Assessing human urinary clomiphene metabolites after consumption of eggs from clomiphene-treated laying hens using chromatographic-mass spectrometric approaches

The anti-estrogen clomiphene is prohibited in sports at all times. Yet, adverse analytical findings (AAFs) have increased since 2011. This is possibly due to improved analytical sensitivity, but also contamination of food of animal origin needs to be taken into consideration as a potential source of drug exposure. For instance, studies with laying hens that received orally administered clomiphene have shown a signifi-cantly increased egg production rate but, as a consequence, eggs were found to incorporate residues of clomiphene.In order to evaluate if the consumption of clomiphene-contaminated eggs can cause an AAF of a doping control sample, eggs obtained from an animal administration study with clomiphene were consumed by human volunteers. Each volunteer ate two eggs, and urine samples were collected and analyzed using routine doping control procedures. Subsequently, additional volunteers received a microdosed clomiphene capsule to compare the excretion profiles.Maximum urinary concentrations of hydroxy-clomiphene (HC) between 80 and 300 pg mL(-1) were detected following the consumption of clomiphene-containing eggs, which would constitute AAFs if observed in athletes' doping control samples. In order to support the differentiation of potential routes of drug exposure, a method was developed which allows for the chromatographic separation of (E)-3-, (Z)-3-, (E)-4-, and (Z)-4-HC using a derivatization step. By comparing the peak areas of these metabolites, characteristic relative distribution patterns were found that assist in identifying AAFs resulting from clomiphene ingested via contaminated eggs and, thus, enable to distinguish clomiphene intake via contaminated eggs from the intake of microdoses or therapeutic dosages, e.g. for doping purposes.& nbsp;(c) 2022 Elsevier B.V. All rights reserved

Molybdenum Cofactor Catabolism Unravels the Physiological Role of the Drug Metabolizing Enzyme Thiopurine S-Methyltransferase

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild -type and knock-out (Tpmt(-/-)) mice as well as human liver cytosol. Urothione levels were quantified by liquid-chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT-genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population-based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin-methylating activity was associated with TPMT activity determined with its drug substrate (6-thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt-/- mice. Urinary urothione concentration in TPMT-deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/*3A) but normal levels in compound heterozygous carriers (TPMT*3A/*3C) as validated in the population-based study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug-and endogenous substrates

Environmentally-induced epigenetic conversion of a piRNA cluster

International audienceTransposable element (TE) activity is repressed in animal gonads by PIWI-interacting RNAs (piRNAs) produced by piRNA clusters. Current models in flies propose that germinal piRNA clusters are functionally defined by the maternal inheritance of piRNAs produced during the previous generation. Taking advantage of an inactive, but ready to go, cluster of P-element derived transgene insertions in Drosophila melanogaster, we show here that raising flies at high temperature (29°C) instead of 25°C triggers the stable conversion of this locus from inactive into actively producing functional piRNAs. The increase of antisense transcripts from the cluster at 29°C combined with the requirement of transcription of euchromatic homologous sequences, suggests a role of double stranded RNA in the production of de novo piRNAs. This report describes the first case of the establishment of an active piRNA cluster by environmental changes in the absence of maternal inheritance of homologous piRNAs

Obesity Alters Endoxifen Plasma Levels in Young Breast Cancer Patients: A Pharmacometric Simulation Approach

Endoxifen is one of the most important metabolites of the prodrug tamoxifen. High interindividual variability in endoxifen steady-state concentrations (CSS,min ENDX ) is observed under tamoxifen standard dosing and patients with breast cancer who do not reach endoxifen concentrations above a proposed therapeutic threshold of 5.97 ng/mL may be at a 26% higher recurrence risk compared with patients with endoxifen concentrations exceeding this value. In this investigation, 10 clinical tamoxifen studies were pooled (1,388 patients) to investigate influential factors on CSS,min ENDX using nonlinear mixed-effects modeling. Age and body weight were found to significantly impact CSS,min ENDX in addition to CYP2D6 phenotype. Compared with postmenopausal patients, premenopausal patients had a 30% higher risk for subtarget CSS,min ENDX at tamoxifen 20 mg per day. In treatment simulations for distinct patient subpopulations, young overweight patients had a 3.1-13.8-fold higher risk for subtarget CSS,min ENDX compared with elderly low-weight patients. Considering ever-rising obesity rates and the clinical importance of tamoxifen for premenopausal patients, this subpopulation may benefit most from individualized tamoxifen dosing.status: publishe