Allosteric modulators of the δ GABAA receptor subtype demonstrate a therapeutic effect in morphine-antinociceptive tolerance and withdrawal in mice.

The present study evaluated the effects of compounds targeting extrasynaptic δ subunit-containing γ-aminobutyric acid type A receptors (δ*-GABAARs) to interrogate the role of tonic inhibition in the development of antinociceptive tolerance caused by repeated morphine administration. We investigated the effect of subchronic or acute treatment with non-steroidal positive allosteric modulators (PAMs) of δ*-GABAARs, such as 2-261, on the morphine-antinociceptive tolerance. Mice were treated twice daily with morphine for 9 days and antinociception was measured using the hot water tail immersion test. Co-treatment with 2-261 and morphine prevented morphine-antinociceptive tolerance and acute administration of 2-261 on day 9 was sufficient to reverse the tolerance. Other compounds with activity at δ*-GABAARs also reversed morphine tolerance, whereas an enaminone that lacked activity at δ*-GABAARs did not. Acute administration of 2-261 did not cause an additive or synergistic antinociceptive effect when combined with an acute submaximal dose of morphine. We then used Cre/LoxP recombination to generate GABAA δ-subunit knockout mice to corroborate the pharmacological results. Observations of male δ-knockout mice demonstrated that the δ*-GABAARs was necessary for 2-261 modulation of both analgesic tolerance and somatic withdrawal symptoms produced by subchronic morphine. While female mice still benefited from the positive effects of 2-261, the δ-subunit was not necessary for these effects, highlighting a distinction of the different pathways that could have implications for some of the sex-related differences seen in human opioid-induced outcomes. Consequently, subtype-specific allosteric modulators of GABAARs may warrant further investigation as pharmacological targets to manage tolerance and withdrawal from opioids.

Behavioral characterization of Co 134444 (3alpha-hydroxy-21-(1'-imidazolyl)-3beta-methoxymethyl-5alpha- pregnan-20-one), a novel sedative-hypnotic neuroactive steroid.

RATIONALE: Neuroactive steroids have been shown to exhibit a wide range of behavioral activities that are similar but not identical to those of benzodiazepines. These activities include anticonvulsant, anxiolytic and sedative-hypnotic effects. OBJECTIVE: The purpose of the present study was to characterize Co 134444 (3alpha-hydroxy-21-(1'-imidazolyl)-3 -methoxymethyl-5alpha-pregnan-20-one), a novel sedative-hypnotic neuroactive steroid, in a variety of behavioral procedures. METHODS: Anticonvulsant effects were determined by the ability to protect against pentylenetetrazol- and maximal electroshock-induced seizures in mice and rats. Anxiolytic-like effects were determined using a punished drinking procedure in rats. Ataxic effects were determined using a horizontal wire procedure in mice and a rotorod procedure in mice and rats. The discriminative stimulus effects were evaluated in rats trained to discriminate pregnanolone from vehicle. RESULTS: Co 134444 exhibited oral anticonvulsant activity against pentylenetetrazol and maximal electroshock with ED50s of 9.8 and 20.6 mg/kg, respectively, in mice and 23.6 and 25.3 mg/kg, respectively, in rats. Anxiolytic-like efficacy was observed at a dose as low as 3.0 mg/kg, PO, in rats. Ataxic effects were observed with rapid onset and short duration. TD50s were 17.4 and 21.2 mg/kg orally in mice in the horizontal wire and rotorod procedures, respectively, and 39.0 mg/kg in rats using the rotorod. Co 134444 completely substituted for pregnanolone as a discriminative stimulus with little effect on response rate. CONCLUSIONS: Co 134444 exhibits a wide variety of behavioral effects; however, its rapid onset and short duration are consistent with its potential use as a sedative-hypnotic drug.

Response-rate suppression in operant paradigm as predictor of soporific potency in rats and identification of three novel sedative-hypnotic neuroactive steroids.

