Acute and chronic effects of the synthetic neuroactive steroid, ganaxolone, against the convulsive and lethal effects of pentylenetetrazol in seizure-kindled mice: comparison with diazepam and valproate

A high-affinity positive modulator of the GABA(A) receptor complex, ganaxolone, is a 3beta-methylated analog of the naturally occurring neuroactive steroid allopregnanolone. In the present study, ganaxolone was tested for its ability to (1) suppress seizures (clonic and tonic) and lethality induced by pentylenetetrazol (PTZ) in PTZ-kindled mice (anticonvulsive effect) and (2) to attenuate the development of sensitization to the convulsive and lethal effects of PTZ in kindled mice (anti-epileptogenic effect) when given as a pretreatment prior to each PTZ injection during kindling acquisition. Two classical antiepileptic drugs, diazepam and valproate, were tested for comparison. All three drugs dose-dependently suppressed tonic seizures and lethality induced by PTZ in kindled mice; only ganaxolone was effective against clonic seizures. Ganaxolone showed anti-epileptogenic properties as it reduced the sensitivity of kindled mice to the convulsive (clonic and tonic seizures) and lethal effects of PTZ. Diazepam showed anti-epileptogenic effects against tonic seizures and lethality, but not clonic seizures; valproate was ineffective in preventing development of any of these effects. Sensitivity to PTZ-induced seizures and lethality was not affected in mice with a history of repeated treatment with ganaxolone, diazepam, or valproate. The drugs had effects on ambulatory activity that ranged from no effect (ganaxolone) through moderate impairment (diazepam) to marked disruption (valproate). Taken together, the results of the present study add to accumulating evidence of the unique anticonvulsive/behavioral profile of neuroactive steroids.     

Antiepileptogenic effects of the novel synthetic neuroactive steroid,ganaxolone, against pentylenetetrazol-induced kindled seizures: Comparison with diazepam and valproate

Pharmacological treatment of epilepsy is often unsatisfactory due to side effects and the lack of drugs that control the progressive epileptogenic process. Modulation of inhibitory γ-aminobutyric acid (GABA)-ergic neurotransmission by synthetic agonists of the neuroactive steroid binding site on the GABA_A receptor complex is one approach toward the identification of improved antiepileptic agents. In this study, antiepileptogenic and anticonvulsive effects of the novel synthetic neuroactive steroid, ganaxolone (3α-hydroxy-3β-methyl-5α-pregnan-20-one), were evaluated in comparison with diazepam and valproate against pentylenetetrazol (PTZ)-induced kindled seizures in mice. Kindled seizures provide a model of the progressive epileptogenic process. Successive administration of 45 mg/kg PTZ on days 1, 3, 5, 8, and 10 resulted in the rapid development of kindled seizures and significant reductions in thresholds for clonic convulsions, tonic convulsions, and lethality induced by PTZ on day 10. Ganaxolone, diazepam, and valproate dose-dependently protected against clonic convulsions induced by acute submaximal dose of PTZ (70 mg/kg). The compounds also dose-dependently suppressed fully kindled seizures and blocked the expression of kindled seizures over successive treatments with PTZ (45 mg/kg). Relative to acute anticonvulsive potencies against 70 mg/kg PTZ, however, ganaxolone was more potent than valproate or diazepam against fully kindled seizures and in blocking the expression of kindled seizures over successive treatments with PTZ. Importantly, only ganaxolone demonstrated antiepileptogenic activity by blocking the development of kindling, as evidenced when PTZ was administered in the absence of anticonvulsant treatments. Both diazepam and valproate failed to prevent development of kindled seizures even at doses that fully suppressed motor expression of seizures during kindling acquisition. Unlike diazepam and valproate, ganaxolone did not impair ambulatory activity within the dose range used in this study. These data, taken in conjunction with other findings on the unique pharmacological actions of ganaxolone, predict an improvement in the pharmacological management of epilepsy with this synthetic neuroactive steroid. Drug Dev. Res. 44:21–33, 1998. © 1998 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Neuroactive steroids attenuate cocaine-induced sucrose intake in rats, but not cocaine-induced hyperactivity in mice

