Phosphorylation state-dependent modulation of spinal glycine receptors alleviates inflammatory pain.

Diminished inhibitory neurotransmission in the superficial dorsal horn of the spinal cord is thought to contribute to chronic pain. In inflammatory pain, reductions in synaptic inhibition occur partially through prostaglandin E2- (PGE2-) and PKA-dependent phosphorylation of a specific subtype of glycine receptors (GlyRs) that contain α3 subunits. Here, we demonstrated that 2,6-di-tert-butylphenol (2,6-DTBP), a nonanesthetic propofol derivative, reverses inflammation-mediated disinhibition through a specific interaction with heteromeric αßGlyRs containing phosphorylated α3 subunits. We expressed mutant GlyRs in HEK293T cells, and electrophysiological analyses of these receptors showed that 2,6-DTBP interacted with a conserved phenylalanine residue in the membrane-associated stretch between transmembrane regions 3 and 4 of the GlyR α3 subunit. In native murine spinal cord tissue, 2,6-DTBP modulated synaptic, presumably αß heteromeric, GlyRs only after priming with PGE2. This observation is consistent with results obtained from molecular modeling of the α-ß subunit interface and suggests that in α3ßGlyRs, the binding site is accessible to 2,6-DTBP only after PKA-dependent phosphorylation. In murine models of inflammatory pain, 2,6-DTBP reduced inflammatory hyperalgesia in an α3GlyR-dependent manner. Together, our data thus establish that selective potentiation of GlyR function is a promising strategy against chronic inflammatory pain and that, to our knowledge, 2,6-DTBP has a unique pharmacological profile that favors an interaction with GlyRs that have been primed by peripheral inflammation.

Antihyperalgesia by α2-GABAA receptors occurs via a genuine spinal action and does not involve supraspinal sites.

Drugs that enhance GABAergic inhibition alleviate inflammatory and neuropathic pain after spinal application. This antihyperalgesia occurs mainly through GABAA receptors (GABAARs) containing α2 subunits (α2-GABAARs). Previous work indicates that potentiation of these receptors in the spinal cord evokes profound antihyperalgesia also after systemic administration, but possible synergistic or antagonistic actions of supraspinal α2-GABAARs on spinal antihyperalgesia have not yet been addressed. Here we generated two lines of GABAAR-mutated mice, which either lack α2-GABAARs specifically from the spinal cord, or, which express only benzodiazepine-insensitive α2-GABAARs at this site. We analyzed the consequences of these mutations for antihyperalgesia evoked by systemic treatment with the novel non-sedative benzodiazepine site agonist HZ166 in neuropathic and inflammatory pain. Wild-type mice and both types of mutated mice had similar baseline nociceptive sensitivities and developed similar hyperalgesia. However, antihyperalgesia by systemic HZ166 was reduced in both mutated mouse lines by about 60% and was virtually indistinguishable from that of global point-mutated mice, in which all α2-GABAARs were benzodiazepine insensitive. The major (α2-dependent) component of GABAAR-mediated antihyperalgesia was therefore exclusively of spinal origin, whereas supraspinal α2-GABAARs had neither synergistic nor antagonistic effects on antihyperalgesia. Our results thus indicate that drugs that specifically target α2-GABAARs exert their antihyperalgesic effect through enhanced spinal nociceptive control. Such drugs may therefore be well-suited for the systemic treatment of different chronic pain conditions.

Antihyperalgesic effect of the GABA(A) ligand clobazam in a neuropathic pain model in mice: a pharmacokinetic-pharmacodynamic study.

