Discovery, Biosynthesis, and Structure Elucidation of Metabolites of a Doping Agent and a Direct Analogue, Tetrahydrogestrinone and Gestrinone, Using Human Hepatocytes

Abstract

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one, THG) is an anabolic androgenic steroid sold to athletes as an undetectable performance enhancer. Being an unapproved substance, no legitimate in vivo human excretion studies could be performed to identify urinary markers of this doping agent. In vitro systems were used as an alternative approach to study the human metabolism of THG and the gestrinone analogue, which is a marketed drug. Incubations of both compounds in the presence of human hepatocytes led to formation of oxidative and glucuroconjugated metabolites. Microgram quantities of the major in vitro metabolites were biosynthesized using human hepatocytes, characterized by HPLC/MS/MS, and their structures elucidated by NMR. Due to high structure similarity, both THG and gestrinone had an analogous in vitro metabolic pathway leading to successive addition of a hydroxyl and a beta-glucuronic acid at C-18. This in vitro metabolite of gestrinone was consistent with a previously reported major but unknown human urinary metabolite. The structure of another metabolite of THG was proposed to be a glucuroconjugate of an oxidative product with a hydroxyl group most likely at C-16epsilon. In vitro information reported therein could significantly impact the identification of new urinary markers of THG for doping control purposes.

Metabolism and excretion of anabolic steroids in doping control—New steroids and new insights

“…In an excretion study with gestrinone, two unidentified gestrinone metabolites were detected as well as the parent drug as glucuronide conjugates [148,149]. Based upon the mass spectrometric data from an in-vitro study, one metabolite was identified as 18-hydroxy-gestrinone [139]. In another study 2,16␤-dihydroxygestrinone was found as the major metabolite, as well as gestrinone and 6␣-hydroxygestrinone ( Fig.…”

“…The oral contraceptive gestrinone (13␤-ethyl-17--ethynyl-17-hydroxygona-4,9-11-trien-3-one), commonly used in the treatment of endomentriosis, is prohibited in sports and can be used in the synthesis of THG [18,135,139]. In an excretion study with gestrinone, two unidentified gestrinone metabolites were detected as well as the parent drug as glucuronide conjugates [148,149].…”

Schemes of metabolic patterns of anabolic androgenic steroids for the estimation of metabolites of designer steroids in human urine

“…AAS with conjugated double bonds, such as gestrinone [6,39] and tetrahydrogestrinone (THG) [6,40], do not undergo extensive biotransformation routes; their metabolic pathways include mainly hydroxylations at C-2, C-6, C-16 or even C-18 positions. In the case of trenbolone, metabolism process is restricted to C-17 epimerization [41].…”

“…hydroxy-13,14-dehydro-17,17-dimethyl-18-nor-5␣-androst-2-eno-(2,3-c)-pyrazole 4␣-hydroxy-stanozolol39 Stenbolone(2-methyl-5␣-androst-1-ene-17␤-ol-3-one) 2-methyl-5␣-androst-1-ene-16␣-ol-3,17-dione 2-methyl-5␣-androst-1-ene-16␤-ol-3,17-dione 2-methyl-5␣-androst-1-ene-3z,16z-diol-17-one 2-methyl-5␣-androst-1-ene-3␣-ol-17-one 2-methyl-5␣-androst-1-ene-16z,17␤-ol-3-one 18-hydroxy-2-methyl-5␣-androst-1-ene-3,17-dione40 1-Testosterone (17␤-hydroxy-androst-1-ene-3-one) 5␣-dihydrotestosterone 5␣-androst-1-ene-3␣,17␤-diol 5␣-androst-1-ene-3,17-dione 5␣-androst-1-ene-3␣-ol-17-one 1-epitestosterone…”

uPA+/+-SCID Mouse with Humanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids: Methandienone as a Case Study

“…Liver fractions (2 ), hepatocyte cell cultures (3)(4)(5), and primates (6,7 ) have been used as alternatives to human administration in studies of metabolism. These alternative models are not ideal, however.…”