Rationale: Neuroactive steroids, including the potent anticonvulsants ganaxolone (3α-hydroxy-3β-methyl-5α-pregnan-20-one) and Co 2-1068 (3β-(4acetylphenyl)ethynyl-3α,21-dihydroxy-5β-20-one-21-hemisuccinate), have recently been shown to protect against cocaine-induced seizures. Objectives: The purpose of the present experiments was to determine whether ganaxolone and Co 2-1068 attenuate acute behavioral effects of cocaine unrelated to seizures. Methods: In the first experiment, the locomotor effects of Co 2-1068 (10–100 mg/ kg), pentobarbital (10–100 mg/kg) and haloperidol (0.03–0.3 mg/kg), alone or in combination with cocaine (5.6–30 mg/kg), were determined in mice. In the second experiment, the effects on sucrose intake of ganaxolone (4–16 mg/kg), Co 2-1068 (8–64 mg/kg), pentobarbital (4–32 mg/kg), and haloperidol (0.04–0.4 mg/kg), alone or in combination with cocaine (4–16 mg/kg), were determined in rats. Results: Cocaine caused a dose-related increase in locomotor activity in mice, whereas Co 2-1068, pentobarbital and haloperidol caused dose-related decreases. The dopamine antagonist haloperidol, at a dose that had no effect on activity by itself, but not Co 2-1068 or pentobarbital, attenuated the cocaine-induced increase in locomotor activity. Cocaine, ganaxolone, Co 2-1068, and haloperidol produced dose-related decreases in sucrose intake in rats; the effects of pentobarbital on sucrose intake were variable. As with locomotor effects, haloperidol attenuated the cocaine-induced decrease in sucrose intake. In addition, cocaine-induced decreases in sucrose intake were attenuated by ganaxolone and Co 2-1068. Pentobarbital had no statistically significant effect on the cocaine dose-response function. Conclusions: These results suggest that the interaction of neuroactive steroids with cocaine extends to pharmacologic actions beyond anticonvulsant efficacy, but that the blockade of behavioral effects of cocaine by neuroactive steroids does not apply to all acute behaviors. Received: 7 September 1999 / Final version: 8 November 1999     

Relevance of endogenous 3α-reduced neurosteroids to depression and antidepressant action

The naturally occurring 3α-reduced neurosteroids allopregnanolone and its isomer pregnanolone are among the most potent positive allosteric modulators of γ-aminobutyric acid type A receptors. They play a critical role in the maintenance of physiological GABAergic tone and display a broad spectrum of neuropsychopharmacological properties. We have reviewed existing evidence implicating the relevance of endogenous 3α-reduced neuroactive steroids to depression and to the mechanism of action of antidepressants. A wide range of preclinical and clinical evidence suggesting the antidepressant potential of 3α-reduced neuroactive steroids and a possible involvement of a deficiency and a disequilibrium of neuroactive steroid levels in pathomechanisms underlying the etiology of major depressive disorder have emerged in recent years. Antidepressants elevate 3α-reduced neurosteroid levels in rodent brain, and clinically effective antidepressant pharmacotherapy is associated with normalization of plasma and cerebrospinal fluid (CSF) concentrations of endogenous neuroactive steroids in depressed patients, unveiling a possible contribution of neuroactive steroids to the mechanism of action of antidepressants. In contrast, recent studies using nonpharmacological antidepressant therapy suggest that changes in plasma neuroactive steroid levels may not be a general mandatory component of clinically effective antidepressant treatment per se, but may reflect distinct properties of pharmacotherapy only. While preclinical studies offer convincing evidence in support of an antidepressant-like effect of 3α-reduced neuroactive steroids in rodent models of depression, current clinical investigations are inconclusive of an involvement of neuroactive steroid deficiency in the pathophysiology of depression. Moreover, clinical evidence is merely suggestive of a role of neuroactive steroids in the mechanism of action of clinically effective antidepressant therapy. Additional clinical studies evaluating the impact of successful pharmacological and nonpharmacological antidepressant therapies on changes in neuroactive steroid levels in both plasma and CSF samples of the same patients are necessary in order to more accurately address the relevance of 3α-reduced neuroactive steroids to major depressive disorder. Finally, proof-of-concept studies with drugs that are known to selectively elevate brain neurosteroid levels may offer a direct assessment of an involvement of neurosteroids in the treatment of depressive symptomatology.     

