Characterization of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one as a positive allosteric modulator of GABAB receptors

Abstract

Background and purpose: As baclofen is active in patients with anxiety disorders, GABAB receptors have been implicated in the modulation of anxiety. To avoid the side effects of baclofen, allosteric enhancers of GABAB receptors have been studied to provide an alternative therapeutic avenue for modulation of GABAB receptors. The aim of this study was to characterize derivatives of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as enhancers of GABAB receptors.

Experimental approach: Enhancing properties of rac-BHFF were assessed in the Chinese hamster ovary (CHO)-Galpha16-hGABA(B1a,2a) cells by Fluorometric Imaging Plate Reader and GTPgamma[35S]-binding assays, and in rat hippocampal slices by population spike (PS) recordings. In vivo activities of rac-BHFF were assessed using the loss of righting reflex (LRR) and stress-induced hyperthermia (SIH) models.

Key results: In GTPgamma[35S]-binding assays, 0.3 microM rac-BHFF or its pure enantiomer (+)-BHFF shifted the GABA concentration-response curve to the left, an effect that resulted in a large increase in both GABA potency (by 15.3- and 87.3-fold) and efficacy (149% and 181%), respectively. In hippocampal slices, rac-BHFF enhanced baclofen-induced inhibition of PS of CA1 pyramidal cells. In an in vivo mechanism-based model in mice, rac-BHFF increased dose-dependently the LRR induced by baclofen with a minimum effective dose of 3 mg kg(-1) p.o. rac-BHFF (100 mg kg(-1) p.o.) tested alone had no effect on LRR nor on spontaneous locomotor activity, but exhibited anxiolytic-like activity in the SIH model in mice.

Conclusions and implications: rac-BHFF derivatives may serve as valuable pharmacological tools to elucidate the pathophysiological roles played by GABAB receptors in the central and peripheral nervous systems.

Figure 1

Chemical structures of GABA_B receptor allosteric enhancers, BHFF derivatives and CGP7930.

Figure 2

Effect of allosteric enhancers on GABA-evoked [Ca²⁺]_i. (a) Concentration–response curves for GABA in the absence and in the presence of 1, 3 and 10 μM rac-BHFF. (b) Concentration–response curves of rac-BHFF derivatives in the presence of 10 nM GABA (∼EC₁₀ value). GABA-evoked response in the Chinese hamster ovary (CHO) cell line (10-14) stably expressing hGABA_B(1a,2a) receptor and Gα16 was generated using the Ca²⁺-sensitive dye, Fluo-4AM and a Fluorometric Imaging Plate Reader (FLIPR-96). Responses were measured as peak increase in fluorescence minus basal, normalized to the maximal stimulatory effect induced by 10 μM GABA alone (considered 100%) and 10 nM GABA alone (considered 0%). Each curve represents mean±s.e.mean (bars) of the three concentration–response measurements (each performed in triplicate) from three independent experiments.

Figure 3

Effects of allosteric enhancers on GABA-induced GTPγ[³⁵S] binding in the membranes from CHO-Gα16-hGABA_B(1a,2a) cell line. Concentration–response curves for GABA in the absence and in the presence of 0.03, 0.3, 1, 3 and 10 μM rac-BHFF (a), in the absence and in the presence of 0.03, 0.1, 0.3, 1, 3, 10, 30 μM BHFI (c), in the absence and in the presence of 0.03 and 0.3 μM (+)-BHFF (e), (−)-BHFF (f), BHFHP (g) and CGP7930 (h). Schild-like analysis for the enhancing mode of rac-BHFF (b), and of BHFI (d) using the EC₅₀ values derived from plots in (a, c). Each curve represents mean±s.e.mean (bars) of the three concentration–response measurements (each performed in triplicate) from three independent experiments.

Figure 4

A comparison of concentration–response curves in the GTPγ[³⁵S]-binding assay for rac-BHFF (a) and BHFI (b) in the absence and in the presence of 0.2, 0.6, 1, 6 and 20 μM GABA, and for (+)-BHFF (c), (−)-BHFF (d), BHFHP (e) and CGP7930 (f) in the absence and in the presence of 1 μM GABA. Each curve represents mean±s.e.mean (bars) of the three concentration–response measurements (each performed in triplicate) from three independent experiments.

Figure 5

Effect of rac-BHFF on population spike (PS) inhibition by baclofen. (a) Representative PS obtained in the same hippocampal slice and evoked in the absence and presence of baclofen and rac-BHFF. (b) Concentration–response curves of baclofen for inhibition of PS generated in the absence and presence of rac-BHFF.

Figure 6

Potentiation of baclofen-induced loss of righting reflex (LRR). Male DBA mice received rac-BHFF at doses of 1, 3, 10, 30 and 100 mg kg⁻¹ p.o. (a) or CGP7930 at doses 3, 10, 30 and 100 mg kg⁻¹ p.o. (b) 45 min prior to baclofen (20 mg kg⁻¹, i.p.). LRR was measured nine times every 15 min. Data are expressed as median of number of periods in which LRR was observed (max=9)±interquartiles based on 24 animals per dose from three LRR tests. veh, vehicle (10 mL kg⁻¹ of a 4:1:15 mixture containing Cremophor EL, 1,2-propanediol and distilled water). ^*P<0.05, ^**P<0.01, ^***P<0.001 vs vehicle; Mann–Whitney U-test (one tailed).

Figure 7

Pharmacokinetic studies in the mouse with rac-BHFF. Plasma concentration–time curves of the hydrolysed rac-BHFF in male NMRI mice after i.v. or p.o. routes of rac-BHFF administration.