Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes

Abstract

AbstractVoxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti‐Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high‐performance liquid chromatography (high‐resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro‐conjugation and O‐methylation) were formed by the HepG2 cells in a time‐dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro‐conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono‐hydroxylation, reduction and O‐methylation in different combinations were identified. Voxelotor was also found as glucuro‐conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.