A Rationale for Allopregnanolone Treatment of Alcohol Use Disorders: Basic and Clinical Studies.

For many years, research from around the world has suggested that the neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone or 3α,5α-THP) may have therapeutic potential for treatment of various symptoms of alcohol use disorders (AUDs). In this critical review, we systematically address all the evidence that supports such a suggestion, delineate the etiologies of AUDs that are addressed by treatment with allopregnanolone or its precursor pregnenolone, and the rationale for treatment of various components of the disease based on basic science and clinical evidence. This review presents a theoretical framework for understanding how endogenous steroids that regulate the effects of stress, alcohol, and the innate immune system could play a key role in both the prevention and the treatment of AUDs. We further discuss cautions and limitations of allopregnanolone or pregnenolone therapy with suggestions regarding the management of risk and the potential for helping millions who suffer from AUDs.

Sex and COVID-19: A Protective Role for Reproductive Steroids.

Evidence shows coronavirus disease 2019 (COVID-19)-induced symptom severity and mortality is more frequent in men than in women, suggesting sex steroids may play a protective role. Female reproductive steroids, estrogen and progesterone, and its metabolite allopregnanolone, are anti-inflammatory, reshape competence of immune cells, stimulate antibody production, and promote proliferation and repair of respiratory epithelial cells, suggesting they may protect against COVID-19 symptoms.

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

RATIONALE: The endogenous GABAergic neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP, allopregnanolone) has been proposed to contribute to ethanol actions. Humans synthesize 3alpha,5alpha-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 3alpha,5alpha-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, 3alpha,5alpha-THP, estradiol, and cortisol) were measured in plasma samples by radioimmunoassay. RESULTS: Ethanol significantly increased plasma levels of progesterone, but not 3alpha,5alpha-THP-like immunoreactivity, in women in the luteal phase. Ethanol had no effect on progesterone or 3alpha,5alpha-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: 3alpha,5alpha-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 5beta-reduced GABAergic steroids, and levels of these steroids may be altered in plasma or brain.

Neurosteroids as endogenous inhibitors of neuronal cell apoptosis in aging.

The neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester DHEAS, and allopregnanolone (Allo) are produced in the adrenals and the brain. Their production rate and levels in serum, brain, and adrenals decrease gradually with advancing age. The decline of their levels was associated with age-related neuronal dysfunction and degeneration, most probably because these steroids protect central nervous system (CNS) neurons against noxious agents. Indeed, DHEA(S) protects rat hippocampal neurons against NMDA-induced excitotoxicity, whereas Allo ameliorates NMDA-induced excitotoxicity in human neurons. These steroids exert also a protective role on the sympathetic nervous system. Indeed, DHEA, DHEAS, and Allo protect chromaffin cells and the sympathoadrenal PC12 cells (an established model for the study of neuronal cell apoptosis and survival) against serum deprivation-induced apoptosis. Their effects are time- and dose-dependent with EC(50) 1.8, 1.1, and 1.5 nM, respectively. The prosurvival effect of DHEA(S) appears to be NMDA-, GABA(A)- sigma1-, or estrogen receptor-independent, and is mediated by G-protein-coupled-specific membrane binding sites. It involves the antiapoptotic Bcl-2 proteins, and the activation of prosurvival transcription factors CREB and NF-kappaB, upstream effectors of the antiapoptotic Bcl-2 protein expression, as well as prosurvival kinase PKCalpha/beta, a posttranslational activator of Bcl-2. Furthermore, they directly stimulate biosynthesis and release of neuroprotective catecholamines, exerting a direct transcriptional effect on tyrosine hydroxylase, and regulating actin depolymerization and submembrane actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. These findings suggest that neurosteroids may act as endogenous neuroprotective factors. The decline of neurosteroid levels during aging may leave the brain unprotected against neurotoxic challenges.

Evidence for a role for the neurosteroid allopregnanolone in the modulation of reproductive function in female rats.

The present study investigated the effect of allopregnanolone (5 alpha-pregnan-3 alpha-ol-20-one) or of passive immunoneutralization of brain allopregnanolone, the most potent steroid produced by neurons, on ovulation rate and sexual behavior in female rats. Allopregnanolone was injected intracerebroventricularly in rats on diestrus and proestrus and tests were done on estrus. The intracerebroventricular injection of allopregnanolone significantly decreased the number of oocytes collected on estrus (p < 0.01). To support a physiological involvement, antiserum to allopregnanolone was injected centrally to block the activity of the endogenous neurosteroid. When administered on diestrus and proestrus or only on proestrus, the antiserum was shown to be correlated with a significant increase (p < 0.01) in oocytes retrieved on estrus. In female rats treated with antiserum to allopregnanolone, the lordosis intensity was augmented significantly as compared to controls. Finally, the possible changes of medial basal hypothalamus concentration of allopregnanolone throughout the estrous cycle and at the time of ovulation were investigated. Hypothalamic extracts were eluted on high-pressure liquid chromatography and allopregnanolone concentration was measured by radioimmunoassay. Brain cortex was used as control tissue. Hypothalamic allopregnanolone concentration on proestrus morning and afternoon was found to be significantly lower than in the remaining phases of the estrous cycle (p < 0.01), while no significant changes were observed in brain cortex concentration of allopregnanolone. The present results suggest that hypothalamic allopregnanolone may be involved in the mechanism of ovulation, affecting hormonal and behavioral events.

