Drug‐drug interaction and doping, part 2: An in vitro study on the effect of non‐prohibited drugs on the phase I metabolic profile of stanozolol

Abstract

The present study was designed to provide preliminary information on the potential impact of metabolic drug-drug interaction on the effectiveness of doping control strategies currently followed by the anti-doping laboratories to detect the intake of prohibited agents. In vitro assays based on the use of human liver microsomes and recombinant cytochrome P450 isoforms were developed and applied to characterize the phase I metabolic profile of the prohibited agent stanozolol, both in the absence and in the presence of substances (ketoconazole, itraconazole, miconazole, cimetidine, ranitidine, and nefazodone) not included in the World Anti-Doping Agency (WADA) list of prohibited substances and methods and frequently administered to athletes. The results show that the in vitro model utilized in this study is adequate to simulate the in vivo metabolism of stanozolol. Furthermore, our data showed that ketoconazole, itraconazole, miconazole, and nefazodone caused a marked modification in the production of the metabolic products (3'-hydroxy-stanozolol, 4β-hydroxy-stanozolol and 16β-hydroxy-stanozolol) normally selected by the anti-doping laboratories as target analytes to detect stanozolol intake. On the contrary, moderate variations were registered in the presence of cimetidine and no significant modifications were measured in the presence of ranitidine. This evidence confirms that the potential effect of drug-drug interactions is duly taken into account also in anti-doping analysis.

Drug-drug interactions and masking effects in sport doping: influence of miconazole administration on the urinary concentrations of endogenous anabolic steroids

“…In particular, miconazole is suspected to exert inhibitory activity on CYP450-dependent enzymes involved in steroid metabolic pathways (decrease in hydroxylated steroid formation) and on phase II metabolic conjugation with glucuronic acid. According to what in vitro demonstrated for other steroids [13,16], miconazole administration could be responsible for a decrease in Andro and Etio glucuronidation that reflects in the reduction of concentrations of these steroids in human urine.…”

“…T glucuronide (G), AndroG, EtioG] and of the T/E ratios [38,39]. This effect was ascribed to the inhibition of the 17a-hydroxylase and 17, 20-lyase activities of CYPC17 (conversion of pregnenolone to DHEA and progesterone to ASD) [40], but recent studies have also shown its inhibitory potential on steroid glucuronidation in vitro [13,16]. Figure 1 schematizes androgen biosynthetic and excretion metabolic pathways and underlines the sites of metabolic modulations by ketoconazole and finasteride [41].…”

“…In the previous studies we reported that, similarly to ketoconazole, the azolic antifungal miconazole is also able to inhibit UDP-glucuronosyltransferase and CYP enzymes during in vitro steroid metabolism [13,16], thus stressing the need to consider this kind of DDI in the evaluation of the analytical results in anti-doping analysis. Furthermore, other investigators reported the strong inhibitory effect of miconazole on CYP2C9 and 2C19 as well as on hepatic CYP3A4, 1A2, and 2D6 in vitro [42,43].…”

“…Previous in-vitro studies have shown that DDIs taking place at the level of the phase I and phase II biotransformation and conjugation processes can strongly decrease the formation of the selected target metabolite(s). In particular, it was demonstrated that the antifungal agents ketoconazole, miconazole, itraconazole, and the antidepressant nefazodone, may inhibit the production of hydroxylated and/or carboxylated metabolites of toremifene and stanozolol (respectively included in the classes S4, ''Hormone and Metabolic Modulators'', and S1 ''Anabolic Agents'' of the WADA List) [12,13]. On the other hand, for the detection of substances that can be (1) produced also in some physiological conditions (e.g., boldenone), or (2) present in the sample as metabolic products of not forbidden drugs (e.g., morphine from codeine), threshold values have been set by the WADA [14].…”

Human hepatoma cell lines on gas foaming templated alginate scaffolds for in vitro drug-drug interaction and metabolism studies

“…As far as drug-drug interaction research is concerned, induction of CYP3A4 activity with DXM may lead to a more rapid metabolism of the CY3A4 target drug, in this case testosterone, reducing its half-life and, therefore, the time frame during which the drug could be detected. Inhibition of CYP3A4 activity may lead instead to an increased half-life of testosterone and therefore to an enhancement of its biological effects (Mazzarino et al 2014). This is not only true for testosterone but also for several other substances.…”