title: Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry.
abstract: FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q-TOF mass spectrometer operated in MSEand MSMS modes, twelve different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.
authors.affiliation: Division of Analytical Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, Box 574, SE-75123, Uppsala, Sweden. Electronic address: Annelie.Hansson@farmkemi.uu.se.
authors.forename: Annelie
authors.initials: A
authors.lastname: Hansson
authors.affiliation: Institute of Biochemistry and Center for Preventive Doping Research, German Sport University, Cologne, Germany. Electronic address: Thevis@dshs-koeln.de.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
authors.affiliation: Sports Medicine Research and Testing Laboratory, Salt Lake City, UT, USA. Electronic address: hcox@smrtl.org.
authors.forename: Holly
authors.initials: H
authors.lastname: Cox
authors.affiliation: Sports Medicine Research and Testing Laboratory, Salt Lake City, UT, USA. Electronic address: gmiller@smrtl.org.
authors.forename: Geoff
authors.initials: G
authors.lastname: Miller
authors.affiliation: Sports Medicine Research and Testing Laboratory, Salt Lake City, UT, USA. Electronic address: de@smrtl.org.
authors.forename: Daniel
authors.initials: D
authors.lastname: Eichner
authors.affiliation: Division of Analytical Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, Box 574, SE-75123, Uppsala, Sweden; National Veterinary Institute (SVA), Department of Chemistry, Environment and Feed Hygiene, SE-75189, Uppsala, Sweden. Electronic address: Ulf.Bondesson@sva.se.
authors.forename: Ulf
authors.initials: U
authors.lastname: Bondesson
authors.affiliation: Division of Analytical Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, Box 574, SE-75123, Uppsala, Sweden; National Veterinary Institute (SVA), Department of Chemistry, Environment and Feed Hygiene, SE-75189, Uppsala, Sweden. Electronic address: Mikael.Hedeland@sva.se.
authors.forename: Mikael
authors.initials: M
authors.lastname: Hedeland
mesh_terms: D000818:Animals; D020093:Cunninghamella / Q000737:chemistry / Q000378:metabolism*; D004300:Doping in Sports / Q000517:prevention & control; D005998:Glycine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000032:analysis / Q000378:metabolism*; D007546:Isoquinolines / Q000032:analysis / Q000378:metabolism*; D059625:Liquid-Liquid Extraction / Q000379:methods; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007546:Isoquinolines; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D005998:Glycine; C584543:roxadustat
keywords: Drug metabolism; FG-4592; High resolution mass spectrometry