Synergy between retigabine and GABA in modulating the convulsant site of the GABAA receptor complex

doi:10.1016/S0014-2999(03)01426-2

European Journal of Pharmacology

Volume 464, Issues 2–3, 19 March 2003, Pages 95-100

https://doi.org/10.1016/S0014-2999(03)01426-2 Get rights and content

Abstract

The molecular mechanism underlying the activity of the novel antiepileptic drug retigabine is not yet fully understood. The aim of this study was to investigate whether retigabine interacts directly with the GABA_A receptor complex (γ-aminobutyric acid). Receptor-binding assays were conducted using rat brain membranes. [³H]-t-Butyl-bicyclo-orthobenzoate ([³H]TBOB) was used as a tracer ligand. We determined the effects of GABA and retigabine in the presence of several concentrations of GABA on the binding of [³H]TBOB. GABA inhibited [³H]TBOB binding with an EC₅₀ of 4.8 μM. In the absence of GABA, retigabine inhibited [³H]TBOB with an EC₅₀ of 124 μM and an EC₅₀ of 42 μM in the presence of 2.5 μM GABA. Isobolic analysis revealed that retigabine acts in synergy with GABA in displacing [³H]TBOB. This synergy could be quantified by a molecular model in which GABA and retigabine both allosterically displace [³H]TBOB, and retigabine allosterically enhances the binding of GABA and vice versa with a factor of 4. In summary, we found that retigabine does indeed interact with a site on the GABA_A receptor complex, and this site is positively allosterically coupled with the GABA site. This GABA-positive effect may well contribute to the clinical anticonvulsive effects of retigabine.

Introduction

Retigabine, (D-23129) [N-(2-amino-4-(4-flurobenzylamino)phenyl)carbamid acid ethyl ester], is a new antiepileptic drug. It is effective in a variety of animal models for convulsions (Rostock et al., 1996). At present the drug is in clinical development (Ferron et al., 2002).

The molecular mechanism underlying the activity of retigabine is not yet fully understood. In vitro experiments showed that retigabine activates K⁺ channels Main et al., 2000, Rundfeldt, 1997, Schroder et al., 2001, Tatulian et al., 2001, Wickenden et al., 2000, and thus suppresses neuronal firing (Rogawski, 2000). In addition, it was suggested that an activation of GABA-ergic (γ-aminobutyric acid-ergic) inhibition adds to the clinical anticonvulsive effects of retigabine: an enhancement of GABA-induced inhibitory postsynaptic Cl⁻ currents was reported Otto et al., 2002, Rundfeldt and Netzer, 2000. This GABA-ergic mechanism includes an increase in GABA concentration by an increase of the synthesis of GABA (Kapetanovic et al., 1995) and by an inhibition of its metabolism (Sills et al., 2000). In addition, the GABA-ergic effect is suggested to result from a direct interaction of retigabine with a site on the GABA_A receptor complex (Otto at al., 2002). GABA_A receptors are ligand-gated chloride ion channels. The GABA_A receptor complex comprises binding sites for a variety of compounds, such as benzodiazepines, barbiturates, neuroactive steroids and general anaesthetics; furthermore, a picrotoxin-sensitive convulsant site is present on the complex (review: Mehta and Ticku, 1999). A number of these binding sites are allosterically coupled, resulting in a network of interactions, ultimately regulating the permeability of the Cl⁻ channel Mehta and Ticku, 1999, Korpi et al., 2002. The functional state of the channel can be assayed by measuring the amount of ligand binding to the picrotoxin-sensitive convulsant site Havoundjian et al., 1986, Maksay, 1996, Im and Blakeman, 1991, Korpi et al., 2002. We can therefore measure the effects of allosteric interactions that modulate GABA-ergic neurotransmission using receptor-binding assays on the picrotoxin-sensitive convulsant site. [³H]-t-Butyl-bicyclo-orthobenzoate ([³H]TBOB) can be used as a tracer ligand for the picrotoxin-sensitive convulsant site Lawrence et al., 1985, Van Rijn et al., 1990.

The aim of the present study was to investigate whether retigabine interacts directly with the GABA_A receptor complex and, if so, whether it interacts with the binding of GABA. In vitro receptor binding assays were conducted using well-washed rat brain membranes. We determined the effects of GABA alone and of retigabine in the presence of several concentrations of GABA on the binding of [³H]TBOB. The experimental data were described in two different ways. First, the sigmoid-E_max model was fitted to the data in order to describe the results in terms of additivity using the isobole method Berenbaum, 1989, Greco et al., 1995, Loewe, 1953, Tallarida, 1992, which allows qualitative conclusions (Greco et al., 1995). Next, the allosteric three-ligand molecular model, the cubic ternary complex (Kenakin, 1997), was fitted to the data, which allowed us to describe the observed interaction in quantitative terms as well (Van Rijn et al., 1999).

Section snippets

Preparation of the tissue

This study was performed in accordance with the guidelines of the European Community for the use of experimental animals. Approval of the local ethical committee for animal studies was obtained. Forebrains of Wistar rats [body weight 350±50 g (mean±S.D.)] were used. The brains were homogenised in 9 volumes of 0.32 M sucrose at 0 °C with a Teflon-glass homogeniser. The homogenate was centrifuged at 1000×g for 10 min at 4 °C. The supernatant was decanted and centrifuged at 48,000×g for 30 min at

Fitting the sigmoid-E_max Eq. (1) to the data

Parameter estimates are given in Table 1, data points and best fits are given in Fig. 2. GABA displaced [³H]TBOB with an EC₅₀ in the low micromolar range. Retigabine displaced [³H]TBOB both in the absence and in the presence of GABA. The effective concentrations were in the high micromolar range. The ED₅₀ of retigabine decreased with increasing concentration of GABA. The Hill coefficients were not significantly different from unity.

Isobolic analysis

An isobologram was constructed for 35% binding of [³H]TBOB

Discussion

In this study, we investigated whether retigabine interacts directly with the GABA_A receptor complex and, if so, whether it interferes with the binding of GABA. The effects of retigabine and GABA were measured indirectly using [³H]TBOB as a tracer ligand. Retigabine indeed displaced [³H]TBOB, and GABA shifted the displacement curves of retigabine to the left. Isobolic analysis of these results showed the interaction between retigabine and GABA to be synergistic. The isobole method, however,

Acknowledgements

We thank Dr. B. Pechstein, Viatris, Frankfurt am Main, Germany, for supplying retigabine and Dr. J.P. Zwart for the constructive discussions.

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Synergy between retigabine and GABA in modulating the convulsant site of the GABAA receptor complex

Abstract

Introduction

Section snippets

Preparation of the tissue

Fitting the sigmoid-Emax Eq. (1) to the data

Isobolic analysis

Discussion

Acknowledgements

Epilepsy Res.

Prog. Neurobiol.

Neurochem. Int.

Brain Res. Rev.

Trends Neurosci.

Epilepsy Res.

Eur. J. Pharmacol.

Neuropharmacology

Pharmacol. Res.

Pain

Eur. J. Pharmacol.

Eur. J. Pharmacol.

What is synergy?

Pharmacol. Rev.

Synergy between retigabine and GABA in modulating the convulsant site of the GABA_A receptor complex

Fitting the sigmoid-E_max Eq. (1) to the data