Distribution, metabolism and excretion of a synthetic androgen 7α-methyl-19-nortestosterone, a potential male-contraceptive

Abstract

A synthetic androgen 7α-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in ‘androgen replacement therapy’ for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24 h prior to the injection of tritium-labeled MENT (³H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30 min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6 h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of ³H-MENT was investigated after subcutaneous injection of ³H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72 h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30 h.

Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of ³H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P₄₅₀ (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7α-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187–93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16α-hydroxylated MENT (a polar metabolite) by both rat and human hepatic microsomes; and [iii] 7α-methyl-19-norandrostenedione (a non-polar metabolite) by human hepatic microsomes, were obtained. By employing chemical inhibitors and specific anti-CYP antibodies, ³H-MENT was found to be metabolized specifically by rat CYP 2C11 and 3-hydroxysteroid dehydrogenase (3-HSD) enzymes whereas in humans it was accomplished by CYP 3A4, 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3-HSD enzymes.

Introduction

A synthetic androgen 7α-methyl-19-nortestosterone (MENT) (C₁₉H₂₈O₂) has several potential therapeutic benefits over testosterone (T). The specific advantages of MENT are: (i) it is 10 times more potent than testosterone [1]; (ii) its high potency allows administration of an optimal low daily dose via a sustained-release implant delivery system [2], [3]; (iii) it is resistant to 5α-reduction and therefore, unlike T, its action is not amplified in the prostate and seminal vesicles [1]; (iv) MENT is rapidly absorbed in the body, reaches peak levels by 1–2 h after single intramuscular administration and disappears from blood within 24 h [4]; (v) pharmacokinetic studies have shown that the metabolic clearance rate (MCR) of MENT is much faster than T. The faster MCR can be attributed, in part, to the finding that, in contrast to testosterone, MENT showed no binding to sex hormone binding globulin (SHBG) [5]. Considering these potential advantages of MENT, its beneficial effect on the prostate and its use in the sustained-release formulations such as silastic implants for long-term treatment was established in clinical studies in normal men [2], [3], [6]. Data obtained from these studies led to the conclusion that MENT has a great potential for long-term use as an ‘androgen replacement therapy’ in hypogonadal men and as a hormonal male-contraceptive in normal men.

It is essential for any new chemical entity (NCE) to have appropriate pharmacokinetic properties viz., absorption, distribution, metabolism, and excretion (ADME), in order to establish itself as a therapeutic agent. There is information available on ADME of testosterone, other androgens, and anabolic steroids [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. Some aspects of pharmacokinetics of MENT have been reported [5], [17], [18]. However, the distribution and excretion of MENT have not been studied. Therefore, in the present study, we have investigated the distribution and excretion of ³H-MENT in castrated rats.

Virtually, all androgens are hydrophobic steroids but hepatic metabolism renders them hydrophilic and inactive [19]. It is well established that the cytochrome P₄₅₀ (CYP) family of isozymes present in the microsomal fractions of liver is involved in the oxidative metabolism of structurally diverse xenobiotics, steroids, and carcinogens [20], [21], [22], [23], [24], [25], [26]. A number of recent studies have shown that CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5 are the major CYP isozymes involved in the xenobiotic biotransformation in human liver [23], [27], [28]. Multiple forms of CYP exist in mammalian hepatic microsomes. Levels in the liver and roles toward substrate oxidation by each of the CYP isoforms have been reported to vary [29]. It is also known that each CYP performs specific metabolic function [30] and catalyzes the conversion of testosterone and related steroids to its inactive metabolites [31], [32]. Enzymes that are actively involved in the androgen metabolism have been found to be 5α- and 5β-steroid reductases, 17β-HSDs, 3α- and 3β-HSD. Moreover, hepatic CYP isozymes have been reported to hydroxylate androgens at 6th, 7th, 15th or 16th position in the liver [33]. The role of human CYPs 2C9, 2C19, and 3A4 in the oxidation of testosterone have been established using human liver microsomes [34]. The in vitro metabolism of MENT has been reported by Agarwal and Monder [18], which led to the identification of three of the metabolites. But they have not studied the enzymes and CYP isozymes involved in the MENT metabolism. Therefore, in the present study enzymes involved in MENT metabolism have been identified using in vitro chemoinhibition (by employing chemical inhibitors) and immunoinhibition studies (by specific anti-CYP antibodies).