Ganaxolone: a novel positive allosteric modulator of the GABA(A) receptor complex for the treatment of epilepsy

Ganaxolone is a member of a novel class of neuroactive steroids which modulates the GABA(A) receptor complex (GRC) via an unique recognition site, distinct from those of benzodiazepines and barbiturates. Preclinical data from an array of chemically- and electrically-induced seizure models demonstrate that ganaxolone possesses broad spectrum anticonvulsant activity with potential clinical utility in both generalised and partial seizures, as well as cocaine-induced seizures. Clinical data to date support a favourable safety profile with somnolence, an extension of GABAergic activity, being the most frequently reported adverse event at higher doses. Target indications include infantile spasms and complex partial seizures. Open-label data in paediatric patients with intractable epilepsy suggest that ganaxolone may be effective in treating infantile spasms. A controlled trial utilising an in-patient, monotherapy design demonstrated that ganaxolone effectively decreases complex partial seizure activity compared to placebo. An important area of further evaluation is ganaxolone's potential role in the treatment of women with catamenial epilepsy.     

Neuropsychopharmacological properties of neuroactive steroids in depression and anxiety disorders

Neuroactive steroids modulate neurotransmission through modulation of specific neurotransmitter receptors such as γ-aminobutyric acid type A (GABA_A) receptors. Preclinical studies suggested that neuroactive steroids may modulate anxiety- and depression-related behaviour and may contribute to the therapeutical effects of antidepressant drugs. Attenuations of 3α-reduced neuroactive steroids have been observed during major depression. This disequilibrium can be corrected by successful treatment with antidepressant drugs. However, non-pharmacological antidepressant treatment strategies did not affect neuroactive steroid composition independently from the clinical response. Further research is needed to clarify whether enhancement of neuroactive steroid levels might represent a new therapeutical approach in the treatment of affective disorders. Nevertheless, the first studies investigating the therapeutical effects of exogenously administered dehydroepiandosterone revealed promising results in the treatment of major depression. In addition, in various anxiety disorders alterations of neuroactive steroid levels have been observed. In panic disorder, in the absence of panic attacks, neuroactive steroid composition is opposite to that seen in depression, which may represent counter-regulatory mechanisms against the occurrence of spontaneous panic attacks. However, during experimentally induced panic attacks, there was a pronounced decline in GABAergic neuroactive steroids, which might contribute to the pathophysiology of panic attacks. In conclusion, neuroactive steroids contribute to the pathophysiology of affective disorders and the mechanisms of action of antidepressants. They are important endogenous modulators of depression and anxiety and may provide a basis for the development of novel therapeutic agents in the treatment of affective disorders.     

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 GABA_A 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 3β-position of the endogenous neuroactive steroid, 3α-hydroxy-5α-pregnan-20-one (3α,5α-P). This report describes part of the in vitro and in vivo pharmacological profile of a 3β-substituted analog, 3β-ethenyl-3α-hydroxy-5α-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, IP, 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 roto-rod deficit which was further enhanced by ethanol. In summary, 3β-ethenyl-substituted 3α,5α-P appeared to maintain the pharmacological activities of the endogenous neuroactive steroid with apparent oral activity. Received: 10 September 1996/Final version: 22 April 1997     

