; Emax=396%) selectivity relative to 1 (EC50 > 3000 nM; Emax=839%). Both SGE-001 and SGE-002 have improved pharmacokinetic properties suitable for oral administration (PO F%=17) and (PO F%=27), respectively, compared to allopregnanolone (PO F%=0.5). Moreover, we show that SGE-001 and SGE-002 are potent anti-seizure agents in the PTZ model with minimum effective doses of 3 and 10 mg/kg (IP), respectively.Conclusions: We have generated synthetic GABA PAM neuroactive steroids that are orally bioavailable and can be suitable for the treatment of acute and chronic epilepsy. Moreover, we have generated a series of GABA PAM neuroactive steroids with preference to extra-synaptic ( -containing) over synaptic ( 2-containing) receptors. These novel subtype-preferring compounds will allow us to test directly the anti-seizure efficacy of GABAA potentiating extra-synaptic, in isolation of synaptic receptors in the near future.