Intranasal Allopregnanolone Confers Rapid Seizure Protection: Evidence for Direct Nose-to-Brain Delivery.

Allopregnanolone, a positive modulator of GABAA receptors with antiseizure activity, has potential in the treatment of seizure emergencies. Instillation of allopregnanolone in 40% sulfobutylether-ß-cyclodextrin into the nose in mice rapidly elevated the seizure threshold in the timed intravenous pentylenetetrazol (ED50, 5.6 mg/kg), picrotoxin (ED50, 5.9 mg/kg), and bicuculline seizure tests. The effect peaked at 15 min, decayed over 1 h, and was still evident in some experiments at 6 h. Intranasal allopregnanolone also delayed the onset of seizures in the maximal PTZ test. At an allopregnanolone dose (16 mg/kg) that conferred comparable effects on seizure threshold as the benzodiazepines midazolam and diazepam (both at doses of 1 mg/kg), allopregnanolone caused minimal sedation or motor toxicity in the horizontal screen test whereas both benzodiazepines produced marked behavioral impairment. In addition, intranasal allopregnanolone failed to cause loss-of-righting reflex in most animals, but when the same dose was administered intramuscularly, all animals became impaired. Intranasal allopregnanolone (10 mg/kg) caused a rapid increase in brain allopregnanolone with a Tmax of ~5 min after initiation of the intranasal delivery. High levels of allopregnanolone were recovered in the olfactory bulb (Cmax, 16,000 ng/mg) whereas much lower levels (Cmax, 670 ng/mg) were present in the remainder of the brain. We conclude that the unique ability of intranasal allopregnanolone to protect against seizures without inducing behavioral adverse effects is due in part to direct nose-to-brain delivery, with preferential transport to brain regions relevant to seizures. Benzodiazepines are commonly administered intranasally for acute seizure therapy, including for the treatment of acute repetitive seizures, but are not transported from nose-to-brain. Intranasal allopregnanolone acts with greater speed, has less propensity for adverse effects, and has the ability to overcome benzodiazepine refractoriness. This is the first study demonstrating rapid functional central nervous system activity of a nose-to-brain-delivered steroid. Intranasal delivery circumvents the poor oral bioavailability of allopregnanolone providing a route of administration permitting its evaluation as a treatment for diverse neuropsychiatric indications.

Brexanolone: First Global Approval.

Brexanolone (ZULRESSO™) is an intravenously administered, small molecule, neuroactive steroid GABAA receptor positive allosteric modulator that was developed by Sage Therapeutics under license to the University of California for the treatment of postpartum depression (PPD). The formulation is a mixture of allopregnanolone, an endogenous inhibitory pregnane neurosteroid, and sulfobutylether-beta-cyclodextrin (a solubilizing agent). In mid-March 2019 brexanolone received its first global approval in the USA for the treatment of PPD in adult women. This article summarizes the milestones in the development of brexanolone leading to its first approval for the treatment of adult women with PPD.

Intramuscular allopregnanolone and ganaxolone in a mouse model of treatment-resistant status epilepticus.

Allopregnanolone (5α-pregnan-3α-ol-20-one) and its synthetic 3ß-methyl analog, ganaxolone, are positive allosteric modulators of synaptic and extrasynaptic γ-aminobutyric acid (GABA)A receptors that exhibit antiseizure activity in diverse animal seizure models, including models of status epilepticus (SE). The 2 neuroactive steroids are being investigated as treatments for SE, including as a treatment for SE induced by chemical threat agents. Intramuscular injection is the preferred route of administration in the prehospital treatment of SE. The objective of this study was to assess the efficacy of intramuscular allopregnanolone and ganaxolone in the treatment of SE induced by the chemical threat agent tetramethylenedisulfotetramine (TETS). The test agents were administered 40 minutes after the onset of SE when mice are refractory to treatment. Allopregnanolone and ganaxolone (each at 3 mg/kg) terminated SE in, respectively, 92% and 75% of animals, and prevented mortality in 85% and 50% of animals; the mean times to termination of behavioral seizures were, respectively, 172 ± 16 and 447 ± 52 seconds. In a separate series of experiments, mice were dosed with the neuroactive steroids by intramuscular injection, and plasma and brain levels were sampled at various time points following injection to estimate pharmacokinetic parameters. Plasma Cmax (maximum concentration) values for allopregnanolone and ganaxolone were 645 and 550 ng/mL, respectively. Brain exposure of both steroids was approximately 3-fold the plasma exposure. Two-compartment pharmacokinetic analysis revealed that the central compartment Vd (volume of distribution), CL (clearance), t½ (terminal half-life), and F (intramuscular bioavailability) values for allopregnanolone and ganaxolone were, respectively, 4.95 L/kg 12.88 L/kg/h,16 minutes, 97%, and 5.07 L/kg, 8.35 L/kg/h, 25 minutes, 95%. Allopregnanolone and ganaxolone are effective in the treatment of TETS-induced SE when administered by the intramuscular route. Allopregnanolone is more rapidly acting and modestly more effective, possibly because it has greater potency on GABAA receptors.