Novel neuroactive steroids were evaluated for their effects on operant responding, rotorod motor performance, and electroencephalogram recording in rats. Co 134444, Co 177843, and Co 127501 were compared with the prototypical gamma-aminobutyric acid(A)-positive allosteric modulators triazolam, zolpidem, pentobarbital, pregnanolone, and CCD 3693. Each of the compounds produced a dose-related decrease in response rates under a variable-interval 2-min schedule of positive reinforcement in an operant paradigm. In addition, all compounds produced a dose-related increase in ataxia and significant increases in nonrapid eye movement sleep in this experiment or have been previously reported to do so. Co 134444, Co 177843, and Co 127501 increased nonrapid eye movement sleep at doses that had no effect on rapid eye movement sleep. All of the compounds were more potent at decreasing operant responding than they were at increasing ataxia. Furthermore, the potency of compounds to produce response-rate suppression in an operant paradigm appeared to be a better predictor of soporific potency than did potency in the rotorod assay. The screening for sedative-hypnotic activity resulted in the identification of the novel orally active neuroactive steroids Co 134444, Co 177843, and Co 127501.

Substituted 3beta-phenylethynyl derivatives of 3alpha-hydroxy-5alpha-pregnan-20-one: remarkably potent neuroactive steroid modulators of gamma-aminobutyric acidA receptors.

Neuroactive steroids are positive allosteric modulators of gamma-aminobutyric acidA (GABAA) receptor complexes. Synthetic modification generally does not increase neuroactive steroid potency beyond that of the naturally occurring progesterone metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-P). Recently, it has been shown that introduction of appropriately para-substituted phenylethynyl groups at the 3beta-position of 5beta steroids increases receptor potency. The present report presents the synthesis and pharmacological profile of an analogous series of 5alpha steroids. The most striking feature of this series is the further enhancement of in vitro and in vivo potency obtained. In particular, 3beta-(p-acetylphenylethynyl)-3alpha-hydroxy-5alpha-pr egnan-20-one (Co 152791) was 11-, 16- and 49-fold more potent than 3alpha, 5alpha-P in modulating the binding of [35S]TBPS, [3H]flunitrazepam and [3H]muscimol, respectively, in rat brain membranes (Co 152791 IC50 or EC50 = 2-7.5 nM). Similarly, Co 152791 was 3- to 20-fold more potent than 3alpha,5alpha-P as an inhibitor of [35S]TBPS binding in human recombinant receptor combinations containing alpha1, alpha2, alpha3 or alpha5 and beta2gamma2L subunits (Co 152791 IC50 1.4-5.7 nM). Co 152791 displayed low efficacy and 3alpha,5alpha-P had low potency at alpha4/6beta3gamma2L GABAA receptor complexes. Interestingly, Co 152791 demonstrated remarkable potency as a potentiator of GABA-evoked currents in Xenopus oocytes expressing alpha1beta2gamma2L receptors (EC50 0.87 nM), being 184-fold more potent than 3alpha,5alpha-P. High in vitro potency was also reflected in enhanced in vivo activity in that Co 152791 exhibited exceptional anticonvulsant potency, protecting mice from pentylenetetrazol-induced seizures at a approximately 5-fold lower dose than 3alpha,5alpha-P after i.p. administration (Co 152791 ED50 0.6 mg/kg). Moreover, Co 152791 was orally active (ED50 1.1 mg/kg) and exhibited a therapeutic index of 7 relative to rotorod impairment. The remarkable potency of Co 152791 as a positive allosteric modulator of GABAA receptors may be explained by its interaction with an auxiliary binding pocket in the neuroactive steroid binding site. In addition, modification at the 3beta-position probably hinders metabolism of the 3alpha-hydroxy group contributing to the exceptional anticonvulsant potency of this compound relative to other neuroactive steroids.

Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.

