Elsevier

Biochemical Pharmacology

Volume 93, Issue 3, 1 February 2015, Pages 370-379
Biochemical Pharmacology

GABAA α5 subunit-containing receptors do not contribute to reversal of inflammatory-induced spinal sensitization as indicated by the unique selectivity profile of the GABAA receptor allosteric modulator NS16085

https://doi.org/10.1016/j.bcp.2014.12.010Get rights and content

Abstract

GABAA receptor positive allosteric modulators (PAMs) mediate robust analgesia in animal models of pathological pain. Restoration of diminished spinal GABAA-α2 and -α3 subunit-containing receptor function is a principal contributor to this analgesia, albeit involvement of GABAA-α5-receptors has not been excluded. Thus, we compared NS11394 and TPA023 (PAMs with selectivity/efficacy at GABAA-α2/α3/α5 receptors) with TP003 (a reportedly GABAA-α3 selective PAM) against spinal sensitization. However, in-house electrophysiology studies designed to confirm the selectivity of TPA023 and TP003 for human GABAA receptors did not corroborate published data, with TP003 displaying considerable GABAA-α5 receptor efficacy. Therefore, we identified a novel PAM, NS16085, which possesses negligible efficacy at GABAA-α5 receptors, but with GABAA-α2/α3 efficacy equivalent to NS11394. At the GABAA-α1 receptor the compound gives low level of negative modulation further separating it from the other compounds. Rat pups with carrageenan-induced hindpaw inflammatory hyperalgesia were used to make ex vivo spinal dorsal root-evoked ventral root recordings. Some spontaneous activity and large numbers of spikes to repetitive stimulation of dorsal roots at C-fibre intensity, indicative of wind-up and sensitization were observed. Equimolar concentrations of NS11394, TP003 and NS16085 all attenuated wind-up to a similar degree; TPA023 was clearly less effective. In adult rats, NS16085 (3–30 mg/kg, p.o.) dose-dependently reduced formalin-induced hindpaw flinching with efficacy comparable to NS11394. Thus, potentiation of GABAA-α2 and-α3 receptors is sufficient to depress spinal sensitization and mediate analgesia after inflammatory injury. Positive modulation at GABAA-α5-receptors is apparently dispensable for this process, an important consideration given the role of this receptor subtype in cognitive function.

Introduction

GABAA receptors are distributed throughout the CNS and contribute to a spectrum of disease pathologies. These ligand-gated chloride channels typically consist of two α, two β and one γ subunit, and have specific binding sites through which a range of compounds interact, including the endogenous neurotransmitter GABA, muscimol, barbiturates, neurosteroids, anaesthetics and benzodiazepines such as diazepam [1]. The benzodiazepines and other related compounds function as positive allosteric modulators (PAMs) to enhance GABA-gated currents at receptors containing an α1, α2, α3, or α5 subunit.

Within the spinal dorsal horn GABAA receptors are localized both pre- and post-synaptically [2], [3], enabling GABA to profoundly influence synaptic transmission. Studies using transgenic mice with selected individual point mutations in the four GABAA α-subunits have indicated a robust contribution from GABAA receptors containing α2 and α3 subunits to intrathecal diazepam-mediated analgesia in pathological pain [4], [5]. This is complimented by pharmacological studies performed in rodent pain models with systemically administered subtype-selective GABAA receptor PAMs such as L838,417, TPA023, HZ166 and NS11394 [4], [6], [7], [8], [9], [10]. Whilst both of the above strategies have generally ruled out a contribution of the α1 subunit to PAM-mediated analgesia in these models, the contribution of α5 subunit-containing GABAA receptors is less clear, a finding which could have repercussions for developing novel GABAA receptor based analgesics [11], as this subtype has been implicated in mediating the memory impairing effects of benzodiazepines [12].

To obtain a clearer picture of the contribution played by α5 subunit-containing GABAA receptors to PAM-mediated analgesia, we decided to compare the efficacy of various subtype-selective PAMs on the electrical excitability of spinal cord pain circuits using tissue sourced from rats with carrageenan-induced inflammatory hyperalgesia [13]. When establishing the contribution of individual GABAA subunits to pain signalling, an ex vivo preparation as typified by the hemisected cord preparation used here can have several advantages. Firstly, the uncertainty in ensuring the desired target concentration of a compound is reached in living animals is circumvented. Moreover, the issue of whether a particular compound has active metabolites with different selectivity profiles from the parent is also mitigated. Finally, the associated plasticity changes occurring within spinal pain networks induced in this preparation, termed wind-up, reflect aspects of central sensitization, a process which in itself is intimately linked to a loss of GABAA receptor-mediated neurotransmission after injury [14], [15].

For the current studies, we compared the α2/α3/α5 subtype-selective PAM NS11394 [16], with the α2/α3 subtype-selective PAM TPA023 [17], [18], and the α3 subtype-selective PAM TP003 [19] on spinal wind-up. Aware that it can be difficult to assign absolute values to efficacy selectivity data obtained across labs due to the use of e.g. different cell lines, receptor combinations and/or reference compounds amongst a number of other variables [20], we first characterized the selectivity profiles of these compounds in house, and in doing so realized that they would be sub-optimal for the task at hand. Accordingly, we identified another GABAA-α2/α3 selective compound (NS16085) with a highly unique selectivity profile exhibiting negligible activity at GABAA-α5. Our data show that GABAA receptor PAM-mediated reversal of spinal wind up in rats, requires a certain threshold level of receptor modulation at GABAA α2/α3-containing receptors. Efficacy at GABAA α5-containing receptors does not appear to be necessary for mediating analgesia after inflammatory injury, a key finding which has important implications for developing GABAA receptor based analgesics.

Section snippets

Synthesis and structure of NS16085

NS16085 (4-chloro-3-{6-[5-(2-hydroxypropan-2-yl)-1H-1,3-benzodiazol-1-yl]pyridin-2-yl}benzonitrile) was developed as part of an ongoing internal drug discovery effort within the GABAA receptor PAM field. The structure of NS16085 is shown in Fig. 1 together with TPA023 (5-({[7-tert-butyl-3-(2-fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl]oxy}methyl)-1-ethyl-1H-1,2,4-triazole), TP003 (2-{3-[8-fluoro-7-(2-hydroxypropan-2-yl)imidazo[1,2-a]pyridin-3-yl]phenyl}benzonitrile) and NS11394

In vitro efficacy of GABAA PAMs in oocytes selectively expressing human GABAA receptors

NS16085 was identified as a positive modulator of GABAA receptors as part of an ongoing internal drug discovery effort within this field (Fig. 1). The modulation of GABA-evoked currents at human α1-, α2-, α3- or α5-containing receptors expressed in combination with β2 and γ2s subunits in X. laevis oocytes by all four PAMs shown in Fig. 1 can be seen in Fig. 2, with modulatory effects of diazepam shown within the inset of each panel. For these experiments, a GABAcontrol concentration giving rise

Discussion

In the current series of experiments we set out to examine the relative contribution of α2, α3 and α5 subunit-containing receptors to GABAA PAM mediated depression of spinal pain circuits sensitized by the inflammatory algogen carrageenan. We had planned to use the α3 subtype-selective compound TP003 to dissect out a contribution of α3-containing GABAA receptors to mechanisms contributing to this process. However, in our hands TP003, and to a lesser extent TPA023 displayed only marginal

Funding sources

Spinal cord electrophysiological experiments were run with the support of the Spanish Ministry of Economy and Competitiveness (BFU2012-37905).

Acknowledgements

The technical assistance of Helene Dyhr is gratefully appreciated.

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