Limiting activity at beta1-subunit-containing GABAA receptor subtypes reduces ataxia

Abstract

GABA(A) receptor (R) positive allosteric modulators that selectively modulate GABA(A)Rs containing beta(2)- and/or beta(3)- over beta(1)-subunits have been reported across diverse chemotypes. Examples include loreclezole, mefenamic acid, tracazolate, and etifoxine. In general,"beta(2/3)-selective" GABA(A)R positive allosteric modulators are nonbenzodiazepines (nonBZs), do not show alpha-subunit isoform selectivity, yet have anxiolytic efficacy with reduced ataxic/sedative effects in animal models and humans. Here, we report on an enantiomeric pair of nonBZ GABA(A)R positive allosteric modulators that demonstrate differential beta-subunit isoform selectivity. We have tested this enantiomeric pair along with a series of other beta(2/3)-subunit selective, alpha-subunit isoform-selective, BZ and nonBZ GABA(A) positive allosteric modulators using electrophysiological, pharmacokinetic, and behavioral assays to test the hypothesis that ataxia may be correlated with the extent of modulation at beta(1)-subunit-containing GABA(A)Rs. Our findings provide an alternative strategy for designing anxioselective allosteric modulators of the GABA(A)R with BZ-like anxiolytic efficacy by reducing or eliminating activity at beta(1)-subunit-containing GABA(A)Rs.

Fig. 1.

Structural diversity of GABA_AR allosteric modulators in the present study.

Fig. 2.

Modulation of submaximal (EC₁₀) GABA-evoked currents by 2-261 and 2-262. Top, representative current tracings from two-electrode voltage-clamp recordings in X. laevis oocytes expressing human α₁β₂γ₂ (A) or α₁β₁γ₂ (B) GABA_ARs. Substitution of the β₂- with the β₃-subunit results in a similar response. Control currents for modulation were 10% of maximal current (EC₁₀₀). Drug exposures are indicated by the bars. Bottom, concentration-response relationship for modulation of EC₁₀ currents by 2-261 and 2-262. Data represents mean ± S.E.M. (n = 3–8).

Fig. 3.

α-Subunit selectivity of 2-261 and 2-262. Concentration-response curves for 2-261 and 2-262 across α₁-, α₂-, or α₃-subunit-containing GABA_AR subtypes. The curves depicted (left) show the effect of 2-261 on α₁β₁γ₂ versus α₁β₂γ₂ (A), α₂β₁γ₂ versus α₂β₂γ₂ (B), and α₃β₁γ₂ versus α₃β₂γ₂ (C) GABA_ARs, with the filled and open symbols denoting the presence of β₂- or β₁-subunits, respectively. 2-261 elicits high-efficacy modulation GABA currents at α₁β₂γ₂, α₂β₂γ₂, and α₃β₂γ₂ but not at α₁β₁γ₂, α₂β₁γ₂, or α₃β₁γ₂. 2-262 (right) elicits a similar pattern of responses at α₁β₁γ₂ versus α₁β₂γ₂ (D), α₂β₁γ₂ versus α₂β₂γ₂ (E), and α₃β₁γ₂ versus α₃β₂γ₂ (F), except that greater responses are observed at β₁-subunit-containing GABA_ARs when concentrations exceed 10⁻⁷ M.

Fig. 4.

Electrophysiological shift of GABA by 2-261 and 2-262. GABA concentration-response curves derived from α₁β₂γ₂ (A) versus α₁β₁γ₂ (B) GABA_ARs ± 0.7 μM 2-261. Likewise, the effect of GABA on α₁β₂γ₂ (C) versus α₁β₁γ₂ (D) GABA_ARs ± 3 μM 2-262 is depicted in the bottom panels. GABA responses are expressed as I/I_max, with each data point representing the mean ± S.E.M. (n = 3–4). The dashed line represents the GABA_max current with GABA only.

Fig. 5.

Electrophysiological evidence that 2-261 blocks modulation by 2-262. A, representative voltage-clamp recording illustrating surmountable blockade of 2-262-induced modulation by 2-261 in oocytes expressing human GABA_A α₁β₁γ₂ GABA_ARs. Drugs were applied as indicated by the bars. Dashed line indicates EC₁₀ control current magnitude used for calculating percentage of modulation. B, quantitative analysis of percentage of modulation (mean ± S.E.M.; n = 3).

