Metabolism of "new" anabolic steroids : development of in vitro methodology in metabolite production and analytical techniques

The aim of this on-going WADA-funded project is to develop flexible in vitro procedure to study and predict the metabolic patterns of new AAS with respect to most prominent target compounds for doping control purposes. In vitro synthesis model was applied to a group of selected compounds, in order to examine its applicability to serve as a model to predict metabolic pathways of "new" AAS. In the pilot experiments with methyltestosterone it became evident that the use of combined fraction (both microsomes and S9) is advantageous in the reaction mixture to ensure a maximal enzyme activity. Furthermore, in the experiments with methandienone it could be concluded that the drawback of the method is the lack of co-substrates (PAPS) responsible for the sulphatation process, which is apparently the limiting step in the formation of epimeric metabolites that are formed via sulphate-conjugated transition state. Until now the developed in vitro metabolic reaction model has been applied for gestrinone, dihydrogestrinone, tetrahydrogestrinone, trenbolone and propyltrenbolone. The main observed metabolic pathway was hydroxylation, which all the selected substrates underwent, gestrinone and propyltrenbolone resulting in the formation of 4 and 3 metabolites, respectively. Phase-I reaction of dihydrogestrinone was only scarce, but exceptional, as the only generated metabolite was hydroxylation of hydrogenated compound. In general, this model has proven applicable for the purpose, as every compound tested has undergone phase-I and phase-II reactions at least to some extent. The results have been coherent also to those reported earlier in the literature. In our earlier studies glucuronidation has been successful using the chemically synthesised phase-I metabolites as starting material, but now these both stages of metabolic reactions are combined in one experiment. Aus dem Text (geändert)