Neuroactive Steroids. 2. 3α-Hydroxy-3ß-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5ß-pregnan-20-one (SAGE-217): A Clinical Next Generation Neuroactive Steroid Positive Allosteric Modulator of the (γ-Aminobutyric Acid)A Receptor.

Certain classes of neuroactive steroids (NASs) are positive allosteric modulators (PAM) of synaptic and extrasynaptic GABAA receptors. Herein, we report new SAR insights in a series of 5ß-nor-19-pregnan-20-one analogues bearing substituted pyrazoles and triazoles at C-21, culminating in the discovery of 3α-hydroxy-3ß-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5ß-pregnan-20-one (SAGE-217, 3), a potent GABAA receptor modulator at both synaptic and extrasynaptic receptor subtypes, with excellent oral DMPK properties. Compound 3 has completed a phase 1 single ascending dose (SAD) and multiple ascending dose (MAD) clinical trial and is currently being studied in parallel phase 2 clinical trials for the treatment of postpartum depression (PPD), major depressive disorder (MDD), and essential tremor (ET).

Open-label, proof-of-concept study of brexanolone in the treatment of severe postpartum depression.

OBJECTIVE: Preclinical evidence indicates that rapid changes in levels of allopregnanolone, the predominant metabolite of progesterone, confer dramatic behavioral changes and may trigger postpartum depression (PPD) in some women. Considering the pathophysiology of PPD (i.e., triggered by reproductive steroids), the need for fast-acting, efficacious treatments and the negative consequences of untreated PPD, there is an increasing focus on developing PPD therapies. Brexanolone (USAN; formerly SAGE-547 Injection), a proprietary injectable allopregnanolone formulation, was evaluated as a treatment for severe PPD in a proof-of-concept, open-label study. METHODS: Four women with severe PPD, defined as a baseline 17-item Hamilton Rating Scale for Depression (HAMD) score of ≥20, received brexanolone, titrated to a dose reflecting third-trimester allopregnanolone levels. After a 36-hour maintenance infusion, tapering occurred over 12 hours. Primary outcomes were measures of safety. Secondary outcomes were assessments of efficacy, including HAMD. RESULTS: All enrolled patients completed the study. Fourteen adverse events were reported, of which none was severe. Starting at the first measure after infusion initiation and continuing through Hour 84, mean HAMD total scores were reduced to levels consistent with remission of symptoms. All other efficacy assessments showed similar improvements. CONCLUSIONS: Brexanolone was well tolerated and demonstrated activity in severe PPD. Larger, double-blind trials are needed for further evaluation.

Allopregnanolone preclinical acute pharmacokinetic and pharmacodynamic studies to predict tolerability and efficacy for Alzheimer's disease.