Fig. 6.

Anxiolytic activity of 2-261 and 2-262 in mice. Dose-dependent effects of 2-261 (A) and 2-262 (B) versus alprazolam (0.1 mg/kg i.p.) on time (seconds) spent in the dark in the mouse LD paradigm after per os administration of each drug. The effects are compared with vehicle control. Each bar represents the mean ± S.E.M. time spent in the dark during a 5-min interval (n = 5–42 animals). Statistically significant differences from vehicle at ∗, P < 0.05 and ∗∗, P < 0.01 after ANOVA and post-hoc Dunnett's test.

Fig. 7.

Anxiolytic activity of 2-261 and 2-262 in rats. Dose-dependent effects of 2-261 (A) versus 2-262 (B) on time spent in the open arms in the rat EPM paradigm measured 30 min after intraperitoneal administration of each enantiomer. The effects are compared with vehicle control and diazepam (1.0 mg/kg i.p.). Each bar represents the mean ± S.E.M. of time spent in the open arms during a 5-min interval (n = 8–35 animals). Statistically significant differences from vehicle control at ∗, P < 0.05 and ∗∗, P < 0.01 after ANOVA and post-hoc Dunnett's test.

Fig. 8.

Effect on Rotarod performance by 2-261 and 2-262 in mice. Time course of RR performance in mice after per os administration of various doses of 2-261 (A) versus 2-262 (B). The AD₅₀ values for 2-261 and 2-262 at the time of peak effect were >120 and 43 mg/kg, respectively, as calculated by the method of Litchfield and Wilcoxon (1949) (n = 8–10 mice/time point).

Fig. 9.

Pharmacokinetics of 2-261 and 2-262 in mice. The pharmacokinetic profile of 2-261 (A) and 2-262 (B) in mice where plasma and brain levels (micromolar) are shown at various time (minutes) points after 10 mg/kg p.o. Each data point represents the mean ± S.E.M. levels (n = 4 animals/time point).

Fig. 10.

Activity at α₁β₁γ₂ GABA_ARs is predictive of ataxia. Rank order of apparent maximal percentage of modulation of EC₁₀ GABA-evoked currents by various test compounds in oocytes expressing the α₁β₁γ₂ (A) or α₁β₂γ₂ (B) GABA_AR subtypes. Maximal modulation was defined by that calculated from the concentration-response curves generated for each compound in the oocyte assays or that observed at ≥10 μM when limited by compound solubility. All compounds with efficacy below the threshold (dashed line) at the α₁β₁γ₂-subtype receptor pass the RR test, whereas those with efficacy above the threshold fail the RR test as defined by a calculable AD₅₀. Filled columns, passing RR test; open columns, failing RR test. ∗, L-838,417 causes RR failure, with AD₅₀ = 30 mg/kg i.p. but has no activity at α₁β₁γ₂ or α₁β₂γ₂ GABA_AR subtypes.

Fig. 11.

Anxiolytic activity of 2-313 and 2-148 in mice. Effects of 2-313 (A) and 2-148 (B) versus alprazolam (0.1 mg/kg) on time (seconds) spent dark in the mouse LD paradigm after intraperitoneal administration of each compound. The effects of the test compounds are compared with vehicle control. Each bar represents the mean ± S.E.M. time spent in the dark chamber during a 5-min interval (n = 6–30 animals). Significantly different from vehicle control at ∗, P < 0.05 after ANOVA and post-hoc Dunnett's test.

Fig. 12.

Lack of ethanol potentiation of RR failure by 2-261. The effect of 2-261(30 mg/kg p.o.) versus diazepam (1 mg/kg i.p.) on EtOH (1 g/kg i.p.)-induced RR deficit in mice. The effect is expressed as a percentage of mice passing the RR assay, with 12 mice tested under each condition. Significantly different from diazepam vehicle + EtOH at ∗, P < 0.01 by the Fischer-Yates exact probability test.