In silico and in vitro human metabolism of IOX2, a performance-enhancing doping agent

IOX2 is a potent inhibitor of prolyl hydroxylase 2, a key enzyme in the regulation of hypoxia-inducible factor (HIF) and oxygen homeostasis. As such, it can be used to enhance athletic performance and is currently banned by the World Anti-Doping Agency (WADA). Detection of metabolites is critical to demonstrate drug use in doping. However, there is currently little data on IOX2 human metabolism. Our aim was to identify relevant biomarkers of IOX2 use in humans. For this purpose, IOX2 was incubated with 10-donor-pooled human hepatocytes for 3 h, incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and LC-HRMS/MS data were screened with Compound Discoverer (Thermo Scientific) for a comprehensive identification of IOX2 metabolites. Additionally, IOX2 human metabolites were predicted with GLORYx open-access software (University of Hamburg, Germany) to assist in the LC-HRMS/MS analysis and data mining. Thirteen metabolites were identified, oxidation at the quinolinyl group, O-glucuronidation, and combinations being predominant biotransformations. The results were consistent with previous animal studies and a single case of oral microdose administration. We suggest hydroxyquinolinyl-IOX2 as major biomarker of IOX2 use in biological samples, glucuronide hydrolysis being critical to increase IOX2 and hydroxyquinolinyl-IOX2 detectability in urine.