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.

Positive allosteric modulators of the GABA(A) receptor: differential interaction of benzodiazepines and neuroactive steroids with ethanol.

Endogenous pregnane steroids, such as allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one; 3alpha, 5alpha-P) and pregnanolone (3alpha-hydroxy-5beta-pregnan-20-one; 3alpha,5beta-P), allosterically modulate GABA(A) receptor function and exhibit behavioral effects similar to benzodiazepines, though acting at a distinct recognition site. Inasmuch as some positive allosteric modulators of GABA(A) receptor function exhibit profound interactions with ethanol, the effects of 3alpha,5alpha-P and 3alpha,5beta-P were compared to those of two benzodiazepines, triazolam and diazepam, on the motor function of mice and rats when administered either alone or in combination with ethanol. All four test compounds exhibited dose-related impairment of motor function in the horizontal wire task in mice and the rotorod task in rats. Ethanol caused a marked enhancement of triazolamand diazepam-induced motor impairment. In contrast, ethanol enhanced to a lesser extent the motor impairment induced by both neurosteroids in mice and not at all in rats. All four compounds increased ethanol-induced behavioral sleep time in mice, although the benzodiazepines did so at a much smaller fraction of their ataxic doses as compared to the neurosteroids. As one of the undesired side-effects of therapeutic use of benzodiazepines is their interaction with ethanol, development of neuroactive steroids as drugs may offer therapeutic advantages.

Anxiolytic and anticonvulsant activity of a synthetic neuroactive steroid Co 3-0593.

Endogeneously occurring neuroactive steroids, metabolites of progesterone and deoxycorticosterone, have been shown previously to interact with the GABAA receptor with great specificity in vitro and to have anticonvulsant, anxiolytic and sedative activity in vivo. However, these endogenously occurring steroids are not useful as therapeutic agents due to their potential metabolism to hormonally active steroids and their poor oral bioavailability. In an attempt to develop therapeutic agents which would maintain the pharmacological profiles of endogeneous neuroactive steroids but with increased oral bioavailability and reduced metabolic liability, we explored simple substitutions at the 3 beta-position of the endogenous neuroactive steroid, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P). This report describes part of the in vitro and in vivo pharmacological profile of a 3 beta-substituted analog, 3 beta-ethenyl-3 alpha-hydroxy-5 alpha-pregnan-20-one (Co 3-0593). The compound exhibited anticonvulsant activity against pentylenetrazol-induced seizures in mice and rats (ED50 = 5.6 and 11.5 mg/kg, i.p., respectively). Co 3-0593 showed robust anxiolytic effects, comparable to benzodiazepines in the Geller-Seifter test after both SC and oral administration. Furthermore, the anxiolytic activity was maintained after chronic administration suggesting an absence of tolerance. The compound did not affect the acquisition of a learned response at both anticonvulsant and anxiolytic doses. However, at higher doses the compound showed rotorod deficit which was further enhanced by ethanol. In summary, 3 beta-ethenyl-substituted 3 alpha, 5 alpha-P appeared to maintain the pharmacological activities of the endogenous neuroactive steroid with apparent oral activity.

CCD-3693: an orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat.

An endogenous neuroactive steroid, pregnanolone, and an orally available synthetic analog, CCD-3693, were administered to rats at the middle of their circadian activity phase (6 hr after lights off). Electroencephalogram-defined sleep-wake states, locomotor activity and body temperature were concurrently measured 30 hr before and after treatment. Identical procedures were used to test triazolam and zolpidem. Triazolam (0.1-1.6 mg/kg), zolpidem (2.5-10 mg/kg) and the neuroactive steroids (10-30 mg/kg) produced dose-dependent increases in non-rapid eye movement (NREM) sleep. At this dose and time of day (in which the rats were predominantly awake during the 6 hr before treatment) the neuroactive steroids appeared more intrinsically efficacious in promoting NREM sleep than the benzodiazepine ligands. The neurosteroids did not, however, significantly interfere with rapid eye movement sleep and were more selective in reducing (EEG) wakefulness, with relatively less locomotor activity impairment during waking than triazolam and zolpidem. In addition, the benzodiazepine receptor ligands showed distinct "rebound" wakefulness after the NREM sleep-promoting effect subsided, although the neuroactive steroids did not. In addition, in vitro binding studies and in vivo pharmacological data confirmed that CCD-3693 was orally active in standard tests of anxiety, anticonvulsant, loss-of-righting and passive avoidance.