To develop allopregnanolone as a therapeutic for Alzheimer's disease, we investigated multiple formulations and routes of administration in translationally relevant animal models of both sexes. Subcutaneous, topical (transdermal and intranasal), intramuscular, and intravenous allopregnanolone were bolus-administered. Pharmacokinetic analyses of intravenous allopregnanolone in rabbit and mouse indicated that peak plasma and brain levels (3-fold brain/plasma ratios) at 5min were sufficient to activate neuroregenerative responses at sub-sedative doses. Slow-release subcutaneous suspension of allopregnanolone displayed 5-fold brain/plasma ratio at Cmax at 30min. At therapeutic doses by either subcutaneous or intravenous routes, allopregnanolone mouse plasma levels ranged between 34-51ng/ml by 30min, comparable to published endogenous human level in the third trimester of pregnancy. Exposure to subcutaneous, topical, intramuscular, and intravenous allopregnanolone, at safe and tolerable doses, increased hippocampal markers of neurogenesis including BrdU and PCNA in young 3xTgAD and aged wildtype mice. Intravenous allopregnanolone transiently and robustly phosphorylated CREB within 5min and increased levels of neuronal differentiation transcription factor NeuroD within 4h. Neurogenic efficacy was achieved with allopregnanolone brain exposure of 300-500hr*ng/g. Formulations were tested to determine the no observable adverse effect level (NOAEL) and maximally tolerated doses (MTD) in male and female rats by sedation behavior time course. Sex differences were apparent, males exhibited ≥40% more sedation time compared to females. Allopregnanolone formulated in sulfobutyl-ether-beta-cyclodextrin at optimized complexation ratio maximized allopregnanolone delivery and neurogenic efficacy. To establish the NOAEL and MTD for Allo-induced sedation using a once-per-week intravenous regenerative treatment regimen: In female rats the NOAEL was 0.5mg/kg and MTD 2mg/kg. The predicted MTD in human female is 0.37mg/kg. In male rats the NOAEL and MTD were less than those determined for female. Outcomes of these PK/PD studies predict a safe and efficacious dose range for initial clinical trials of allopregnanolone for Alzheimer's disease. These findings have translational relevance to multiple neurodegenerative conditions.

Neuroactive Steroids. 1. Positive Allosteric Modulators of the (γ-Aminobutyric Acid)A Receptor: Structure-Activity Relationships of Heterocyclic Substitution at C-21.

Neuroactive steroids (NASs) have been shown to impact central nervous system (CNS) function through positive allosteric modulation of the GABA(A) receptor (GABA(A)-R). Herein we report the effects on the activity and pharmacokinetic properties of a series of nor-19 pregnanolone analogues bearing a heterocyclic substituent at C-21. These efforts resulted in the identification of SGE-516, a balanced synaptic/extrasynaptic GABA(A) receptor modulator, and SGE-872, a selective extrasynaptic GABA(A) receptor modulator. Both molecules possess excellent druglike properties, making them advanced leads for oral delivery of GABA(A) receptor modulators.

Pharmacological profile of a 17ß-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 17ß-acetyl side chain with an isoxazole bioisostere. OBJECTIVES: UCI-50027 (3-[3α-hydroxy-3ß-methyl-5α-androstan-17ß-yl]-5-(hydroxymethyl)isoxazole) was designed as an orally active neuroactive steroid specifically targeted at the gamma-aminobutyric acid(A) receptor (GABAAR). METHODS: UCI-50027 was tested in vitro in Xenopus oocytes expressing human GABAARs 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 α1ß2γ2L, α2ß1γ2L, and α4ß3δ GABAARs. 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 GABAAR PAM that has an improved separation between anticonvulsant/anxiolytic and rotarod effects, potent activity as an anticonvulsant and anxiolytic when compared to ganaxolone.

The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids.

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50∼70 µM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈20 µM.

Role of the neurosteroid allopregnanolone in the hyperalgesic behavior induced by painful nerve injury in rats.

The neurosteroid allopregnanolone (AP) influences the excitability of the central nervous system by acting as a positive allosteric modulator of γ-aminobutyric acid type A (GABA(A)) receptors. Here, we investigated the role of AP and its therapeutic potential in rats that showed hyperalgesic behavior after undergoing spinal nerve ligation (SNL). AP levels measured in the spinal cord and brain of rats that underwent SNL were greater than the corresponding levels in control animals. More importantly, spinal AP levels in hyperalgesic rats were lower than those in the rats that did not develop hyperalgesia following SNL; in contrast, brain AP levels were comparable among these groups. No differences in serum AP levels were observed among the groups. In addition, intrathecal exogenous administration of AP showed the antihyperalgesic effects in hyperalgesic rats after SNL. These findings suggest that changes in spinal AP biosynthesis are involved in the pathogenesis of neuropathic pain following peripheral nerve injury, and pharmacological manipulation of this phenomenon may provide a potential therapeutic target for neuropathic pain.

