Identification of Hypoxia-inducible factor (HIF) stabilizer roxadustat and its possible metabolites in thoroughbred horses for doping control.
Abstract: Hypoxia-inducible factor (HIF) stabilizer belongs to a novel class of pharmacologically active substances, which are capable of inducing the endogenous erythropoietic system. The transcriptional activator HIF has been shown to significantly increase blood hemoglobin and is well set for the treatment of anemia resulting from chronic kidney disease. This research work reports a comprehensive study of the most popular HIF stabilizer roxadustat and its metabolites in thoroughbred horse urine after oral administration. The plausible structures of the detected metabolites were postulated using liquid chromatography-high-resolution mass spectrometry. Under the experimental condition 13 metabolites (7 phase I, 1 phase II, and 5 conjugates of phase I metabolism) were positively detected (M1-M13). The major phase I metabolites identified were formed by hydroxylation. Dealkylated and hydrolyzed phase I metabolites were also observed in this study. In phase II, a glucuronic acid conjugate of roxadustat was detected as the major metabolite. The sulfonic acid conjugates were observed to be formed from phase I metabolites. The characterized in vivo metabolites can potentially serve as target analytes for doping control analysis; hence, the result is an important tool for assessing its use and abuse in competitive sport.
© 2021 John Wiley & Sons, Ltd.
Publication Date: 2021-02-23 PubMed ID: 33569900DOI: 10.1002/dta.3014Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Validation Study
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The article discusses a study on the HIF stabilizer, roxadustat, which enhances the body’s red blood cell production and can be used to treat anemia derived from chronic kidney disease. It details how roxadustat and its metabolites are identified in horse urine, highlighting the possibility of using these findings to prevent doping in competitive sports.
Exploration of HIF Stabilizers
- Researchers look into hypoxia-inducible factor (HIF) stabilizers, a new class of pharmacologically active substances. These stabilizers have the ability to stimulate the body’s natural erythropoietic system, a process that involves the production of red blood cells.
- The study primarily focuses on roxadustat, the most widely recognized HIF stabilizer. Roxadustat has been found to significantly escalate the level of blood hemoglobin, making it an effective treatment for anemia resulting from chronic kidney disease.
Roxadustat Metabolites in Thoroughbred Horses
- In this research, the team seeks to identify roxadustat and its metabolites in the urine of thoroughbred horses after it has been orally administered. This is done through the use of liquid chromatography-high-resolution mass spectrometry.
- While the experiment identified 13 metabolites overall (7 from phase I metabolisms, 1 from phase II, and 5 conjugates that had undergone phase I metabolism), they could only be certain about the metabolites M1-M13.
- The majority of the phase I metabolites were found to have undergone hydroxylation. Other metabolic mechanisms such as dealkylation and hydrolyzation were also observed amongst the phase I metabolites.
- The main metabolite from phase II metabolism is a glucuronic acid conjugate of roxadustat. There were also sulfonic acid conjugates identified, believed to have originated from phase I metabolites.
Implications for Doping Control
- The metabolites of roxadustat detected in the horses can serve as target analytes for doping control tests. As these drugs can enhance the production of red blood cells, there’s potential for misuse in the sporting world, particularly competitive horse racing, since higher red blood cell count can theoretically improve the performance of an athlete.
- By establishing a way to test for the use of these substances, this research provides a vital tool for ensuring the integrity of competitive sports.
Cite This Article
APA
Mathew B, Philip M, Perwad Z, Karatt TK, Caveney MR, Subhahar MB, Karakka Kal AK.
(2021).
Identification of Hypoxia-inducible factor (HIF) stabilizer roxadustat and its possible metabolites in thoroughbred horses for doping control.
Drug Test Anal, 13(6), 1203-1215.
https://doi.org/10.1002/dta.3014 Publication
Researcher Affiliations
- Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
- Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
- Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
- Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
- Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
- Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
- Equine Forensic Unit, Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
MeSH Terms
- Administration, Oral
- Animals
- Chromatography, Liquid / methods
- Doping in Sports / prevention & control
- Female
- Glycine / analogs & derivatives
- Glycine / analysis
- Glycine / metabolism
- Glycine / urine
- Horses
- Hypoxia-Inducible Factor-Proline Dioxygenases / antagonists & inhibitors
- Isoquinolines / analysis
- Isoquinolines / metabolism
- Isoquinolines / urine
- Male
- Substance Abuse Detection / methods
- Tandem Mass Spectrometry / methods
References
This article includes 23 references
- Elliott S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport.. Br J Pharmacol 2008;154(3):529-541.
- Jelkmann W, Lundby C. Blood doping and its detection.. Blood 118(9):2395-2404.
- Jelkmann W. Features of blood doping.. Dtsch Z Sportmed 2016;67:255-262.
- WADA (World Anti-Doping Agency). The prohibited list.. 2020.
- FEI (Fédération Équestre Internationale). The prohibited substances list.. 2020.
