Preclinical characterization of zuranolone (SAGE-217), a selective neuroactive steroid GABAA receptor positive allosteric modulator

Abstract

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABA_A receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABA_A receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α₁β₂γ₂, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABA_A receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABA_A receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram β-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABA_A receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABA_A receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.

Keywords: GABA(A) receptor; Neuroactive steroid; Positive allosteric modulator.

Fig. 1.. Zuranolone potentiated human GABA_A receptor subtypes representative of both synaptic and extrasynaptic receptors.

A. Representative traces from synaptic (α₁β₂γ₂) and extrasynaptic (α₄β₃δ) GABA_A receptors expressed in Ltk or CHO cells, respectively, showing potentiation of GABA-evoked currents. Solid lines indicate administration of a submaximal GABA concentration (2 μM), dashed lines indicate administration of zuranolone, nM. Scale bars = 500 pA, 4 sec. B. Dose-response curves for both receptor types, vertical lines indicate SEM, dotted curves indicate 95% confidence interval.

Fig. 2.. Zuranolone increased potency and E_max of the orthosteric partial agonist P4S at the α₁β₂γ₂ GABA_A receptor.

The E_max of P4S, a GABA_A receptor partial agonist, was 25% of the maximal GABA (100 μM) response, which was doubled by the addition of 200 nM zuranolone to the bath solution. The EC₅₀ for P4S was reduced by nearly half (44.5 μM–25.2 μM) by addition of 200 nM zuranolone. GABA_A receptors were expressed in Ltk cells.

Fig. 3.. Zuranolone exhibits synergistic activity with the benzodiazepine diazepam.

A. Concentration-response curves of zuranolone, DZP, and a combination at α1β2γ2-expressing Ltk cells. Percent potentiation is plotted relative to the amplitude of GABA (2 μM) alone. The horizontal dotted line corresponds to the effect level plotted in the isobologram. B. Isobologram of the 150% effect level. The concentration of DZP applied alone required to reach this effect was 360 nM, the concentration of zuranolone applied alone was 315 nM. When coapplied with 100 nM DZP, only 10 nM of zuranolone was required potentiate the current greater than 150%. C. Concentration-response curves of zuranolone, DZP, and a combination at α₄β₃δ-expressing cells. Percent potentiation is plotted relative to the maximum GABA response.

Fig. 4.. GABA_A receptor currents were increased by acute treatment with zuranolone when measured after a 30–50 min washout period.

A_i. Sample recordings from dentate granule cells in mouse brain slices show larger tonic current after pre-treatment with 1 μM zuranolone compared to control treated cells. A_ii. Tonic current density was approximately doubled following pre-treatment with 1 μM zuranolone. B_i. Example of averaged sIPSC recordings normalized to amplitude in vehicle control (black) or after a 15 min exposure of 1 μM zuranolone followed by 30–50 min washout (gray) demonstrating a prolongation of sIPSC decay. B_ii. sIPSC amplitude was not significantly changed in cells after zuranolone pre-treatment. B_iii. sIPSC decay time was significantly prolonged after pre-treatment with 1 μM zuranolone.

Fig. 5.. Zuranolone exhibited oral bioavailability consistent with once or twice daily dosing with exposures that cover behaviorally-active concentrations in mice.

A. Mean plasma concentrations and pharmacokinetic parameters following a single IP or PO administration of 10 mg/kg zuranolone. Brain to plasma ratios were obtained by single point at 1 h post dosing. B. PO administration 60 min prior to systemic PTZ injection protected mice from exhibiting seizures with a minimum effective dose of 1 mg/kg, corresponding to a plasma concentration of 74 ng/mL and brain concentration of 143 ng/g.

Fig. 6.. Zuranolone elicits changes in β-band EEG power in rat.

A. Averaged β-band EEG power plotted across time (post-administration) in response to vehicle, and 0.3, 3, and 20 mg/kg zuranolone in rat. Arrows indicate peak for the 3 and 20 mg/kg dose groups. Dotted line indicates 1 h time point. B. Box- and-whisker plot summarizing β-band EEG power at 1 h across distinct zuranolone concentrations. C. Cumulative time (min) spent in sleep during each hour. D. Plasma zuranolone concentrations were 22, 301, and 1466 ng/mL at 60 min post dosing for 0.3, 3.0, and 20 mg/kg zuranolone, respectively from rats designated for pharmacokinetic analysis.