Partial agonism by 3alpha,21-dihydroxy-5beta-pregnan-20-one at the gamma-aminobutyric acidA receptor neurosteroid site.

3alpha,21-Dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) and 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-P) have full efficacy as allosteric modulators of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to sites on the gamma-aminobutyric acid (GABA) type A receptor complex (GRC). Relative to 3alpha,5alpha-P and 5alpha-THDOC, 3alpha,21-dihydroxy-5beta-pregnan-20-one (5beta-THDOC) has limited efficacy as an allosteric modulator of [35S]TBPS binding. Interactions between 3alpha,5alpha-P, 5alpha-THDOC and 5beta-THDOC were examined to determine whether these neuroactive steroids share a common site for modulation of the GRC. The concentration-response curves for both 3alpha,5alpha-P and 5alpha-THDOC modulation of [35S]TBPS binding to brain and recombinantly derived GRCs are shifted rightward in the presence of various concentrations of 5beta-THDOC. Similarly, 5beta-THDOC modulates GABA-evoked Cl- currents with low efficacy and inhibits the potentiation of GABA-evoked Cl- currents by 3alpha,5alpha-P. Furthermore, behavioral studies reveal that 5beta-THDOC antagonizes 3alpha,5alpha-P-induced loss of the righting reflex in mice at a dose that has no effect alone. These results represent the first demonstration of antagonist-like actions of a neuroactive steroid on the GRCs at levels ranging from the receptor to animal behavior and suggest the existence of partial agonist neurosteroids.

Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-affinity, steroid modulator of the gamma-aminobutyric acid(A) receptor.

Ganaxolone (CCD 1042) is a 3beta-methyl-substituted analog of the endogenous neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one. Ganaxolone inhibited binding of the gamma-aminobutyric acid (GABA)A receptor-chloride channel ligand t-[35S]butylbicyclophosphorothionate (IC50 of 80 nM) and enhanced binding of the benzodiazepine site ligand [3H]flunitrazepam (EC50 of 125 nM) and the GABA site ligand [3H]muscimol (EC50 of 86 nM), consistent with activity as a positive allosteric modulator of the GABA(A) receptor. Electrophysiological recordings showed that, whereas nanomolar concentrations of ganaxolone potentiated GABA-evoked chloride currents in Xenopus oocytes expressing the human GABA(A) receptor subunits alpha1beta1gamma2L, alpha2beta1gamma2L or alpha3beta1gamma2L, direct activation of chloride flux occurred to a limited extent only at micromolar concentrations. Ganaxolone was effective in nontoxic doses against clonic convulsions induced by s.c. pentylenetetrazol administration in mice and rats (ED50 values of 4.3 and 7.8 mg/kg i.p., respectively). Ganaxolone also exhibited potent anticonvulsant activity against seizures induced by s.c. bicuculline (ED50 of 4.6 mg/kg i.p.), i.p. TBPS (ED50 of 11.7 mg/kg i.p.) and i.p. aminophylline (ED50 of 11.5 mg/kg i.p.) in mice. Although ganaxolone effectively blocked tonic seizures induced by maximal electroshock in mice (ED50 of 29.7 mg/kg i.p.), it did so only at doses that produced ataxia on the Rotorod (TD50 of 33.4 mg/kg i.p.). Conversely, ganaxolone was a potent anticonvulsant against fully kindled stage 5 seizures induced by corneal kindling in rats (ED50 of 4.5 mg/kg i.p.), producing these effects at doses well below those that resulted in ataxia (TD50 of 14.2 mg/kg i.p.). The seizure threshold, as determined by an increase in the dose of i.v. infused pentylenetetrazol required to induce clonus, was also significantly elevated by nontoxic doses of ganaxolone in mice. In summary, these data indicate that ganaxolone is a high-affinity, stereoselective, positive allosteric modulator of the GABA(A) receptor complex that exhibits potent anticonvulsant activity across a range of animal procedures. The profile of anticonvulsant activity obtained for ganaxolone supports clinical evaluation of this drug as an antiepileptic therapy with potential utility in the treatment of generalized absence seizures as well as simple and complex partial seizures.