- IFHA (International Federation of Horseracing Authorities). International screening limits.. 2020.
- Thevis M, Geyer H, Thomas A, Schanzer W. Trafficking of drug candidates relevant for sports drug testing: detection of non-approved therapeutics categorized as anabolic and gene doping agents in products distributed via the internet.. Drug Test Anal 2011;3(5):331-336.
- Reichel C. Recent developments in doping testing for erythropoietin.. Anal Bioanal Chem 2011;401(2):463-481.
- Koury MJ, Haase VH. Anaemia in kidney disease: harnessing hypoxia responses for therapy.. Nat Rev Nephrol 2015;11(7):394-410.
- Ge RL, Witkowski S, Zhang Y. Determinants of erythropoietin release in response to short-term hypobaric hypoxia.. J Appl Physiol (1985) 2002;92(6):2361-2367.
- Levine BD. Intermittent hypoxic training: fact and fancy.. High Alt Med Biol 2002;3(2):177-193.
- Bosman DR, Osborne CA, Marsden JT, Macdougall IC, Gardner WN, Watkins PJ. Erythropoietin response to hypoxia in patients with diabetic autonomic neuropathy and non-diabetic chronic renal failure.. Diabet Med 2002;19(1):65-69.
- Gupta N, Wish JB. Hypoxia-inducible factor prolyl hydroxylase inhibitors: a potential new treatment for anemia in patients with CKD.. Am J Kidney Dis 2017;69(6):815-826.
- Akizawa T, Iwasaki M, Otsuka T, Reusch M, Misumi T. Roxadustat treatment of chronic kidney disease-associated anemia in Japanese patients not on dialysis: a phase 2, randomized, double-blind, placebo-controlled trial.. Adv Ther 2019;36(6):1438-1454.
- Besarab A, Chernyavskaya E, Motylev I. Roxadustat (FG-4592): correction of anemia in incident dialysis patients.. J am Soc Nephrol 2016;27(4):1225-1233.
- Parmar DV, Kansagra KA, Patel JC. Outcomes of desidustat treatment in people with anemia and chronic kidney disease: a phase 2 study.. Am J Nephrol 2019;49(6):470-478.
- Bernhardt WM, Wiesener MS, Scigalla P. Inhibition of prolyl hydroxylases increases erythropoietin production in ESRD.. J am Soc Nephrol 2010;21(12):2151-2156.
- Flamme I, Oehme F, Ellinghaus P, Jeske M, Keldenich J, Thuss U. Mimicking hypoxia to treat anemia: HIF-stabilizer BAY 85-3934 (molidustat) stimulates erythropoietin production without hypertensive effects.. PLoS One 2014;9(11):1-14, e111838.
- Hansson A, Thevis M, Cox H. 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.. J Pharm Biomed Anal 2017;134:228-236.
- Eichner D, Van Wagoner RM, Brenner M. Implementation of the prolyl hydroxylase inhibitor roxadustat (FG-4592) and its main metabolites into routine doping controls.. Drug Test Anal 2017;9:1768-1778(11-12):1768-1778.
- Subhahar MB, Singh J, Albert PH, Kadry AM. Pharmacokinetics, metabolism and excretion of celecoxib, a selective cyclooxygenase-2 inhibitor, in horse.. J Vet Pharmacol Therap 2019:1-7.
- Subhahar MB, Abdul KKK, Philip M. Detection and identification of ACP-105 and its metabolites in equine urine using LC/MS/MS after oral administration.. Drug Test Anal 2020:1-19.
- Philip M, Mathew B, Tajudheen KK, Zubair P, Subhahar MB, Abdul KKK. Metabolic studies of HIF stabilizers IOX2, IOX3, and IOX4 (in vitro) for doping control.. Drug Test Anal 2021:1-23.
Citations
This article has been cited 4 times.- Ishii H, Shibuya M, Kusano K, Sone Y, Kamiya T, Wakuno A, Ito H, Miyata K, Sato F, Kuroda T, Yamada M, Leung GN. Pharmacokinetic Study of Vadadustat and High-Resolution Mass Spectrometric Characterization of its Novel Metabolites in Equines for the Purpose of Doping Control. Curr Drug Metab 2022;23(10):850-865.
- Creangă EC, Ott C, Nicolae AC, Drăgoi CM, Stan R. Roxadustat as a Hypoxia-Mimetic Agent: Erythropoietic Mechanisms, Bioanalytical Detection, and Regulatory Considerations in Sports Medicine. Curr Issues Mol Biol 2025 Sep 9;47(9).
- Jain S, Patil R, Sharma S. Bioanalysis, Analysis, Chemistry, and Pharmacological Aspects of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors. Curr Top Med Chem 2025;25(12):1451-1466.
- Mahajan R, Samanthula G, Srivastava S, Asthana A. A critical review of Roxadustat formulations, solid state studies, and analytical methodology. Heliyon 2023 Jun;9(6):e16595.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
