Clobazam and Its Active Metabolite N-desmethylclobazam Display Significantly Greater Affinities for α2- versus α1-GABAA–Receptor Complexes

Henrik Sindal Jensen,Kathryn Nichol,Deborah Lee,Bjarke Ebert,Uwe Rudolph

doi:10.1371/journal.pone.0088456

Henrik Sindal Jensen, Kathryn Nichol + Show 3 more

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https://doi.org/10.1371/journal.pone.0088456

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Abstract

Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α1-subunit–selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α2 subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α1, α2, α3, or α5), β2, and γ2 subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α2- vs. α1-receptor complexes, a difference not observed for CLN, for which no distinction between α2 and α1 receptors was observed. Our experiments with ZOL confirmed the high preference for α1 receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB.

Highlights

GABAA receptors are the principal inhibitory neurotransmitterreceptor system in the mammalian brain
Clobazam (OnfiH; CLB), a structurally unique 1,5-BZD, was approved by the US Food and Drug Administration (FDA) in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older, based on results from a Phase III randomized controlled study [13]
Flumazenil as the tracer; Kd values were 0.43–1.24 nM and Bmax values were 188–300 fmol/mg across the 4 GABAA-receptor complexes (Figure 1B and Table 2). These Kd values are in agreement with previously published data for combinations of a1, a2, a3, and a5 subunits with b3 and c2 expressed in mouse fibroblast L(tk2) cells [22]

Summary

Introduction

GABAA receptors are the principal inhibitory neurotransmitterreceptor system in the mammalian brain. GABAA receptors are hetero-oligopentameric complexes that are members of the pentameric, ligand-gated family ( known as cys-loop family). A subset of GABAA-receptor modulators bind to the allosteric site situated at the a- and c-subunit interface, termed the benzodiazepine (BZD) site. The variety of possible GABAA-receptor subunit combinations suggests that subunit-specific compounds may allow for clinical tuning of desired therapeutic effects [2], [3], [4], [5]. Such subtype selectivity toward the GABAA a1 subunit is linked to the clinical effectiveness of zolpidem as a sedative [6], [7]. Preclinical studies with knock-in mice carrying single amino acid point–

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