Facilitation of spinal GABAergic inhibition with benzodiazepines (BZDs) reverses pain sensitization in animals; however, the use of BZDs in man is limited by their sedative effect. The antihyperalgesic effects of GABA(A) agonists are mediated by GABA(A) receptors containing α2 subunits, whereas sedation is linked to α1 subunit-containing receptors. α2 and α3 selective GABA(A) receptor modulators have been tested in animals but are not yet available for use in human beings. Clobazam is a 1,5-BZD, which exhibits less cognitive side effects than other benzodiazepines. Here, we studied its antihyperalgesic effects in a mouse model of neuropathic pain. Clobazam showed a dose-dependent antihyperalgesic effect in the chronic constriction injury (CCI) model of neuropathic pain, peaking at 1 hr after administration and lasting for 4 hr with no relevant sedation at a dose of 3 mg/kg. At higher doses, the antihyperalgesic effect was stronger, but sedation became significant. The blood and brain kinetics of clobazam were linear over the range of doses tested with a short half-life of the parent compound and a ready penetration of the blood-brain barrier. Clobazam blood concentrations decreased rapidly, falling below the limit of detection at 120 min. after drug application. Its main metabolite, N-desmethyl-clobazam, showed more delayed and prolonged pharmacokinetics, partly explaining why antihyperalgesia persisted when clobazam was no longer detectable in the blood. Considering its therapeutic margin and its pharmacokinetic properties, clobazam would be a valuable compound to assess the role of the GABAergic pathway in pain transmission in human beings.

HZ166, a novel GABAA receptor subtype-selective benzodiazepine site ligand, is antihyperalgesic in mouse models of inflammatory and neuropathic pain.

Diminished GABAergic and glycinergic inhibition in the spinal dorsal horn contributes significantly to chronic pain of different origins. Accordingly, pharmacological facilitation of GABAergic inhibition by spinal benzodiazepines (BDZs) has been shown to reverse pathological pain in animals as well as in human patients. Previous studies in GABA(A) receptor point-mutated mice have demonstrated that the spinal anti-hyperalgesic effect of classical BDZs is mainly mediated by GABA(A) receptors containing the α2 subunit (α2-GABA(A) receptors), while α1-GABA(A) receptors, which mediate the sedative effects, do not contribute. Here, we investigated the potential analgesic profile of HZ166, a new partial BDZ-site agonist with preferential activity at α2- and α3-GABA(A) receptors. HZ166 showed a dose-dependent anti-hyperalgesic effect in mouse models of neuropathic and inflammatory pain, triggered by chronic constriction injury (CCI) of the sciatic nerve and by subcutaneous injection of the yeast extract zymosan A, respectively. This antihyperalgesic activity was antagonized by flumazenil and hence mediated via the BDZ-binding site of GABA(A) receptors. A central site of action of HZ166 was consistent with its pharmacokinetics in the CNS. When non-sedative doses of HZ166 and gabapentin, a drug widely used in the clinical management of neuropathic pain, were compared, the efficacies of both drugs against CCI-induced pain were similar. At doses producing already maximal antihyperalgesia, HZ166 was devoid of sedation and motor impairment, and showed no loss of analgesic activity during a 9-day chronic treatment period (i.e. no tolerance development). These findings provide further evidence that compounds selective for α2- and α3-GABA(A) receptors might constitute a novel class of analgesics suitable for the treatment of chronic pain.

GABAergic analgesia: new insights from mutant mice and subtype-selective agonists.

Gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the brain where it regulates many physiological functions including sleep, anxiety, reward and memory formation. GABAergic neurons and ionotropic GABA(A) receptors are also found in the spinal cord dorsal horn where they control the propagation of pain signals from the periphery to higher central nervous system areas. Recent evidence indicates that diminished inhibitory control at this site is a major factor in chronic pain syndromes. So far, this knowledge could not be translated into clinical pain therapy, probably because of the widespread actions of GABA in the central nervous system. The identification of GABA(A) receptor subtypes responsible for spinal antihyperalgesic effects has recently opened new avenues for the development of subtype-selective modulators of GABA(A) receptors. First results raise hopes that such compounds will be active against inflammatory and neuropathic pain but devoid of many of the side-effects of the established benzodiazepine-like drugs.