Analgesia and unwanted benzodiazepine effects in point-mutated mice expressing only one benzodiazepine-sensitive GABAA receptor subtype

William T Ralvenius,Mario A Acuña,Hanns Ulrich Zeilhofer,Uwe Rudolph,Dietmar Benke

doi:10.1038/ncomms7803

Abstract

Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (α1, α2, α3 and α5) of GABAA receptors (GABAAR). When applied to the spinal cord, they alleviate pathological pain; however, insufficient efficacy after systemic administration and undesired effects preclude their use in routine pain therapy. Previous work suggested that subtype-selective drugs might allow separating desired antihyperalgesia from unwanted effects, but the lack of selective agents has hitherto prevented systematic analyses. Here we use four lines of triple GABAAR point-mutated mice, which express only one benzodiazepine-sensitive GABAAR subtype at a time, to show that targeting only α2GABAARs achieves strong antihyperalgesia and reduced side effects (that is, no sedation, motor impairment and tolerance development). Additional pharmacokinetic and pharmacodynamic analyses in these mice explain why clinically relevant antihyperalgesia cannot be achieved with nonselective BDZs. These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain.

Highlights

Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (a1, a2, a3 and a5) of GABAA receptors (GABAAR)
With the use of triple GABAAR point-mutated mice, we were able to perform a systematic analysis on the GABAAR subtypes best targeted for optimal antihyperalgesia and least-pronounced side effects; we provide an explanation why classical BDZs are largely devoid of clinically relevant analgesic properties
The [3H]flumazenil data are consistent with a high prevalence of GABAARs containing two different a subunits[14,15] and with a model of the GABAAR assembly in which nonpointmutated a subunits have a higher probability for interaction with the g2 subunit than histidine to arginine (H-R) point-mutated subunits[16]

Summary

Introduction

Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (a1, a2, a3 and a5) of GABAA receptors (GABAAR). We have previously demonstrated that local spinal application of BDZ site ligands that positively modulate GABAAR function alleviates neuropathic and inflammatory pain in rodents[5] Translation of these results to routine systemic pain treatment, requires a separation of desired antihyperalgesia from unwanted side effects. Selective tool compounds that would allow addressing these questions pharmacologically are still lacking[13] For this reason, we have generated triple GABAAR point-mutated mice, in which only a single GABAAR subtype remains BDZ-sensitive. We designate these mice hereafter as HRRR, short for a1H;a2R;a3R;a5R (that is, a mouse in which only a1GABAARs are left BDZ-sensitive) and, as RHRR, RRHR, RRRH for mouse lines, in which either only a2, a3 or a5 subunits remain BDZ-sensitive In these mice, the action of the classical normally nonselective BDZ agonist DZP should mimic the action of fully subtype-selective compounds. With the use of triple GABAAR point-mutated mice, we were able to perform a systematic analysis on the GABAAR subtypes best targeted for optimal antihyperalgesia and least-pronounced side effects; we provide an explanation why classical (nonselective) BDZs are largely devoid of clinically relevant analgesic properties

Methods

Results

Conclusion