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.

Anxiolytic properties of endogenously occurring pregnanediols in two rodent models of anxiety.

Certain endogenously occurring 3 alpha-hydroxylated, 5-reduced pregnane steroids act at a specific site on the GABAA receptor complex (GRC) to modulate the effects of GABA at its receptor. Modulators that potentiate GABA at the GABAA receptor often possess anxiolytic properties. The anxiolytic potential of four 5-reduced, 3 alpha, 20-pregnanediols, differing only in the stereochemical orientation of the steroid A-ring and the 20-hydroxyl group, were tested in the Vogel test following intracerebroventricular (ICV) administration. The effects of these pregnanediols were compared to those of their 20-ketone analogues, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha, 5 beta-P). All four pregnanediols tested significantly enhanced punished drinking at doses ranging from 10 to 60 micrograms. The rank order of potency based on the minimum effective dose (MED) observed was 5 alpha-pregnan-3 alpha,20 alpha-diol = 5 beta-pregnan-3 alpha,20 alpha-diol > 5 beta-pregnan-3 alpha,20 beta-diol > 5 alpha-pregnan-3 alpha, 20 beta-diol. 3 alpha,5 beta-P and 3 alpha,5 alpha-P enhanced punished responding when administered at 2.5 and 5 micrograms, respectively. 3 beta,5 alpha-P which is inactive at the GRC was also inactive (up to 100 micrograms) in the Vogel test. The benzodiazepine control diazepam was efficacious when administered at 2.5 micrograms. 5 alpha-Pregnan-3 alpha,20 alpha-diol was further tested in the mouse elevated plus-maze model following systemic administration where it was found to be active in a dose range of 10-40 mg/kg IP. These results raise the possibility that in addition to 3 alpha,5 alpha-P and 3 alpha,5 beta-P, some of their endogenously occurring pregnanediol metabolites may also influence physiological processes related to anxiety via the GRC.

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.

Loreclezole modulates [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding via a distinct site on the GABAA receptor complex.

The allosteric modulation of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) and [3H]flunitrazepam binding was utilized to evaluate the actions of loreclezole at the GABAA receptor complex in the rat brain. Loreclezole was observed to allosterically inhibit the binding of [35S]TBPS in a dose-dependent manner with micromolar potency (IC50 = 1 microM). Loreclezole was found to have an additive effect on neuroactive steroid modulation of [35S]TBPS binding, but merely potentiated the effect of Ro5-4864 (4"-chlorodiazepam) modulation of [35S]TBPS binding. These observations suggest that loreclezole modulates [35S]TBPS binding through a site independent of the neuroactive steroid and Ro5-4864 sites on the GABAA receptor complex. The enhancement of [3H]flunitrazepam binding to the benzodiazepine receptor by loreclezole as well as the effect of loreclezole on CL218872/[3H]flunitrazepam dose-response curves suggest that loreclezole does not act through the benzodiazepine site on the GABAA receptor complex, nor does it selectively modulate benzodiazepine receptor subtypes. The potency of loreclezole as and inhibitor of [35S]TBPS binding in rat brain was regionally dependent and GABA-sensitive. Loreclezole modulation of [35S]TBPS binding showed greater potency and GABA sensitivity in the cerebellum and thalamus when compared to other brain regions such as the cortex, hippocampus and striatum. This finding is consistent with previous reports of the selectivity of loreclezole for GABAA receptor complex's containing beta 2 and beta 3 subunits. These beta subunit isoforms predominate in the cerebellum and thalamus. Collectively the evidence suggests that loreclezole modulates [35S]TBPS and [3H]flunitrazepam binding through a site distinct from benzodiazepine, neuroactive steroid, Ro5-4864 and GABA sites on the GABAA receptor complex.

