Epilepsia 46(6): 819-827

Anticonvulsant Activity of Androsterone and Etiocholanolone

^{Epilepsy Research Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA;}

^{Faculty of Medicine and Surgery, A.O.U. G. Martino, University of Messina, Messina, Italy}

Corresponding author: Michael A. Rogawski, M.D., Ph.D., Epilepsy Research Section, Porter Neuroscience Research Center, NINDS, NIH, Building 35, Room 1C-1002, 35 Convent Drive MSC 3702, Bethesda, MD 20892-3702, Telephone: 301-496-8013, Fax: 775-249-7715, E-mail: vog.hin@ikswagor.leahcim

Summary

Purpose

Men with epilepsy often have sexual or reproductive abnormalities that are attributed to alterations in androgen levels, including subnormal free testosterone. Levels of the major metabolites of testosterone – androsterone (5α-androstan-3α-ol-17-one; 5α, 3α-A), a neurosteroid that acts as a positive allosteric modulator of GABA_A receptors, and its 5β-epimer etiocholanolone (5β-androstan-3α-ol-17-one; 5β, 3α-A) – may also be reduced in epilepsy. 5α 3α-A has been found in adult brain and both metabolites, which can also be derived from androstenedione, are present in substantial quantities in serum along with their glucuronide and sulfate conjugates. This study sought to determine whether these endogenous steroid metabolites can protect against seizures.

Methods

The anticonvulsant activity of 5α 3α-A and 5β, 3α-A was investigated in electrical and chemoconvulsant seizure models in mice. The steroids were also examined for activity against extracellularly-recorded epileptiform discharges in the CA3 region of the rat hippocampal slice induced by perfusion with 55 μM 4-aminopyridine (4-AP).

Results

Intraperitoneal injection of 5α, 3α-A protected mice in a dose-dependent fashion from seizures in the following models (ED₅₀, dose in mg/kg protecting 50% of animals): 6 Hz electrical stimulation (29.1), pentylenetetrazol (43.5), pilocarpine (105), 4-AP (215), and maximal electroshock (224). 5β, 3α-A was also active in the 6 Hz and pentylenetetrazol models, but was less potent (ED₅₀ values, 76.9 and 139 mg/kg, respectively), whereas epiandrosterone (5α,3β-A) was inactive (ED₅₀, ≤300 mg/kg). 5α, 3α-A (10–100 μM) also inhibited epileptiform discharges in a concentration-dependent fashion in the in vitro slice model, whereas 5β, 3α-A was active but of lower potency and 5α, 3β-A was inactive.

Conclusions

5α, 3α-A and 5β, 3α-A have anticonvulsant properties. Although of low potency, the steroids are present in high abundance and could represent endogenous modulators of seizure susceptibility.

Keywords: Androsterone, Etiocholanolone, Epiandrosterone, Pentylenetetrazol, Pilocarpine, 4-Aminopyridine, 6-Hz model, Seizure, Mouse

Summary

Male sex steroid hormones (“androgens”) were recognized in the early part of the last century (1). Androsterone (5α-androstan-3α-ol-17-one; 5α,3α-A), the first androgen to be identified, was isolated from urine in 1931 and chemically synthesized four years later at the same time as the first synthesis of testosterone (2). 5α,3α-A and its 5β-epimer etiocholanolone (5β,3α-A) are the major excreted metabolites of testosterone (3–5). The first step in testosterone metabolism is its irreversible reduction by 5α-reductase isoenzymes, which leads to the formation of the biologically potent androgen 5α-dihydrotestosterone (Fig. 1). Testosterone is also a good substrate for liver 5β-reductase (6), which synthesizes 5β-dihydrotestosterone. The reductions at the 5-position are followed by sequential 3α- and 17β-reductions resulting in the synthesis of 5α,3α-A and 5β,3α-A. The latter two reactions are catalyzed by 3α-hydroxysteroid dehydrogenase (3α-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD), respectively (Fig. 1). 5α,3α-A and 5β,3α-A can also be derived from the metabolism of androstenedione through 5α- and 5β-reduction, followed by the action of 3α-HSD. Substantial quantities of both compounds (~3–4 mg/24 h in young men) are excreted in the urine as 3-glucuronide and 3-sulfate conjugates (7–10).

