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.

Characterization of the anxiolytic properties of a novel neuroactive steroid, Co 2-6749 (GMA-839; WAY-141839; 3alpha, 21-dihydroxy-3beta-trifluoromethyl-19-nor-5beta-pregnan-20-one), a selective modulator of gamma-aminobutyric acid(A) receptors.

The purpose of this study was to evaluate the effects of a novel neuroactive steroid, Co 2-6749 (GMA-839; WAY-141839; 3alpha, 21-dihydroxy-3beta-trifluoromethyl-19-nor-5beta-pregnan-20-one), on gamma-aminobutyric acid(A) receptors in vitro and to define its anxiolytic-like effects and side effect profile in vivo. Co 2-6749 fully inhibited [(35)S]t-butylbicyclophosphorothionate binding in rat brain cortical membranes with an IC(50) value of 230 nM and in human gamma-aminobutyric acid(A) receptor subunit combinations of alpha1beta2gamma2L, alpha2beta2gamma2L, alpha3beta2gamma2L, alpha4beta3gamma2L, alpha5beta2gamma2L, and alpha6beta3gamma2L receptors (IC(50) values of 200, 200, 96, 2300, 210, and 2000 nM). Rats were trained in a Geller-Seifter operant conflict paradigm. Co 2-6749 caused a dose-related increase in punished responding with a minimum effective dose of 1.6 mg/kg, p.o., a wide therapeutic index relative to a decrease in unpunished responding and relative to ataxia, and no tolerance. Additionally, ethanol caused less than a 2-fold shift to the left in the dose-response function of Co 2-6749 in the rotorod procedure in rats. In a pigeon conflict paradigm, punished responding was maximally increased to 784% of vehicle control by 30 mg/kg, p.o., with a 2-h duration and no effect on unpunished responding at this dose. Similarly, punished responding in squirrel monkeys was maximally increased to 1774% of control by 10 mg/kg, p.o., with no effect on unpunished responding at this dose. With robust anxiolytic-like activity across species, a large separation between anxiolytic-like effects and sedation/ataxia, a minimal interaction with ethanol, a lack of tolerance, and apparent oral bioavailability, Co 2-6749 makes an ideal candidate for development as a novel anxiolytic 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.

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.

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.

3 alpha-Hydroxy-3 beta-(phenylethynyl)-5 beta-pregnan-20-ones: synthesis and pharmacological activity of neuroactive steroids with high affinity for GABAA receptors.

Neuroactive steroids that allosterically modulate GABAA receptors have potential uses as anticonvulsants, anxiolytics, and sedative-hypnotic agents. Recently, a series of pregnanes substituted with simple alkyl groups at the 3 beta-position were synthesized and found to be active in vitro. The present report describes the synthesis of a series of substituted 3 alpha-hydroxy-3 beta-(phenylethynyl)pregnan-20-ones and their in vitro structure-activity relationship determined by their potency for inhibition of [35S]TBPS binding in rat brain membranes. Appropriate substitution of the phenyl group results in ligands with particularly high affinity for the neuroactive steroid site on GABAA receptors (e.g., 4-acetyl 28, IC50 10 nM). The potency of selected steroids was confirmed electrophysiologically in oocytes expressing cloned human GABAA alpha 1 beta 2 gamma 2L receptors (e.g., compound 28, EC50 6.6 nM). Consistent with their in vitro activity, some of the 3 beta-(phenylethynyl)-substituted steroids displayed anticonvulsant activity in the pentylenetetrazol (PTZ) and maximal electroshock (MES) tests following ip administration in mice. Notably, the 3 beta-[(4-acetylphenyl)ethynyl]-19-nor derivative 36 demonstrated an attractive anticonvulsant profile (PTZ and MES ED50 values of 2.8 and 9.2 mg/kg, respectively). A new pharmacophore for the neuroactive steroid site of GABAA receptors is proposed based upon the high affinity of certain substituted 3 beta-(phenylethynyl) steroids.

In vitro and in vivo activity of 16,17-dehydro-epipregnanolones: 17,20-bond torsional energy analysis and D-ring conformation.

PURPOSE: Certain neuroactive pregnane steroids (also known as "epalons") are allosteric modulators of the GABA, receptor and have been shown to be potent anticonvulsants, anxiolytics, sedative/hypnotics, and anesthetic agents. The purpose of this study was to calculate the structural consequences of introduction of a double bond in the 16,17-position and to determine if this modification would selectively reduce sedative activity, but maintain the potent anticonvulsant activity of neuroactive steroids. METHODS: We have studied the biochemical and behavioral effects of introducing a 16,17 double bond into the naturally occurring neuroactive steroids, 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) and three synthetic neuroactive steroid derivatives, 3 alpha-hydroxy-3 beta-methyl-5 alpha-pregnan-20-one (3 alpha,3 beta Me,5 alpha-P), 3 alpha-hydroxy-5 alpha-androstane (3 alpha, 5 alpha-A), and alphaxalone (3 alpha,5 alpha-11-one-P). RESULTS: The 16-ene analogs of most of these neuroactive steroids were found to be 7- and 16-fold less potent in inhibiting [35S]TBPS binding to GABAA receptors and in a similar fashion, had reduced anticonvulsant and sedative potency in proportional amounts. The exception was the androstane (3 alpha,5 alpha-A) without a 17-acetyl group, that had virtually identical IC50 and ED50 values for the saturated and unsaturated derivatives. Calculation of the torsional energy profile for each of the 17-acetyl side chain conformations showed that the conformational energy minima found in the alpha,beta-unsaturated keto systems, produce an orientation of the 20-keto group that is rotated by 165 degrees when compared to the non-conjugated acetyl group (determined by X-ray crystallography and its minimum energy conformation). CONCLUSIONS: The modified orientation of the 20-keto group of neuroactive steroids containing a 16-ene, provides an explanation for their decreased biological activity overall, but did not lead to an enhanced protective index.