Two naturally occurring metabolites of progesterone, 3 alpha-hydroxy-5 alpha- and 5 beta-pregnan-20-one (1 and 2), are potent allosteric modulators of the GABAA receptor. Their therapeutic potential as anxiolytics, anticonvulsants, and sedative/hypnotics is limited by rapid metabolism. To avoid these shortcomings, a series of 3 beta-substituted derivatives of 1 and 2 was prepared. Small lipophilic groups generally maintain potency in both the 5 alpha- and 5 beta-series as determined by inhibition of [35S]TBPS binding. In the 5 alpha-series, 3 beta-ethyl, -propyl, -trifluoromethyl and -(benzyloxy)methyl, as well as substituents of the form 3 beta-XCH2, where X is Cl, Br, or I or contains unsaturation, show limited efficacy in inhibiting [35S]TBPS binding. In the 5 beta-series, the unsubstituted parent 2 is a two-component inhibitor, whereas all of the 3 beta-substituted derivatives of 2 inhibit TBPS via a single class of binding sites. In addition, all of the 3-substituted 5 beta-sterols tested are full inhibitors of [35S]TBPS binding. Electrophysiological measurements using alpha 1 beta 2 gamma 2L receptors expressed in oocytes show that 3 beta-methyl- and 3 beta-(azidomethyl)-3 alpha-hydroxy-5 alpha-pregnan-20-one (6 and 22, respectively) are potent full efficacy modulators and that 3 alpha-hydroxy-3 beta-(trifluoromethyl)-5 alpha-pregnan -20-one (24) is a low-efficacy modulator, confirming the results obtained from [35S]TBPS binding. These results indicate that modification of the 3 beta-position in 1 and 2 maintains activity at the neuroactive steroid site on the GABAA receptor. In animal studies, compound 6 (CCD 1042) is an orally active anticonvulsant, while the naturally occurring progesterone metabolites 1 and 2 are inactive when administered orally, suggesting that 3 beta-substitution slows metabolism of the 3-hydroxyl, resulting in orally bioavailable steroid modulators of the GABAA receptor.

3 alpha-Hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970): a partial agonist at the neuroactive steroid site of the gamma-aminobutyric acidA receptor.

Neuroactive steroids bind to a unique site on the gamma-aminobutyric acidA (GABAA) receptor complex and allosterically modulate the binding of convulsant ([35S]t-butylbicyclophosphorothionate, [35S]TBPS), GABA ([3H]muscimol), and benzodiazepine ([3H]flunitrazepam) site ligands. In rat cortical membranes, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) is a full agonist at the steroid site, inhibiting 96% of specific [35S]TBPS binding and enhancing [3H]flunitrazepam and [3H]muscimol binding 95% and 69% above control levels, respectively. In contrast, the synthetic steroid 3 alpha-hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970) has limited efficacy for modulating the binding of [35S]TBPS (44% inhibition), [3H]flunitrazepam (41% enhancement), and [3H]muscimol (< 10% enhancement). In competition experiments, Co 2-1970 (10 microM) reduced the apparent potency of 3 alpha, 5 alpha-P by 7-17-fold for modulating the binding of these radioligands in rat cortical membranes, suggesting that it has partial agonist properties. Because cortical membranes contain a heterogeneous population of receptors, Co 2-1970 was examined in recombinant GABAA receptors stably expressed in human embryonic kidney 293 cells. Co 2-1970 inhibited [35S]TBPS binding with limited efficacy (39-65% inhibition) in the five receptor combinations examined and, at 10 microM, reduced the apparent potency of 3 alpha, 5 alpha-P 57-fold for inhibiting [35S]TBPS binding to alpha 1 beta 1 gamma 2L receptors. To verify these findings functionally, the effects of 3 alpha, 5 alpha-P and Co 2-1970 were examined electrophysiologically in Xenopus oo-cytes expressing alpha 1 beta 1 gamma 2L receptors. Co 2-1970 showed limited efficacy potentiation of GABA-evoked chloride currents relative to 3 alpha, 5 alpha-P (28% and 86% of the GABA maximum current, respectively). Moreover, Co 2-1970 produced a concentration-dependent antagonism of the 3 alpha, 5 alpha-P-induced potentiation that was associated with a reduction in the apparent affinity of 3 alpha, 5 alpha-P (11-fold at 10 microM Co 2-1970). Taken together, these data indicate that Co 2-1970 is a partial agonist at the neuroactive steroid site associated with GABAA receptors.