Preclinical pharmacokinetics, dose proportionality, gender difference and protein binding study of 16-dehydropregnenolone, an antihyperlipidemic agent, in rats.

OBJECTIVES: This manuscript addresses key pharmacokinetic issues in support of the development of a potent candidate lipid-lowering drug molecule, 16-dehydropregnenolone (DHP). METHODS: Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay for simultaneous estimation of DHP and its metabolites, including 5-pregnene-3ß-ol-16, 17-epoxi-20-one (M(1) ) was validated in male and female Sprague-Dawley rat plasma and applied to different studies. Pharmacokinetic studies of DHP after intravenous and oral administration were carried out to assess any gender effect. Dose-proportionality after oral administration was assessed at three dose levels. Protein binding was estimated using the modified charcoal adsorption method. KEY FINDINGS: Rapid elimination of DHP from the systemic circulation resulted in a comparatively lesser systemic exposure in male compare to female rats. The area under the curve (AUC) after oral administration in males was significantly different to females. The large volume of distribution and low degree of protein binding suggest extensive distribution of DHP. An increase in the oral dose led to a disproportionate change in peak concentration (C(max) ) and AUC, indicating variable absorption. However, the dose-normalized AUC and C(max) at two dose levels were not found to be statistically different. CONCLUSIONS: The extent of conversion of DHP to M(1) was higher after oral administration in male rats but was insignificant in female rats. DHP showed low systemic oral bioavailability and exhibited dose-independent pharmacokinetics and gender differences.

Allopregnanolone, a GABAA receptor agonist, decreases gonadotropin levels in women. A preliminary study.

Animal studies suggest regulatory effects on the hypothalamic-pituitary-gonad axis by allopregnanolone, an endogenous gamma-aminobutyric acid A (GABA(A)) receptor agonist. Elevated levels of allopregnanolone in women with hypothalamic amenorrhea have been seen. Isoallopregnanolone is an isomer to allopregnanolone, but without GABA(A) receptor effects. The purpose of this study was to investigate effects of allopregnanolone and isoallopregnanolone on gonadotropin levels in healthy women of fertile age. Ten women were given allopregnanolone and five women isoallopregnanolone intravenously in follicular phase. Repeated blood samples were drawn during the test day. Main outcomes were changes in serum levels of follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol, and progesterone. Serum-FSH decreased between 5 and 105 min after the allopregnanolone injection (F(16,144)=2.18, p=0.008). Serum-LH was reduced between 5 and 35 min following the allopregnanolone injection (F(16,144)=2.63, p=0.001). Serum-oestradiol and -progesterone were not significantly changed after allopregnanolone injections. No effect on gonadotropin levels were seen after administration of isoallopregnanolone. Allopregnanolone reduces FSH and LH levels in women and the effect might be mediated via a specific GABA(A) receptor activation since isoallopregnanolone lacked this effect. Although the number of women was small, the results suggest a regulatory mechanism on the hypothalamic-pituitary-gonadal axis by allopregnanolon.

Kinetic analysis of voltage-dependent potentiation and block of the glycine alpha 3 receptor by a neuroactive steroid analogue.

