Metabolic studies of hypoxia‐inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control

• Published in: Drug testing and analysis 2021-04, Vol.13 (4), p.794-816
• Main Authors: Philip, Moses, Mathew, Binoy, Karatt, Tajudheen K., Perwad, Zubair, Subhahar, Michael Benedict, Karakka Kal, Abdul Khader
• Format: Article
• Language: English
• Subjects: equine liver microsomes; Hypoxia; hypoxia‐inducible factor; IOX2; IOX3; IOX4; Metabolism; Metabolites
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.3000

The transcriptional activator hypoxia‐inducible factor (HIF) is a vital arbitrator in the performance of cellular responses lacking oxygen supply in aerobic organisms. Because these compounds are capable of enhancing the organism's capacity for molecular oxygen transport, they possess great potential for abuse as a performance‐enhancing agent in sports. A comprehensive study of the metabolic conversion of the most popular HIF stabilisers such as IOX2, IOX3 and IOX4 using equine liver microsomes (in vitro) is reported. The parents and their metabolites were identified and characterised by liquid chromatography–mass spectrometry in negative ionisation mode using a QExactive high‐resolution mass spectrometer. Under the current experimental condition, a total of 10 metabolites for IOX2 (three phase I and seven phase II), nine metabolites for IOX3 (four phase I and five phase II) and five metabolites for IOX4 (three phase I and two phase II) were detected. The outcome of the present study is as follows: (1) all the three IOX candidates are prone to oxidation, results in subsequent monohydroxylated, and some dihydroxylated metabolites. (2) Besides oxidation, there is a possibility of hydrolysis and de‐alkylation, which results in corresponding carboxylic acid and amide, respectively. (3) The glucuronide and sulphate conjugate of the parent drugs as well as the monohydroxylated analogues were observed in this study. The characterised in vitro metabolites can potentially serve as target analytes for doping control analysis. A comprehensive study of the metabolic conversion of the most popular HIF stabilizers such as IOX2, IOX3, and IOX4 using equine liver microsomes (in vitro) is reported. Under the current experimental condition, a total of ten metabolites for IOX2, nine for IOX3, and five for IOX4 were detected. The major phase I metabolites identified were formed by hydroxylation. Hydrolysed metabolites were as well observed in the study. The glucuronic acid and sulfonic acid conjugates were detected as phase II metabolites.