Effect of the neuroactive steroid α-THDOC on staircase test behavior in mice

This study examined the effect of the neuroactive steroid 3α, 5α-tetrahydrodeoxycorticosterone (α-THDOC) as compared to the benzodiazepines diazepam and midazolam and the barbiturate phenobarbital on the number of rearing events and the number of steps ascended in the mouse staircase test. The benzodiazepines, phenobarbital and α-THDOC all reduced rearing activity at doses that did not affect climbing. The rearing-suppression effect of the benzodiazepines and α-THDOC, but not of phenobarbital, was blocked by the benzodiazepine antagonist flumazenil. It appears that, although such neuroactive steroids, like barbiturates, bind to distinct sites within the chloride ion channel of the gamma-aminobutyric acid type A (GABA_A) receptor complex, α-THDOC behavioral activity is modulated by the benzodiazepine recognition site. Received: 19 October 1995 / Final version: 1 July 1996     

Sex differences in the acquisition of intravenously self-administered cocaine and heroin in rats

Endogenous pregnane steroids, such as allopregnanolone (3α-hydroxy-5α-pregnan-20-one; 3α, 5α-P) and pregnanolone (3α-hydroxy-5β-pregnan-20-one; 3α,5β-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 3α,5α-P and 3α,5β-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 triazolam- and 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. Received: 24 March 1998/Final version: 12 May 1988     

In vivo and in vitro hyperbaric studies in mice suggest novel sites of action for ethanol

 The present study uses increased atmospheric pressure as an ethanol antagonist to test the hypothesis that allosteric coupling pathways in the GABA_A receptor complex represent initial sites of action for ethanol. This was accomplished using behavioral and in vitro measures to determine the effects of pressure on ethanol and other GABAergic drugs in C57BL/6 and LS mice. Behaviorally, exposure to 12 times normal atmospheric pressure (ATA) of a helium-oxygen gas mixture (heliox) antagonized loss of righting reflex (LORR) induced by the allosteric modulators ethanol and pentobarbital, but did not antagonize LORR induced by the direct GABA agonist 4,5,6,7-tetrahydroisoxazolo-pyridin-3-ol (THIP). Similarly, exposure to 12 ATA heliox antagonized the anticonvulsant effects verses isoniazid of ethanol, diazepam and pentobarbital. Biochemically, exposure to 12 ATA heliox antagonized potentiation of GABA-activated ³⁶Cl^– uptake by ethanol, flunitrazepam and pentobarbital in LS mouse brain preparations, but did not alter GABA-activated ³⁶Cl^– uptake per se. In contrast to its antagonist effect versus other allosteric modulators, pressure did not antagonize these behavioral or in vitro effects induced by the neuroactive steroid, 3α-hydroxy-5β-pregnan-20-one (3α,5β-P). These findings add to evidence that pressure directly and selectively antagonizes drug effects mediated through allosteric coupling pathways. The results fit predictions, and thus support the hypothesis that allosteric coupling pathways in GABA_A receptors represent initial sites of action for ethanol. Collectively, the results suggest that there may be common physicochemical and underlying structural characteristics that define ethanol sensitive regions of receptor proteins and/or their associated membranes that can be identified by pressure within (e.g., GABA_A) and possibly across (e.g., GABA_A, NMDA, 5HT₃) receptors. Received: 15 December 1997 / Final version: 23 July 1998     

Neuroactive steroids and affective disorders

Neuroactive steroids modulate neurotransmission through modulation of specific neurotransmitter receptors such as gamma-aminobutyric acid type A (GABA(A)) receptors. Preclinical studies suggested that neuroactive steroids may modulate anxiety and depression-related behaviour and may contribute to the therapeutical effects of antidepressant drugs. Attenuations of such neuroactive steroids have been observed during major depression and in several anxiety disorders, suggesting a pathophysiological role in such psychiatric conditions. In panic disorder patients a dysequilibrium of neuroactive steroid composition has been observed, which may represent a counterregulatory mechanism against the occurrence of spontaneous panic attacks. Furthermore, alterations of 3alpha-reduced pregnane steroids during major depression were corrected by successful treatment with antidepressant drugs. However in contrast, non-pharmacological antidepressant treatment strategies did not affect neuroactive steroid composition. In addition, changes in neuroactive steroid concentrations after mirtazapine therapy occurred independently from the clinical response, thereby suggesting that changes in neuroactive steroid concentrations more likely reflect direct pharmacological effects of antidepressants rather than clinical improvement in general. Nevertheless, the effects of antidepressant pharmacotherapy on the composition of neuroactive steroids may contribute to the alleviation of certain depressive symptoms, such as amelioration of anxiety, inner tension or sleep disturbances. Moreover, first studies investigating the therapeutical effects of dehydroepiandrosterone revealed promising results in the treatment of major depression. In conclusion, neuroactive steroids are important endogenous modulators of depression and anxiety and may provide a basis for development of novel therapeutic agents in the treatment of affective disorders.     