Determination of progesterone and some of its neuroactive ring A-reduced metabolites in human serum.

A method for the separation and assay of some ring A-reduced metabolites of progesterone (pregnanediones and pregnanolones) is described. Serum was extracted with an organic solvent, and the extract chromatographed using high performance liquid chromatography (HPLC). A total of 50 fractions was collected for each sample and split using a stream splitter so that 30% was collected in counting vials for recovery while 70% was collected in test tubes which were assayed by radioimmunoassay. An antiserum raised in our laboratory to progesterone-3-CMO-BSA cross-reacted with five of these compounds (5alpha- and 5beta-dihydroprogesterone, 3alpha- and 3beta-5alpha-tetrahydroprogesterone, and 3beta, 5beta-tetrahydroprogesterone). Since pregnenolone eluted with 5alpha, 3beta-tetrahydroprogesterone, pregnenolone was assayed separately and its effect subtracted. Using this method it was shown that picogram to nanogram/ml amounts of these metabolites are present in all human sera. Levels in men were comparable to those of women in the follicular phase of the menstrual cycle. 5alpha-Dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone rose substantially in the luteal phase of the menstrual cycle and all rose considerably during pregnancy.

Prediction of estrus cyclicity in Asian elephants (Elephas maximus) through estimation of fecal progesterone metabolite: development of an enzyme-linked immuno-sorbent assay.

Asian elephants (Elephas maximus), prominent "flagship species", are listed under the category of endangered species (EN - A2c, ver. 3.1; IUCN Red List 2009) and there is a need for their conservation. This requires understanding demographic and reproductive dynamics of the species. Monitoring reproductive status of any species is traditionally being carried out through invasive blood sampling and this is restrictive for large animals such as wild or semi-captive elephants due to legal, ethical, and practical reasons. Hence, there is a need for a non-invasive technique to assess reproductive cyclicity profiles of elephants, which will help in the species' conservation strategies. In this study, we developed an indirect competitive enzyme linked immuno-sorbent assay (ELISA) to estimate the concentration of one of the progesterone-metabolites i.e., allopregnanolone (5 alpha-P-3OH) in fecal samples of Asian elephants. We validated the assay which had a sensitivity of 0.25 microM at 90% binding with an EC(50) value of 1.37 microM. Using female elephants, kept under semi-captive conditions in the forest camps of Mudumalai Wildlife Sanctuary, Tamil Nadu and Bandipur National Park, Karnataka, India, we measured fecal progesterone-metabolite (5 alpha-P-3OH) concentrations in six animals and showed their clear correlation with those of serum progesterone, measured by a standard radio-immuno assay. Statistical analyses using a Linear Mixed Effect model showed a positive correlation (P<0.1) between the profiles of fecal 5 alpha-P-3OH (range: 0.5-10 microg/g) and serum progesterone (range: 0.1-1.8 ng/mL). Therefore, our studies show, for the first time, that the fecal progesterone-metabolite assay could be exploited to predict estrus cyclicity and to potentially assess the reproductive status of captive and free-ranging female Asian elephants, thereby helping to plan their breeding strategy.

The relationship of allopregnanolone immunoreactivity and HPA-axis measures to experimental pain sensitivity: Evidence for ethnic differences.

In animal models, allopregnanolone (ALLO) negatively modulates the hypothalamic-pituitary-adrenal (HPA) axis and has been shown to exert analgesic effects. The purpose of this study was to assess the relationship between plasma ALLO immunoreactivity (ALLO-ir), HPA-axis measures, and pain sensitivity in humans. Forty-five African Americans (21 men, 24 women) and 39 non-Hispanic Whites (20 men, 19 women) were tested for pain sensitivity to tourniquet ischemia, thermal heat, and cold pressor tests. Plasma ALLO-ir, cortisol, and beta-endorphin concentrations were taken following an extended rest period. Lower concentrations of ALLO-ir were associated with increased pain tolerance to all three pain tests and increased pain threshold to the thermal heat pain task in the non-Hispanic Whites only (rs=-.35 to -.49, ps<.05). Also, only in the non-Hispanic Whites was cortisol associated with thermal heat tolerance (r=+.39, p<.05) and threshold (r=+.50, p<.01) and cold pressor tolerance (r=+.32, p<.05), and were beta-endorphin concentrations associated with cold pressor tolerance (r=+.33, p<.05). Mediational analyses revealed that higher cortisol levels mediated the relationship between lower ALLO-ir and increased thermal heat pain threshold in the non-Hispanic Whites only. These results suggest that lower ALLO-ir concentrations are associated with decreased pain sensitivity in humans, especially in non-Hispanic Whites, and that this relationship may be mediated by HPA-axis function.