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.

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.

Cloning and characterization of the human GABAA receptor alpha 4 subunit: identification of a unique diazepam-insensitive binding site.

Benzodiazepines modulate gamma-aminobutyric acid (GABA)-evoked chloride currents through a specific binding site at the GABAA receptor-chloride channel complex. The heterogeneity of diazepam-sensitive benzodiazepine binding sites (type I and type II) has been identified by pharmacological approaches both with native receptors and recombinant receptors coexpressing alpha, beta and gamma subunits. In addition, two distinguishable diazepam-insensitive benzodiazepine sites are found, spatially distributed between cerebral cortical and cerebellar regions. Coexpression of alpha 6 with beta 2 and gamma 2L subunits creates a pharmacologically similar benzodiazepine receptor to the diazepam-insensitive site observed in cerebellum, however, there is no evidence regarding the possible subunit combination forming the DI site in cerebral tissues. Here we report the cloning of the human alpha 4 cDNA and its pharmacology by coexpression of this alpha 4 subunit with beta 2 and gamma 2L subunits. This recombinant receptor complex showed a high affinity for the previously described benzodiazepine partial agonist bretazenill, the pyrazoloquinoline compounds CGS-9895 and CGS-9896, as well as the inverse agonists DMCM (methyl 6,7-dimethoxy 4-ethyl-beta-carboline-3-carboxylate) and Ro15-4513 as determined by [3H]Ro15-4513 binding. However, it is insensitive to the benzodiazepine type I selective compounds CL218.872 (3-methyl-6-[3-(trifluoromethyl)[phenyl]-1,2,4-triazolo[4.3-b]pyridazine ) and zolpidem as well as the benzodiazepine full agonists diazepam, halazolam and midazolam. In addition, the benzodiazepine receptor ligands DMCM, beta-CCE (beta-carboline-3-carboxylate ethyl ester), Beta-CCM (beta-carboline-3-carboxylate methyl ester), FG-7142, CGS-9895 and CGS-9896 showed 7 to 10 times higher affinity for alpha 4 beta 2 gamma 2L. The pharmacology of the alpha 4 beta 2 gamma 2L receptor complex appears to resemble those of the diazepam-insensitive site found in the cerebral cortex. Our study thus suggests that this subpopulation of diazepam-insensitive GABAA receptors may be composed of alpha 4 beta 2 gamma 2L subunits.

Comparative behavioral characterization of the neuroactive steroids 3 alpha-OH,5 alpha-pregnan-20-one and 3 alpha-OH,5 beta-pregnan-20-one in rodents.

Pregnan steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, two endogenous neuroactive steroid isomers, 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), were studied for differences in their pharmacological properties using behavioral assays. 3 alpha,5 alpha-P and 3 alpha,5 beta-P were similar in their potencies and efficacies in blocking pentylenetetrazol-induced seizures in mice (ED50: 3 alpha, 5 alpha-P = 2.8 mg/kg and 3 alpha,5 beta-P = 3.0 mg/kg). Similarly, both neuroactive steroids produced roto-rod deficits within the same range of potency (TD50: 3 alpha,5 alpha-P = 18.8 mg/kg and 3 alpha,5 beta-P = 21.2 mg/kg). However, in animal models of anxiety, subtle differences were observed between the two isomers. In both the light/dark transition test and elevated plus-maze, 3 alpha,5 beta-P was more efficacious than 3 alpha,5 alpha-P, though both compounds had similar potencies. In the Geller-Seifter test, 3 alpha,5 beta-P was more potent and efficacious than 3 alpha,5 alpha-P. Neither compound had significant effects on unpunished responding within the dose range tested. Both compounds produced similar biphasic curves in the locomotor test. All together, the data indicate that 3 alpha,5 alpha-P and 3 alpha,5 beta-P have similar anticonvulsant activity, but the 5 beta-isomer possesses more potent and efficacious anxiolytic properties than the 5 alpha-isomer.

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)

Differential effects of 4'-chlorodiazepam on expressed human GABAA receptors.

The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABAA receptor cDNAs were determined. Cotransfection of human alpha 2, beta 1, and gamma 2 subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t-[35S]butylbicyclophosphorothionate ([35S]-TBPS) binding to the GABA-activated chloride channel. This site is found on GABAA receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the alpha subunit was further demonstrated as substitution of either alpha 1 or alpha 3 for the alpha 2 subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [35S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of alpha 2 and beta 1 alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [35S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the alpha and beta polypeptides.