Investigation of the anticonvulsive effect of acute immobilization stress in anxious Balb/cByJ mice using GABAA-related mechanistic probes

Rationale A disordered regulation of neuroactive steroids release in response to acute stress could induce GABAergic dysfunctions underlying anxiety disorders. Objectives First, we conducted studies indicating that a short immobilization stress in anxious Balb/cByJ mice produced an anticonvulsive effect. Second, the effects of different positive allosteric modulators (etifoxine, progesterone, clonazepam, and allopregnanolone) of GABA_A receptors were compared in a mouse model mimicking the disruption of the acute stress-induced neuroactive steroids release with finasteride (types I and II 5α-reductase inhibitor). Results The acute stress-induced anticonvulsive effect, expressed by the threshold dose of t-butylbicyclophosphorothionate-producing clonic seizures, was time-dependent. The extent of the enhancement of acute stress-induced anticonvulsive effect was lowered in the presence of finasteride. The same effect was observed with PK11195, which behaves as an antagonist of the peripheral benzodiazepine receptor in the dose range used in this study. Picrotoxin reduced the acute stress anticonvulsive effect, proving that this effect operates through the GABA_A receptor. Contrary to progesterone (up to 30 mg/kg), etifoxine (50 mg/kg), allopregnanolone (10 mg/kg), and clonazepam (10 μg/kg) inhibited the finasteride effect in stressed animals. The effect of etifoxine was blocked in the presence of finasteride and picrotoxin combined in stressed animals. Conclusions These findings support the hypothesis suggesting an involvement of neuroactive steroids in the anticonvulsive effect of restraint stress. The dual and complementary mechanisms of action of etifoxine (directly on the GABA_A receptor and indirectly via the neuroactive steroids) may represent a therapeutic benefit in the treatment of various anxiety disorders with abnormal production of neuroactive steroids.     

Pharmacological profile of a 17β-heteroaryl-substituted neuroactive steroid

Rationale

In order to improve upon the pharmacological properties of the neuroactive steroid ganaxolone, it was used as the starting point in the design of novel neurosteroids that replace the 17β-acetyl side chain with an isoxazole bioisostere.

Objectives

UCI-50027 (3-[3α-hydroxy-3β-methyl-5α-androstan-17β-yl]-5-(hydroxymethyl)isoxazole) was designed as an orally active neuroactive steroid specifically targeted at the gamma-aminobutyric acid(A) receptor (GABA_AR).

Methods

UCI-50027 was tested in vitro in Xenopus oocytes expressing human GABA_ARs and in vivo as an anticonvulsant, for ataxic effects and for anxiolytic activity.

Results

In vitro, UCI-50027 dose-dependently enhanced the activity of GABA at human α₁β₂γ_2L, α₂β₁γ_2L, and α₄β₃δ GABA_ARs. Consistent with its action as a positive allosteric modulator (PAM), it had no direct activity in the absence of GABA. UCI-50027 protected against acute pentylenetetrazol (PTZ)-induced convulsions with an ED₅₀ of 6 mg/kg p.o. In the rotarod (RR) paradigm in mice, the AD₅₀ (the ataxic dose where half of the animals fail the RR test) was found to be 38 mg/kg p.o., giving a therapeutic index (TI = RR AD₅₀/PTZ ED₅₀)～6 versus 2.8 for ganaxolone. In the mouse-elevated plus maze (EPM) model for anxiety, UCI-50027 showed a minimum effective dose (MED) ≤0.3 mg/kg p.o. Thus, the TI (TI = RR AD₅₀/EPM MED) for the compound as an anxiolytic is ≥127 versus 3.3 for ganaxolone.

