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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 GABAA agonists are mediated by GABAA receptors containing α2 subunits, whereas sedation is linked to α1 subunit-containing receptors. α2 and α3 selective GABAA 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.
Diminished synaptic inhibition in the spinal cord critically contributes to central sensitization, a key phenomenon in chronic inflammatory and neuropathic pain. The role of glycinergic and γ-aminobutyric acid (GABA)ergic neurons in this process has been widely described [1, 2]. Therefore, facilitation of the spinal GABAergic input is a rational approach to compensate for diminished inhibitory pain control. In fact, the antihyperalgesic effect of several GABAA agonists such as muscimol or of positive allosteric modulators such as diazepam has been demonstrated in animals .
In human beings, research on the analgesic effect of BZDs is scarce and controversial. In clinical research, diazepam has been used as an active placebo in a study seeking to demonstrate the analgesic effect of fentanyl in patients with chronic non-cancer neuropathic pain . On the other hand, clonazepam is widely used in practice to treat neuropathic pain and has demonstrated efficacy in myofascial pain, temporomandibular joint dysfunction, cancer-related neuropathic pain and in stomatodynia when used topically [5-8]. However, the use of BZDs in chronic pain is rather limited by their side effects, such as sedation, memory impairment and dependence.
Advances in the understanding of the molecular diversity of GABAA receptors have suggested that the therapeutic index might become improved through the development of subtype-selective or partial BDZ-site agonists [9-12].
Benzodiazepine-sensitive GABAA receptors contain at least one of the following α subunits α1, α2, α3 or α5, together with two β subunits and a γ2 subunit in a 2:2:1 stoichiometry [13, 14]. Work in GABAA receptor point-mutated mice has shown that the sedative action of BDZs is mainly mediated by GABAA receptors containing α1 subunits , whereas α2- and α3-containing GABAA receptors were found to be responsible for the anxiolytic properties  and largely responsible for the spinal antihyperalgesic actions of classical BDZs [3, 17]. In animals, α1-sparing (non-sedative) BDZ agonists showed an antihyperalgesic activity in inflammatory and neuropathic pain models without losing efficacy after repeated treatment [17, 18]. Such compounds are under clinical development but are not yet available for use in human beings .
Clobazam is a 1–5 BZD prescribed in all forms of anxiety and in epilepsy. It seems to exert less cognitive and psychomotor side effects compared with clonazepam and lorazepam in a wide range of pharmacodynamic tests in man [20, 21]. Therefore, clobazam may be a suitable compound to test the antihyperalgesic effect of GABAA agonists in exploratory pain studies in human beings. Although an antihyperalgesic action of clobazam in mice is likely, it has not been proven so far. In a set of experiments, we therefore investigated the antihyperalgesic and sedative effects of clobazam in a neuropathic pain model in mice and correlated this to its pharmacokinetic properties.
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Our results confirm that like other non-selective GABAA ligands such as diazepam, clobazam has an antihyperalgesic effect in the CCI model in mice. Clobazam exhibited a dose-dependent effect, which was well correlated to its blood and brain concentrations. After oral intake, clobazam rapidly appeared in the systemic circulation and in the brain (peak value at Tmax 25 and 30 min. in blood and brain, respectively), demonstrating a fast absorption and distribution with ready penetration of the blood–brain barrier. Its half-life in mice was markedly shorter than the half-life in human beings (39 min. versus 16–50 hr) and similar to previously reported values . In the systemic circulation, the Tmax of the main metabolite N-desmethyl-clobazam was 102(±73) min., and its half-life 264 (±51) min. after a dose of 3 mg/kg and in similar ranges after 10 mg/kg.
To quantify the antihyperalgesic effect of clobazam and to compare it to its pharmacokinetics, we calculated the %MPE and showed that it was reached at 1 hr, which is consistent with the pharmacokinetic profile of the parent compound (Tmax = 30 min. in the brain). However, the persistence of the effect for 4 hr, although plasma and brain concentrations of clobazam were undetectable after 2 hr despite a sensitive analytical method, suggests a possible role of N-desmethyl-clobazam. In fact, the time course of the antihyperalgesic effect was best matched when the plasma levels of clobazam and of N-desmethyl-clobazam were taken into account. Besides, N-desmethylclobazam was tested in vitro on cultured cerebral neurons of rats and exhibited a dose-dependent enhancement of GABA-activated currents identical to that of clobazam .
As expected, in this mouse model of neuropathic pain, clobazam had a strong and significant antihyperalgesic effect. The magnitude of this effect was comparable to the effect of diazepam ) and of the α2/α3 – subtype-selective BZD-site ligand HZ166 . Similar to what is described in the literature for diazepam  and α2–α3 selective compounds [9, 17, 18], clobazam did not modify the response of the non-injured paw, suggesting that a facilitation of GABAA receptor-mediated inhibition at the spinal cord level is involved in the observed antihyperalgesia.
Clobazam is a non-selective BZD, which appears to show relatively little cognitive and psychomotor side effects at therapeutic doses in human beings. Unlike other non-selective BZDs such as diazepam or clonazepam, which possess a 1–4 chemical structure, clobazam has a 1–5 chemical structure which may contribute to its better side effect profile . However, the selectivity of clobazam has not been determined. The reduced effect on psychomotor performance of clobazam (10 and 20 mg) compared with clonazepam (0.5 and 1 mg) was demonstrated in healthy volunteers  as well as, compared with lorazepam (1 mg), in anxious patients [20, 21]. Our experiment corroborates these findings because the activity of the mice, recorded for 1 hr from the peak effect, was not significantly decreased at the dose of 3 mg/kg, which already displayed a notable antihyperalgesic effect. When the doses were increased up to 10 mg/kg or higher, the sedation became significant.
The use of selective compounds targeting the α2 and α3 subunits of the GABAA receptor would give an opportunity to separate the desired antihyperalgesic effects from the unwanted sedative effects, which are mainly mediated by α1-containing GABAA receptors. Such compounds were successfully tested in rats (L-838,417, for example ). They were, however, not further developed in human beings for pharmacokinetic reasons such as a poor bioavailability and short half-lives. More recently, these positive results were replicated with HZ166, a novel 8-substituted triazolo- and imidazobenzodiazepine, which also showed a better pharmacokinetic profile .
Compounds that have entered clinical trials include TPA023 and more recently MRK-409 [27, 28]. However, the development of MRK-409 was terminated because, unlike in pre-clinical tests, it failed to exhibit anxiolytic activity at non-sedative doses .
In summary, clobazam exhibits pronounced antihyperalgesic activity at a dose that does not cause significant sedation. As long as subtype-selective compounds suitable for use in human beings are lacking, clobazam is a good candidate for proof-of-principle studies addressing antihyperalgesic properties of BZDs in human experimental pain models.