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FIG. 1

Metabolic pathways for conversion of testosterone and androstenedione to androsterone (5α-androstan-3α-ol-17-one; 5α,3α-A) and etiocholanolone (5β-androstan-3α-ol-17-one; 5β,3α-A). 5α-Reductase (5α-R) and 5β-reductase (5β-R) catalyze the rate-limiting irreversible initial steps, which are followed by sequential reductions by 3α-hydroxysteroid dehydrogenase (3α-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD). 5α,3α-A and 5β,3α-A are conjugated by gucuronidation in the liver. 5α,3α-A has been identified as a substrate for human dehydroepiandrosterone (DHEA) sulfotransferase and some studies have indicated that 5α,3α-A sulfate may be more abundant in serum than the glucuronide (44,53).

5α,3α-A has an identical reduced A-ring as the neurosteroids allopregnanolone (5α,3α-P) and allotetrahydrodeoxycorticosterone (5α,3α-THDOC), which are potent positive allosteric modulators of GABA_A receptors (11,12). 5β,3α-A is analogous to pregnanolone (5β,3α-P) and tetrahydrodeoxycorticosterone (5β,3α-THDOC), which also have activity at GABA_A receptors, but are less potent (11,13). In fact, 5α,3α-A has been reported to interact with GABA_A receptors in vitro in a fashion similar to other neurosteroid positive GABA_A receptor modulators. Thus, 5α,3α-A enhances muscimol and flunitrazepam binding and inhibits t-butyl bicyclophosphorothionate binding in rat brain membranes, and also enhances muscimol-stimulated ^Cl^{flux in intact neurons (}14,15). Furthermore, in electrophysiological studies, 5α,3α-A potentiates GABA_A receptor chloride currents (15–19). Neurosteroids, including 5α,3α-P and 5α,3α-THDOC and their 5β-epimers, that act as similar positive GABA_A receptor modulators, have anticonvulsant properties in animal models (11,12,20–23) and recently we have demonstrated that they also protect against epileptiform activity in an in vitro brain slice system (24). Consequently, 5α,3α-A and 5β,3α-A could potentially have anticonvulsant properties although as far as we are aware this has only previously been demonstrated for 5α,3α-A in regard to ruthenium red convulsions in cats (25) and 3-mercaptoprionic acid seizures in Syrian hamsters (26).

There is accumulating evidence that neurosteroids with GABA_A receptor modulating activity serve as endogenous regulators of seizure susceptibility (27). Men with temporal lobe epilepsy often have impaired sexual function, including diminished testicular efficiency and loss of libido in association with reduced androgen levels (28,29). The deficiency of androgens is attributed to the effects of antiepileptic medications and also to the suppression of the hypothalamic-pituitary-gonadal axis by recurrent seizures (30–32). If androgen metabolites have anti-seizure properties, alterations in androgen levels caused by antiepileptic drugs or inadequately controlled seizures could be relevant to seizure control. In fact, there is evidence that long-term antiepileptic drug therapy is associated with markedly reduced urinary excretion of both 5α,3α-A and 5β,3α-A (3,33). In the present study we characterize for the first time the anticonvulsant profile of these steroids in several animal seizure models and also in an in vitro slice model previously shown to be sensitive to neurosteroids (24). For comparison, we examined the activity 5α,3β-A (epiandrosterone) which is predicted to have very weak activity at GABA_A receptors (11). Our results indicate that the 3α-epimers are effective anticonvulsants and the structure-activity comparisons indicate that this likely occurs through positive modulation of GABA_A receptors.

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