title: Characterization of the biotransformation pathways of clomiphene, tamoxifen and toremifene as assessed by LC-MS/(MS) following in vitro and excretion studies.
abstract: The use of selective oestrogen receptor modulators has been prohibited since 2005 by the World Anti-Doping Agency regulations. As they are extensively cleared by hepatic and intestinal metabolism via oxidative and conjugating enzymes, a complete investigation of their biotransformation pathways and kinetics of excretion is essential for the anti-doping laboratories to select the right marker(s) of misuse. This work was designed to characterize the chemical reactions and the metabolizing enzymes involved in the metabolic routes of clomiphene, tamoxifen and toremifene. To determine the biotransformation pathways of the substrates under investigation, urine samples were collected from six subjects (three females and three males) after oral administration of 50 mg of clomiphene citrate or 40 mg of tamoxifen or 60 mg of toremifene, whereas the metabolizing enzymes were characterized in vitro, using expressed cytochrome P450s and uridine diphosphoglucuronosyltransferases. The separation, identification and determination of the compounds formed in the in vivo and in vitro experiments were carried out by liquid chromatography coupled with mass spectrometry techniques using different acquisition modes. Clomiphene, tamoxifen and toremifene were biotransformed to 22, 23 and 18 metabolites respectively, these phase I reactions being catalyzed mainly by CYP3A4 and CYP2D6 isoforms and, to a lesser degree, by CYP3A5, CYP2B6, CYP2C9, CYP2C19 isoforms. The phase I metabolic reactions include hydroxylation in different positions, N-oxidation, dehalogenation, carboxylation, hydrogenation, methoxylation, N-dealkylation and combinations of them. In turn, most of the phase I metabolites underwent conjugation reaction to form the corresponding glucuro-conjugated mainly by UGT1A1, UGT1A3, UGT1A4, UGT2B7, UGT2B15 and UGT2B17 isoenzymes.
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197 Rome, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Mazzarino
authors.forename: Mariangela
authors.initials: M
authors.lastname: Biava
authors.forename: Xavier
authors.initials: X
authors.lastname: de la Torre
authors.forename: Ilaria
authors.initials: I
authors.lastname: Fiacco
authors.forename: Francesco
authors.initials: F
authors.lastname: Botrè
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001711:Biotransformation; D002853:Chromatography, Liquid / Q000379:methods*; D002996:Clomiphene / Q000378:metabolism / Q000493:pharmacokinetics* / Q000652:urine; D065702:Cytochrome P-450 CYP2B6; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D003640:Dealkylation; D004300:Doping in Sports; D005260:Female; D014453:Glucuronosyltransferase / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D013629:Tamoxifen / Q000378:metabolism / Q000493:pharmacokinetics* / Q000652:urine; D053719:Tandem Mass Spectrometry / Q000379:methods*; D017312:Toremifene / Q000378:metabolism / Q000493:pharmacokinetics* / Q000652:urine; D000099148:UGT1A1 Enzyme
chemical_list: D001189:Aryl Hydrocarbon Hydroxylases; D002996:Clomiphene; D065702:Cytochrome P-450 CYP2B6; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D014453:Glucuronosyltransferase; D010089:Oxidoreductases, N-Demethylating; D013629:Tamoxifen; D017312:Toremifene; D000099148:UGT1A1 Enzyme; C450260:CYP2C9 protein, human; C585599:CYP2B6 protein, human; C045793:CYP2C19 protein, human; C510164:CYP3A5 protein, human; C510163:CYP3A4 protein, human