We examined the actions of a carboxylated analogue of pregnanolone ((3alpha,5beta)-20-oxopregnane-3-carboxylic acid; 3alphaCOOH5betaP) on receptors composed of glycine receptor alpha3 subunits, expressed in Xenopus oocytes. This analogue both inhibits and potentiates this receptor; potentiation increases with more negative membrane potentials while block increases with less negative membrane potentials. We used a second analogue ((3alpha,5beta)-3-hydroxymethylpregnan-20-one; 3alphaCH(2)OH5betaP) to examine the mechanism for voltage-dependent potentiation. This analogue potentiates but does not block the glycine alpha3 receptor. Steady-state responses and current relaxations following voltage jumps support the idea that the voltage dependence of potentiation indirectly arises from a voltage dependence for channel activation by glycine, rather than an intrinsic voltage dependence for potentiation. Potentiation results from a slowing of the channel deactivation rate. In the absence of steroid, at a low [glycine] current relaxations after a voltage jump show two exponential components, with a weighted average time constant of approximately 425 ms (-50 mV, 22 degrees C). The rate for channel deactivation increases at more negative potentials (e-fold per 170 mV) whereas activation decreases (e-fold per 230 mV). The probability a channel is active at a high [glycine] is greater than 0.9. The addition of 10 microM 3alphaCH(2)OH5betaP decreases the deactivation rate by 6.3-fold (-50 mV). Voltage-dependent block by 3alphaCOOH5betaP is consistent with simple open-channel block, with voltage dependence reflecting interactions of the charge on the analogue with the electrical field. Block and unblock have equal but opposite dependence on membrane potential, and the charge on 3alphaCOOH5betaP senses approximately 70% of the membrane field at the blocking site. The apparent forward rate for block, however, is very slow (2 x 10(5) m(-1) s(-1)).

Studies of pharmacokinetic and pharmacodynamic properties of isoallopregnanolone in healthy women.

RATIONALE: The pharmacokinetics and behavioral effects of isoallopregnanolone (3beta-hydoxy-5alpha-pregnan-20-one) in women are not known. OBJECTIVES: Allopregnanolone (3alpha-hydoxy-5alpha-pregnan-20-one) is a well-known neurosteroid, acting via the GABA(A) receptor in the human brain. The naturally occurring progesterone metabolite isoallopregnanolone is the 3beta-stereoisomer of allopregnanolone. Prior studies have concluded that isoallopregnanolone has no effect on the GABA(A) receptor. However, an antagonistic effect of isoallopregnanolone to allopregnanolone on the GABA(A) receptor has been shown in animal and in vitro studies. The purpose of this study was to evaluate the pharmacokinetics and behavioral effects of isoallopregnanolone in humans. MATERIALS AND METHODS: Six healthy women were given three increasing doses of isoallopregnanolone intravenously in the follicular phase. Repeated blood samples for analyses of isoallopregnanolone and allopregnanolone concentrations were drawn. Saccadic eye movement variables, self-rated sedation, and mood rating scales were used during the test day. A Likert scale for prospective symptoms was used to measure daily fluctuations during the ongoing menstrual cycle. RESULTS: Exogenously administered isoallopregnanolone produced a dose-dependent increase in the serum concentration of isoallopregnanolone. In parallel, there was also a rise in the allopregnanolone concentration. There was a decrease in saccadic eye movement variables, but no effect was found on self-rated sedation or mood and no changes were seen in prospective symptoms during the menstrual cycle. CONCLUSIONS: After administration of isoallopregnanolone at a cumulative dose of 0.20 mg/kg, no adverse effects were observed. There is a metabolism of isoallopregnanolone to allopregnanolone, most likely explaining the effects on the saccadic eye movements.

Persistence of tolerance to the anaesthetic effect of allopregnanolone in male rats.

Both acute and chronic tolerance can develop to allopregnanolone-a gamma-aminobutyric acid (GABA)-modulatory progesterone metabolite. Here we investigated if acute tolerance to allopregnanolone persisted for 1 or 2 days after the induction and thus could be the initial part of chronic tolerance. Male rats were anaesthetised with allopregnanolone (i.v) to the deep anaesthesia level of the silent second (SS), which is an EEG burst suppression of 1 s or more. They were divided into four groups: SS1-anaesthesia to the first silent second; LAn (long anaesthesia)-90 min anaesthesia at the SS level; SS2;D1-90 min anaesthesia and SS induction 1 day later; SS2;D2-90 min anaesthesia and SS induction 2 days later. Allopregnanolone concentrations in tissue and serum were analysed. Levels of the GABAA receptor alpha2, alpha4, gamma2(S+L) and delta subunits mRNAs were analysed by in situ hybridisation. Acute tolerance was induced during the 90 min anaesthesia. Tolerance persisted for 1 day, since the dose of allopregnanolone needed to induce a new SS anaesthesia was increased after 1 day. The level of alpha4 subunit mRNA expression in the ventral posteriomedial nucleus of thalamus was negatively related to the tolerance parameters, the SS dose of allopregnanolone and DeltaSS (SS dose difference between days). Allopregnanolone threshold anaesthesia lasting 90 min induces acute tolerance that persisted for at least 1 day, which could be used as the start of a chronic tolerance. The alpha4 subunit may be involved in allopregnanolone caused effects in the brain.