Conclusions

UCI-50027 is an orally active neuroactive steroid with pharmacological activity consistent with a GABA_AR PAM that has an improved separation between anticonvulsant/anxiolytic and rotarod effects, potent activity as an anticonvulsant and anxiolytic when compared to ganaxolone.     

Protective efficacy of neuroactive steroids against cocaine kindled-seizures in mice

Neuroactive steroids demonstrate pharmacological actions that have relevance for a host of neurological and psychiatric disorders. They offer protection against seizures in a range of models and seem to inhibit certain stages of drug dependence in preclinical assessments. The present study was designed to evaluate two endogenous and one synthetic neuroactive steroid that positively modulate the gamma-aminobutyric acid (GABA(A)) receptor against the increase in sensitivity to the convulsant effects of cocaine engendered by repeated cocaine administration (seizure kindling). Allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), pregnanolone (3alpha-hydroxy-5beta-pregnan-20-one) and ganaxolone (a synthetic derivative of allopregnanolone 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one) were tested for their ability to suppress the expression (anticonvulsant effect) and development (antiepileptogenic effect) of cocaine-kindled seizures in male, Swiss-Webster mice. Kindled seizures were induced by daily administration of 60 mg/kg cocaine for 5 days. All of these positive GABA(A) modulators suppressed the expression of kindled seizures, whereas only allopregnanolone and ganaxolone inhibited the development of kindling. Allopregnanolone and pregnanolone, but not ganaxolone, also reduced cumulative lethality associated with kindling. These findings demonstrate that some neuroactive steroids attenuate convulsant and sensitizing properties of cocaine and add to a growing literature on their potential use in the modulation of effects of drugs of abuse.     

Moderate doses of ethanol fail to increase plasma levels of neurosteroid 3α-hydroxy-5α-pregnan-20-one-like immunoreactivity in healthy men and women

Rationale: The endogenous GABAergic neuroactive steroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP, allopregnanolone) has been proposed to contribute to ethanol actions. Humans synthesize 3α,5α-THP, but its role in response to systemic administration of ethanol is unclear. Objective: The present study aims to determine the effect of a moderate dose of ethanol on progesterone and 3α,5α-THP concentrations in plasma samples of healthy male and female subjects and to determine if these levels are related to the subjective effects of ethanol. Females were tested in both the follicular and luteal phases of the menstrual cycle. Methods: Healthy men (N=9) and women (N=12) aged 21–35 participated in the study. Men participated in two sessions on which they received ethanol (0.8 g/kg) or placebo. Women participated in four sessions on which they received ethanol (0.7 g/kg) or placebo during the follicular and luteal phases of their cycle. Subjective states and mood were measured by standardized self-report questionnaires and a measure of psychomotor performance. Steroid levels (progesterone, 3α,5α-THP, estradiol, and cortisol) were measured in plasma samples by radioimmunoassay. Results: Ethanol significantly increased plasma levels of progesterone, but not 3α,5α-THP-like immunoreactivity, in women in the luteal phase. Ethanol had no effect on progesterone or 3α,5α-THP-like immunoreactivity levels in women in the follicular phase or in men, and it did not increase cortisol in men or women. Ethanol also did not affect estradiol in men or women. Conclusions: 3α,5α-THP-like immunoreactivity levels in human plasma are not increased following moderate ethanol consumption, suggesting that circulating levels of progesterone or its tetrahydro-reduced metabolites do not play a major role in ethanol action. However, the possibility remains that ethanol increases endogenous brain production of GABAergic neurosteroids without affecting plasma levels. Moreover, humans synthesize 5β-reduced GABAergic steroids, and levels of these steroids may be altered in plasma or brain.     