Characterization of peripheral benzodiazepine type sites in a cultured murine BV-2 microglial cell line.

It is known that the density of peripheral benzodiazepine receptors (PBR) increases after brain damage. Astrocytes are among the cell types where PBR ligand binding has been detected and may be involved in the response to neuronal injury and regeneration. Consistent with the hypothesis, the apparent density of PBR sites in astrocytes is increased by both cytokines and neurotoxins. However, microglia, the resident macrophages which represent 5-15% of glial cell populations have not been evaluated for the presence of the PBR. In the present study, we report the presence of [3H]Ro5-4864 binding in microglial cells. In particular, we used BV-2 cells, an immortalized cell line of murine microglial cells. High affinity binding of [3H]Ro5-4864 to a single site was detected in membranes prepared from BV-2 cells (KD = 4.4 nM, Bmax = 3,800 fmoles/mg protein). Various ligands for the PBR displaced [3H]Ro5-4864 binding with the following rank order of potencies: PK11195 = Ro5-4864 > FGIN-1-27 > triazolam = diazepam > beta-pro-pyl-beta-carboline-3-carboxylate = clonazepam > lorazepam = flurazepam >> chlordiazepoxide = clorazepate. Subcellular fractionationstudies indicate that the majority of the Ro5-4864 binding sites is in the mitochondrial fraction. The remainder is found in nonmitochondrial cell fractions. The [3H]Ro5-4864 binding observed on intact cells had characteristics similar to those found on membranes. The presence of a high density of PBRs in these cells establish the basis for additional investigations into their possible functional role, if any, in the microglial response to neuronal injury.

Soporific effect of the neurosteroid pregnanolone in relation to the substance's plasma level: a pilot study.

The soporific effect of the neuroactive steroid pregnanolone, a metabolite of progesterone, and its relationship with plasma concentrations was assessed in 18 young, healthy, male volunteers for 2 h after administration of a single dose of pregnanolone prepared in two different formulations. Sedation was measured as sleep propensity and power increase in the low frequency delta band of the quantified EEG, based on 5-min polygraphic (EEG, EOG, EMG) recordings under resting conditions, which were performed immediately before, and 30, 60, 90, and 120 min after intake of the study drug. With both formulations, there was a time-dependent increase in plasma concentration of pregnanolone with highest values 1-2 h postdosing. The model of short polygraphic recordings under resting conditions demonstrated soporific effects of pregnanolone. Compared to predosing baseline the number of sleep attempts and the time asleep increased after treatment with a peak 60 min postdosing. Quantitative EEG analysis revealed an increase of absolute amplitude in the delta frequency range with a comparable temporal pattern. Correlations between the soporific effect and plasma concentrations of pregnanolone suggest that the effects were drug-related, although this has to be replicated with placebo control.

Modulation of human recombinant GABAA receptors by pregnanediols.