Evidence for a role of neurosteroids in modulation of diurnal changes and acute stress-induced corticosterone secretion in rats.

The neurosteroid allopregnanolone has been shown to be a potent ligand of gamma-aminobutyric acid (GABA)-A receptors and enhances its receptor-mediated inhibitory events. Since central GABA plays a major inhibitory role, via GABA-A receptors, in hypothalamic-pituitary-adrenal (HPA) function in rats, the present study has evaluated the effect of passive immunoneutralization of allopregnanolone on diurnal changes in corticosterone secretion and acute stress-induced corticosterone secretion in rats. In the first protocol, four groups of male rats (prepubertal, fertile, castrated adult and aged) and three groups of female rats (prepubertal, fertile at different phases of the estrous cycle and aged) were studied. Rats were injected intracerebroventricularly (i.c.v.) with 10 microliters anti-allopregnanolone serum or 10 microliters normal rabbit serum (control) 24 h before exposure to an acute cold swimming stress, and sacrificed either before stress or after 5 min stress. In the second protocol, fertile male or female rats at diestrus II were injected i.c.v. with anti-allopregnanolone serum or normal rabbit serum and sacrificed on the following day at 10.00 or 18.00. Truncal blood samples were collected for measuring plasma corticosterone. Our results showed that there was no significant difference in basal plasma corticosterone levels between antiserum-treated and control rats of both sexes. However, in male rats, central injection of antiserum to allopregnanolone significantly potentiated plasma corticosterone response to stress in prepubertal and adult fertile rats as well as in castrated rats. Likewise, in female rats, the stress response of plasma corticosterone was enhanced by passive immunoneutralization of allopregnanolone in prepubertal and fertile rats throughout the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Compounds in the urinary pH 1 extract reacting with corticosteroid and tetrahydrocorticosteroid antisera. Separation of a nonpolar substance with affinity to a tetrahydroaldosterone antiserum.

In human urinary pH 1 extracts prepared for the aldosterone-18-glucuronide estimation, several other substances are present, crossreacting not only with aldosterone antisera, but also with various corticosteroid and tetrahydrocorticosteroid antisera. Aldosterone was measured before and after chromatographic purification. Further characterization of the non-aldosterone immunoreactive material was made by immunological analysis of paper chromatogram eluats. Pregnancy, and administration of ACTH, dexamethasone, and metopirone led to a change of excretion in the antigenic equivalents. A method for the separation of the antigenic material is described. For structural elucidation the gaschromatography-mass spectrometry (GC-MS) method was applied.

Reproductive steroids during maturation in a primitive teleost, the Pacific herring (Clupea harengus pallasi).

Concentrations of reproductive steroids were measured in the plasma of captive Pacific herring, Clupea harengus pallasi, (1) prior to ovulation and milt production, (2) during the periovulatory and newly milt-producing period, (3) during a "ripe" holding period after ovulation and during milt production, and (4) before and after spawning. 17alpha,20beta-Dihydroxyprogesterone (17,20beta-P), despite being present only in low concentrations in the unconjugated (free) form (<10 ng/ml), is likely to be the maturation inducing steroid in females and was associated with the initiation of milt production in males since its levels are elevated coincidentally with these events. Glucuronated 17,20beta-P, free 17alpha-hydroxyprogesterone (17-P), and free and glucuronated 3alpha, 17alpha-dihydroxy-5beta-pregnan-20-one (3alpha,17-P-5beta) were present in high concentrations (140-250 ng/ml) in periovulatory females and newly milt-producing males. This steroid pattern suggests that the low levels of 17,20beta-P are due to glucuronation and competitive conversion of its precursor, 17-P, to free and glucuronated 3alpha, 17-P-5beta. Glucuronated testosterone was the principal steroid in preovulatory and premilt-producing fish (200-350 ng/ml), coincident with similar levels of glucuronated 11-ketotestosterone in males. After ovulation females did not spawn synchronously until 2 months later, which may be partially due to reduced environmental cues in the captive situation, while male fish released milt sporadically throughout the ripe holding period. Steroidal indicators of readiness to spawn in females or males were not detected. Rather, levels of all steroids gradually decreased in ripe holding fish (<30 ng/ml) to reach even lower levels (<1 ng/ml) after spawning. We suggest that "runniness" of gametes is a distinctive characteristic of females that are ready to spawn, but that this may result from relaxation of sphincter muscles rather than being an additional maturational step.