Pharmacokinetic and behavioral effects of allopregnanolone in healthy women

Rationale The behavioral effects of allopregnanolone (3α-hydroxy-5α-pregnan-20-one) in women are not known.Objective Allopregnanolone, a neuroactive steroid secreted by the mammalian ovary, exerts its anesthetic, anxiolytic, and sedative/hypnotic effects through potentiation of GABA_A receptors. The purpose of this study was to evaluate the behavioral effects of allopregnanolone in healthy women.Methods Ten healthy women were given three increasing intravenous doses of allopregnanolone in the follicular phase of the menstrual cycle. Saccadic eye movement parameters and visual analogue scales of sedation were used to evaluate the behavioral response of allopregnanolone. Repeated blood samples for analyses of allopregnanolone were drawn throughout the study day.Results Exogenously administered allopregnanolone decreases saccadic eye movement parameters and increases subjective ratings of sedation that correlate with increased serum concentrations of this neuroactive steroid.Conclusion The behavioral effects of allopregnanolone are similar to that of its 5β-stereoisomer, pregnanolone (3α-hydroxy-5β-pregnan-20-one). Apart from fatigue and mild nausea, allopregnanolone given in a cumulative dose of 0.09 mg/kg did not have any adverse effects.     

Neuroactive steroids, negative affect, and nicotine dependence severity in male smokers

Rationale Nicotine administration alters neuroactive steroids in rodent models, and serum levels of the neuroactive steroid DHEAS (dehydroepiandrosterone sulfate) appear to be higher in smokers. These molecules may be relevant to tobacco addiction and affective symptoms.Objectives This study aims to investigate DHEAS, allopregnanolone, pregnenolone, and other steroids in male smokers to determine potential associations with nicotine dependence severity and negative affect.Materials and methods Allopregnanolone and pregnenolone serum levels were determined by gas chromatography/mass spectrometry, while DHEAS and other steroid levels were determined by radioimmunoassay in 28 male smokers. Correlational analyses were performed to determine potential associations with rating measures, including the Fagerstrom Test for Nicotine Dependence (FTND), the addiction subscale of the Ikard Smoking Motivation Questionnaire (ISMQ), the craving item on the Reasons to Smoke (RTS) Questionnaire, and the negative affect and craving subscales of the Shiffman–Jarvik Withdrawal Questionnaire.Results DHEAS levels were inversely correlated with the negative affect subscale of the Shiffman–Jarvik Withdrawal Questionnaire (r=−0.60, p=0.002) and the RTS craving item (r=−0.43, p=0.03), and tended to be inversely correlated with the FTND scores (r=−0.38, p=0.067) and the ISMQ addiction subscale (r=−0.38, p=0.059), adjusting for age. Allopregnanolone levels were positively correlated with cotinine levels (r=0.57, p=0.006); pregnenolone levels tended to be positively correlated with cotinine levels (r=0.40, p=0.066).Conclusions DHEAS levels were inversely correlated with negative affect and craving measures, and may predict nicotine dependence severity. Allopregnanolone levels were positively correlated with cotinine levels, suggesting that this neuroactive steroid may be upregulated in smokers. Neuroactive steroids may represent novel smoking cessation agents.     

The effect ofN-alkyloxycarbonyl group on the anticonvulsant activities ofN-alkyloxycarbonyl-α-amino-N-methylsuccinimides

In connection with the development of new anticonvulsant agents with a broad spectrum, we found thatN-Cbz-α-amino-N-alkylsuccinimides showed significant anticonvulsant activities, and the pharmacological activities of these compounds were dependent on their stereochemistry andN-substituted alkyl group. These results prompted us to define the effects of other functional group on the anticonvulsant activities of these compounds. Therefore a series ofN-alkoxycarbonyl-α-amino-N-methylsuccinimide were prepared fromN-Cbz-aspartic acid and were evaluated with their anticonvulsant activities against the MES and PTZ tests, in order to define the effect ofN-substituted alkoxy carbonyl group with the anticonvulsant activities. From these studies, it was found that all the testedN-alkoxycarbonyl-α-amino-N-methylsuccinimides exhibited significant anticonvulsant activities in the PTZ test and were not active in the MES test. The most active compound in the PTZ test was (S)N-ethoxycarbonyl-α-amino-N-methylsuccinimide. We found that the pharmacological activities in the PTZ test were dependent on theirN-alkoxycarbonyl groups. They follow as such; The order of anticonvulsant activities for (R) series as evaluated by ED₅₀ wasN-phenoxycarbonyl=N-4-nitrobenzyloxycarbonyl>N-ethoxycarbonyl >N-allyloxycarbonyl>N-tert. butoxycarbonyl compound; For the (S) seriesN-ethoxycarbonyl >N-phenoxycarbonyl>N-allyloxycarbonyl compound. From the above results, it was conceivable thatN-substituted alkoxycarbonyl group had certain effects on the anticonvulsant activities ofN-alkoxycarbonyl-α-amino-N-methylsuccinimides.     