Utilising two point voltage-clamp techniques on Xenopus laevis oocytes expressing human (alpha 1 beta 1 gamma 2L) recombinant GABAA receptors, the GABA modulatory actions of six naturally occurring neurosteroids have been determined and compared with those of known positive allosteric modulators. The anaesthetic steroids 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one produced a concentration-dependent enhancement of the GABA-evoked current. The maximal enhancement of the agonist-induced response produced by these steroids was intermediate between that of pentobarbitone and diazepam, but much greater than that caused by bretazenil. For both the 5 alpha and 5 beta steroid a reduction of the 20 ketone group to form either the corresponding 20 alpha or 20 beta hydroxy steroid produced, in all cases, a reduction in potency and a decrease in the maximal effect. The relationship of steroid structure to these two parameters is considered. The influence of the alpha subtype (alpha x beta 1 gamma 2L, where x = 1, 2 or 3) for the behaviourally active 5 alpha-pregnan-3 alpha,20 alpha-diol is also determined. Although the maximal effect of the steroid is not influenced by the alpha subtype, the alpha 2-containing receptor exhibits a modest decrease (approximately 6-fold) in potency compared to alpha 1- and alpha 3-containing receptors. The results described here are discussed in relation to the distinct behavioural actions of the neurosteroids.

Influence of the estrus cycle on the discrimination of apparent neuroactive steroid site subtypes on the gamma-aminobutyric acidA receptor complex in the rat.

Estrus cycle-related changes in gamma-aminobutyric acid (GABA)A receptor complex (GRC) sensitivity to modulation by reduced progesterone metabolites is suggestive of a possible mechanism for maintaining brain homeostasis in the presence of fluctuating levels of these neuroactive metabolites. In addition, certain endogenously occurring pregnanediols are selective for apparent neuroactive steroid site subtypes discriminated by the progesterone metabolite 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) on the GRC. Thus, it was of interest to evaluate the influence of gender and the estrus cycle on the ability of 3 alpha,5 beta-P and its 20-reduced analog 5 beta-pregnan-3 alpha,20 beta-diol to differentiate neuroactive steroid site subtypes. Neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to the GRC in rats during estrus, diestrus and after ovariectomy (OVX) was measured in washed cortical P2 homogenates in the presence or absence of exogenous GABA. During diestrus, the inability of 5 beta-pregnan-3 alpha,20 beta-diol to allosterically modulate [35S]TBPS binding in the absence of GABA coincides with the inability of 3 alpha,5 beta-P to modulate [35S]TBPS binding with high potency. In contrast, the addition of GABA to the assay produced high potency inhibition of [35S]TBPS binding by each steroid. Remarkably, although findings in diestrus and OVX homogenates were no different from those observed in males, the proportions and IC50 values of the two sites discriminated by 3 alpha,5 beta-P in [35S]TBPS binding assays during the estrus phase were significantly different from male, OVX and diestrus rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Potency of lipid and protein formulation of 5 alpha-pregnanolone at induction of anaesthesia and the corresponding regional brain distribution.

We have studied the anaesthetic potencies of 5 alpha-pregnanolone albumin solution (PAS) and 5 alpha-pregnanolone Intralipid emulsion (PLE) at equivalent concentrations in male rats using an EEG threshold method. The criterion of anaesthesia was burst suppression of the EEG of 1 s or more (the "silent second" (SS)) as a sign of deep anaesthesia. The potency of the two formulations was assessed by comparing the threshold doses of 5 alpha-pregnanolone at three dose rates (1.0, 2.0 and 3.0 mg kg-1 min-1). We found that SS was initiated in all rats after infusions of PAS, while no SS could be induced in rats after infusion of PLE at a larger dose. A higher concentration of 5 alpha-pregnanolone was found in all brain and peripheral tissues of PAS-treated rats than in those treated with PLE. In rats with PAS-induced anaesthesia (3.0 mg kg-1 min-1), the highest concentrations were detected in striatum (mean 19.40 (SD 1.21) ng mg-1). Although there was a small insignificant reduction in threshold doses with dose rates at 2.0-3.0 mg kg-1 min-1, the tissue concentrations in striatum, frontal cortex and occipital cortex were found to be significantly increased. We conclude that PAS was more potent than PLE in inducing anaesthesia. Brain distribution of 5 alpha-pregnanolone varied regionally in a manner similar to the variation in GABAA receptor sensitivity to this neuroactive steroid.

Benzodiazepines and peptides stimulate pregnenolone synthesis in brain mitochondria.

