Prediction of Drug-Drug-Gene Interaction Scenarios of (E)-Clomiphene and Its Metabolites Using Physiologically Based Pharmacokinetic Modeling.

Clomiphene, a selective estrogen receptor modulator (SERM), has been used for the treatment of anovulation for more than 50 years. However, since (E)-clomiphene ((E)-Clom) and its metabolites are eliminated primarily via Cytochrome P450 (CYP) 2D6 and CYP3A4, exposure can be affected by CYP2D6 polymorphisms and concomitant use with CYP inhibitors. Thus, clomiphene therapy may be susceptible to drug-gene interactions (DGIs), drug-drug interactions (DDIs) and drug-drug-gene interactions (DDGIs). Physiologically based pharmacokinetic (PBPK) modeling is a tool to quantify such DGI and DD(G)I scenarios. This study aimed to develop a whole-body PBPK model of (E)-Clom including three important metabolites to describe and predict DGI and DD(G)I effects. Model performance was evaluated both graphically and by calculating quantitative measures. Here, 90% of predicted Cmax and 80% of AUClast values were within two-fold of the corresponding observed value for DGIs and DD(G)Is with clarithromycin and paroxetine. The model also revealed quantitative contributions of different CYP enzymes to the involved metabolic pathways of (E)-Clom and its metabolites. The developed PBPK model can be employed to assess the exposure of (E)-Clom and its active metabolites in as-yet unexplored DD(G)I scenarios in future studies.

Stereoselective quantification of phase 1 and 2 metabolites of clomiphene in human plasma and urine.

Clomiphene citrate is first line therapy of female infertility but is also frequently abused by athletes. Human biotransformation of clomiphene results in numerous phase 1 and phase 2 metabolites. The involvement of the polymorphic cytochrome P450 2D6 leads to a high inter-individual variability. To comprehensively investigate clomiphene metabolism in vivo we established a highly sensitive and specific UPLC-MS/MS method for the stereoselective quantification of clomiphene and its phase 1 and phase 2 metabolites in plasma and urine. Reference compounds and stable isotope labelled internal standards were synthesized in-house. High-throughput sample preparation was done by protein precipitation. Analytes were separated by UPLC on a C18 column (1.8 µm, 2.1 * 100 mm) using a gradient of 0.1% formic acid in acetonitrile in 0.1% aqueous formic acid and detected by positive ESI-MS/MS in MRM mode. The lower limit of quantification was below 1 nM for all analytes. The method was validated according to recent guidelines. However, due to absorption effects during sampling the quantification of metabolites in urine was limited to phase 2 metabolites. The method was successfully applied to determine the pharmacokinetic of (E)- and (Z)-clomiphene and 14 metabolites following a single dose of 100 mg clomiphene citrate in 3 healthy subjects and proofed to be an essential tool to comprehensively investigate the human biotransformation of clomiphene.

The formation of estrogen-like tamoxifen metabolites and their influence on enzyme activity and gene expression of ADME genes.

Tamoxifen, a standard therapy for breast cancer, is metabolized to compounds with anti-estrogenic as well as estrogen-like action at the estrogen receptor. Little is known about the formation of estrogen-like metabolites and their biological impact. Thus, we characterized the estrogen-like metabolites tamoxifen bisphenol and metabolite E for their metabolic pathway and their influence on cytochrome P450 activity and ADME gene expression. The formation of tamoxifen bisphenol and metabolite E was studied in human liver microsomes and Supersomes™. Cellular metabolism and impact on CYP enzymes was analyzed in upcyte® hepatocytes. The influence of 5 µM of tamoxifen, anti-estrogenic and estrogen-like metabolites on CYP activity was measured by HPLC MS/MS and on ADME gene expression using RT-PCR analyses. Metabolite E was formed from tamoxifen by CYP2C19, 3A and 1A2 and from desmethyltamoxifen by CYP2D6, 1A2 and 3A. Tamoxifen bisphenol was mainly formed from (E)- and (Z)-metabolite E by CYP2B6 and CYP2C19, respectively. Regarding phase II metabolism, UGT2B7, 1A8 and 1A3 showed highest activity in glucuronidation of tamoxifen bisphenol and metabolite E. Anti-estrogenic metabolites (Z)-4-hydroxytamoxifen, (Z)-endoxifen and (Z)-norendoxifen inhibited the activity of CYP2C enzymes while tamoxifen bisphenol consistently induced CYPs similar to rifampicin and phenobarbital. On the transcript level, highest induction up to 5.6-fold was observed for CYP3A4 by tamoxifen, (Z)-4-hydroxytamoxifen, tamoxifen bisphenol and (E)-metabolite E. Estrogen-like tamoxifen metabolites are formed in CYP-dependent reactions and are further metabolized by glucuronidation. The induction of CYP activity by tamoxifen bisphenol and the inhibition of CYP2C enzymes by anti-estrogenic metabolites may lead to drug-drug-interactions.