Etomidate and other non-barbiturates.

It is today generally accepted that anesthetics act by binding directly to sensitive target proteins. For certain intravenous anesthetics, such as propofol, barbiturates, and etomidate, the major target for anesthetic effect has been identified as the gamma-aminobutyric acid type A (GABA(A)) receptor, with particular subunits playing a crucial role. Etomidate, an intravenous imidazole general anesthetic, is thought to produce anesthesia by modulating or activating ionotropic Cl(-)-permeable GABA(A) receptors. For the less potent steroid anesthetic agents the picture is less clear, although a relatively small number of targets have been identified as being the most likely candidates. In this review, we summarize the most relevant clinical and experimental pharmacological properties of these intravenous anesthetics, the molecular targets mediating other endpoints of the anesthetic state in vivo, and the work that led to the identification of the GABA(A) receptor as the key target for etomidate and aminosteroids.

A novel chemical delivery system comprising an ocular sustained release formulation of a 3alpha, 17alpha, 21-trihydroxy-5beta-pregnan-20-one-BIS-5-fluorouracil [correction of flourouracil] codrug.

Directly compressed sustained release pellets were prepared from material consisting of a molecule of 3alpha, 17alpha, 21-trihydroxy-5beta-pregnan-20-one (trihydroxy steroid, THS) covalently linked via carbonate moieties to two molecules of 5-flourouracil (5FU) to form a novel THS-BIS-5FU codrug for the treatment of angiogenesis. Dissolution and drug release was tested in vitro in 0.1M phosphate buffer (pH 7.4), human serum, and vitreous humor. The results suggest that neat THS-BIS-5FU codrug pellets are useful for sustained release ocular delivery of the parent compounds, and that the unique physicochemical properties of the codrug allow slow dissolution and rapid release of the two parent drugs. This codrug formulation is regarded as a "chemical delivery" system that involves dissolution of the codrug as the rate-limiting step followed by rapid hydrolysis of the carbonate ester linkages to release the parent drugs via sustained delivery.

Oral progesterone decreases saccadic eye velocity and increases sedation in women.

The aim of this study was to investigate the neurophysiological and behavioural effects of a single dose of progesterone in women. Allopregnanolone is a metabolite of progesterone and a potent positive modulator of the GABA(A) receptor and produces sedative and anxiolytic effects. This study was designed to examine the effect of oral progesterone and the metabolite allopregnanolone in women. Women (n=15) in their follicular phase received oral progesterone (400mg) or placebo. Dependent measures included plasma levels of progesterone and allopregnanolone, saccadic eye velocity (SEV), subjective ratings (visual analogue scales), and reaction time. Administration of progesterone decreased SEV and increased sedation. This effect is probably due to enhanced GABA activity.

GABA(A) receptor changes in acute allopregnanolone tolerance.

To study acute tolerance, rats were anesthetized with interrupted i.v. allopregnanolone infusions where the "silent second" in the electroencephalogram (EEG) was the target. Animals were killed either directly at the first silent second or at the silent second level after 30 or 90 min of anaesthesia. Acute tolerance was demonstrated at 90 min of anaesthesia as earlier shown. In situ hybridization showed a decreased expression of the gamma-aminobutyric acid(A) (GABA(A)) receptor subunit alpha4mRNA amount in the thalamus ventral-posteriomedial nucleus of the tolerant rats. A parallel change in the abundance of the alpha4 subunit was detected with immunohistochemistry. The increase in maintenance dose rate (MDR) was significantly negatively correlated with the alpha4mRNA in the thalamus ventral-posteriomedial nucleus, and positively correlated with alpha2mRNA in different hippocampal subregions. There was also a positive relationship between the alpha1mRNA amounts in the different hippocampal subregions, with significant differences between groups. These changes in GABA(A) receptor subunits mRNA expression and protein (alpha4) might be of importance for the development of acute tolerance to allopregnanolone.