Reinforcing and discriminative stimulus effects of the neuroactive steroids pregnanolone and Co 8-7071 in rhesus monkeys

  Rationale and Objectives: The present study was designed to assess possible abuse-related effects of the endogenous neuroactive steroid pregnanolone (3α-hydroxy-5β-pregnan-20-one) and the orally bioavailable, water-soluble neuroactive steroid pro-drug Co 8-7071 (3α,21-dihydroxy-3β-trifluoromethyl-5β-pregnan-20-one, 21-hemisuccinate). Methods: Four rhesus monkeys were prepared with chronic intravenous (IV) catheters and trained to press a lever under a ten-response fixed-ratio (FR) schedule of methohexital injection (0.1 mg/kg per injection). Three rhesus monkeys were trained to discriminate intragastric infusions of pentobarbital (10 mg/kg) from saline infusions under a FR5 schedule of stimulus-shock termination. Results: At least two doses of pregnanolone (0.003–0.1 mg/kg per injection) maintained injections per session above saline levels in the four monkeys tested, whereas Co 8-7071 (0.01–1.0 mg/kg per injection) maintained injections per session above saline levels in two of four monkeys at relatively low levels of injections per session. In rhesus monkeys trained to discriminate pentobarbital, IV pregnanolone injections (0.1–1.7 mg/kg, 5-min presession) dose-dependently reproduced the discriminative stimulus effects of pentobarbital in all monkeys tested. Intravenous administration of Co 8-7071 (1–10 mg/kg, 5-min presession) resulted in a dose-dependent increase to >80% pentobarbital-appropriate responding in two of three monkeys tested. Following intragastric infusions of Co 8-7071 (1.0–30 mg/kg), ≥80% pentobarbital-appropriate responding occurred in one out of three monkeys at 10 mg/kg when administered 60 min before the session. When administered 120 min before the session, however, 10–30 mg/kg Co 8-7071 reproduced the discriminative stimulus effects of pentobarbital in each of the three monkeys tested. Conclusions: These data demonstrate barbiturate-like abuse-related effects that differed between two pregnane steroids. Whereas pregnanolone functioned as a reinforcer, suggesting that this compound has abuse potential, Co 8-7071 did not, despite having pentobarbital-like discriminative effects. Received: 17 November 1998 / Final version: 22 January 1999     

Human interferon-α increases immobility in the forced swimming test in rats

Objectives: We examined the immobility of the forced swimming test induced in an animal model by human interferon (IFN), which has often been reported to induce depression in clinical use. Methods: In the present study, we examined the effects of human IFNs on results of the forced swimming test in rats. Results: Single intravenous (IV) administration of human IFN-α (6×10⁴ IU/kg), but not of human IFN-β or -γ, significantly increased immobility time in the forced swimming test in rats. Repeated administration of human IFN-α (6×10³ IU/kg) also significantly increased the immobility time. On the other hand, none of the rat IFNs (rat IFN-α, -β and -γ, 6×10⁴ IU/kg, IV) changed the immobility time. Neither human IFNs nor rat IFNs changed the locomotor activity of rats. Conclusions: These findings suggest that human IFN-α has a greater potential for inducing increase of the immobility in the rat forced swimming test than human IFN-β and -γ, and that the effect of human IFN-α might not be mediated through IFN-α/β receptors. Received: 2 June 1999 / Final version: 16 July 1999