Mitochondria isolated from rat brain were found to cleave cholesterol to produce pregnenolone, the precursor for hormonal steroids, at a mean rate of 21.0 pmol pregnenolone.mg protein-1.min-1. This rate-limiting step in steroidogenesis was significantly stimulated by PK 11195 (1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide) and Ro5 4864 (4'-chlorodiazepam), ligands which bind to peripheral benzodiazepine receptors with high affinity. Low-affinity ligands for the peripheral benzodiazepine receptor such as Ro15 1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5 alpha][1,4] benzo-3-carboxylate) and clonazepam had no significant effect on the rate of pregnenolone synthesis. Furthermore, the rank order of potency of these compounds as inhibitors of [3H]Ro5 4864 binding was identical to the rank order for steroid production. Since the 86-amino acid peptide diazepam binding inhibitor is also thought to bind to the peripheral benzodiazepine receptor, four fragments of this peptide, a random sequence and steroidogenesis activator peptide were also evaluated for their ability to interact with peripheral benzodiazepine receptors and to stimulate steroidogenesis in rat brain mitochondria. Steroidogenesis activator peptide and two fragments of diazepam binding inhibitor significantly stimulated pregnenolone biosynthesis. In contrast to the peripheral benzodiazepine receptor ligands, no correlation between peptide potency in displacing [3H]Ro5 4864 binding and steroidogenesis was observed.

Selective actions of certain neuroactive pregnanediols at the gamma-aminobutyric acid type A receptor complex in rat brain.

Certain endogenous pregnanediols (5 alpha-pregnan-3 alpha,20 alpha-diol and 5 beta-pregnan-3 alpha,20 beta-diol) were observed to have limited efficacy as allosteric modulators of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) and [3H]flunitrazepam binding to sites on the gamma-aminobutyric acid (GABA)A receptor complex in rat brain. In contrast, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) have full efficacy. Moreover, 3 alpha,5 beta-P but not 3 alpha,5 alpha-P recognizes high (nanomolar) and low (micromolar) affinity neuroactive steroid sites in these allosteric modulatory assays. The concentration-response curve for 3 alpha,5 alpha-P modulation of [35S]TBPS binding was shifted rightward in the presence of these pregnanediols and GABA. The maximum shift produced by these pregnanediols never exceeded the concentration-response curve obtained with 3 alpha,5 alpha-P alone in the absence of GABA. Additionally, neither 5 alpha-pregnan-3 alpha,20 alpha-diol nor 5 beta-pregnan-3 alpha,20 beta-diol had any effect on the site recognized by 3 alpha,5 alpha-P in the absence of GABA. The difference in the affinities of the two apparent sites (29 nM versus 152 nM in the presence and absence of GABA, respectively) recognized by 3 alpha,5 alpha-P is only approximately 5-fold. In contrast, the difference between the high (30 nM) and low (7 microM) affinity sites discriminated by 3 alpha,5 beta-P is > 200-fold. Thus, the selective interaction between the high affinity site recognized by 3 alpha,5 beta-P and these pregnanediols can be clearly observed. A saturating concentration of 5 beta-pregnan-3 alpha,20 beta-diol selectively eliminated the high affinity component recognized by 3 alpha,5 beta-P, whereas 5 alpha-pregnan-3 alpha,20 alpha-diol did not completely abolish the high affinity site. 5 alpha-Pregnan-3 alpha,20 alpha-diol recognized only a portion of the high affinity sites discriminated by 3 alpha,5 beta-P, relative to 5 beta-pregnan-3 alpha,20 beta-diol, whereas the two pregnanediols recognized a similar population of sites mediating 3 alpha,5 alpha-P inhibition of [35S]TBPS binding. Collectively, these studies provide evidence that the limited efficacy of certain pregnanediols as allosteric modulators of [35S]TBPS binding may be explained in part by selectivity for the high affinity site recognized by 3 alpha,5 beta-P.(ABSTRACT TRUNCATED AT 400 WORDS)