In vitromodels for metabolic studies of small peptide hormones in sport drug testing

Abstract

Peptide hormones represent an emerging class of potential doping agents. Detection of their misuse is difficult due to their short half-life in plasma and rapid elimination. Therefore, investigating their metabolism can improve detectability. Unfortunately, pharmacokinetic studies with human volunteers are often not allowed because of ethical constraints, and therefore alternative models are needed. This study was performed in order to evaluate in vitro models (human liver microsomes and S9 fraction) for the prediction of the metabolism of peptidic doping agents and to compare them with the established models. The peptides that were investigated include desmopressin, TB-500, GHRP-2, GHRP-6, hexarelin, LHRH and leuprolide. Several metabolites were detected for each peptide after incubation with human liver microsomes, S9 fraction, and serum, which all showed endopeptidase and exopeptidase activity. In vitro models from different organs (liver vs. kidney) were compared, but no significant differences were recorded. Deamidation was not observed in any of the models and was therefore evaluated by incubation with α-chymotrypsin. In conclusion, in vitro models are useful tools for forensic and clinical analysts to detect peptidic metabolic markers in biological fluids.

Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine

“…These substances are excreted in urine as parent compounds and/or as metabolites/degradation products at sub-nanogram-per-milliliter levels, but cannot be measured by immunological methods because of low molecular weight and a lack of efficient immune response. Mass spectrometry-based techniques were used for singleanalyte approaches by Okano et al [25] to detect GHRP-2 and its main metabolite in urine, by Gil et al [13] to detect GHRP-6 in plasma, by Esposito et al [34] and Thomas et al [28] to detect desmopressin and its analogues in blood and urine, and for multi-analyte screening to simultaneously detect desmopressin, luteinizing-hormone-releasing hormone (LH-RH), GHRPs, and their major metabolites in human urine [29,33,38].…”

Identification of gonadotropin‐releasing hormone metabolites in greyhound urine

“…In vitro and in vivo studies in different species suggest that metabolic proteolysis at virtually every peptide bond in GnRH is possible. Incubation of GnRH in human serum produced an array of C‐terminal fragments spanning GnRH 2–10 to GnRH 7–10 . These fragments as well as GnRH 2–9 and N‐terminal fragments spanning GnRH 1–9 to GnRH 1–6 were produced by incubation with human liver microsomes or the S9 fraction of human liver or kidney .…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…In the absence of elimination studies and, thus, knowledge about potential metabolites of the full‐length MGF, spiked urine samples were used to estimate the LOD for the intact analyte (0.25 ng/mL) after adaptation and extension of an existing initial testing method. As excretion study samples are frequently a limiting factor in anti‐doping research, options to simulate metabolic reactions have gained increasing attention at least for peptidic drugs of lower molecular mass …”