Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high-resolution mass spectrometer for the purpose of doping control.
Abstract: IOX4 is a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF-PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono-hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post-administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high-resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O-desbutyl IOX4, O-desbutyl IOX4 glucuronide, four mono-hydroxylated IOX4, N-oxidized IOX4, and N-oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O-desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post-administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h.
© 2021 John Wiley & Sons, Ltd.
Publication Date: 2021-10-21 PubMed ID: 34612014DOI: 10.1002/dta.3172Google Scholar: Lookup
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Summary
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The research article details a comprehensive metabolic study of IOX4, a substance used to treat anemia, which is prohibited for use in horses by major horse racing and equestrian authorities. Using a high-resolution mass spectrometer, the researchers investigated the metabolic effects of IOX4 in horse plasma and urine, uncovering a total of ten metabolites in urine and three in plasma.
Overview of Research
- The researchers conducted a thorough analysis of the metabolic effects of IOX4 in the plasma and urine of horses. This study was deemed necessary because IOX4, despite its medical usage in treating anemia, is strongly forbidden in equine sports due to the potential for misuse in enhancing performance.
- The researchers administered IOX4 to horses using a nasoesophageal method at a dose of 500mg/day for three consecutive days. The metabolites present after administration were then analysed both in vitro (in the lab) and in vivo (in the body).
Metabolite Analysis
- Using a homogenized horse liver for the in vitro study, the researchers discovered a total of four metabolites of IOX4 (three mono-hydroxylated IOX4 and one IOX4 glucuronide).
- The in vivo study involved a comprehensive analysis of post-administration plasma and urine samples, powered by liquid chromatography/electrospray ionization high-resolution mass spectrometry. The primary objective was to identify potential metabolites and establish the corresponding detection times for each.
- In conclusion, ten metabolites (IOX4 glucuronide, IOX4 glucoside, O-desbutyl IOX4, O-desbutyl IOX4 glucuronide, four mono-hydroxylated IOX4, N-oxidized IOX4, and N-oxidized IOX4 glucoside) were found in urine samples, while three metabolites (glucuronide, glucoside, and O-desbutyl) were found in plasma samples post-administration of IOX4.
Detection and Quantification Method
- A quantification method for the detection of free and conjugated IOX4 metabolites in urine and plasma was also developed through a procedure the researchers referred to as a “biphase enzymatic hydrolysis”.
- This method was applied to samples after administration helping to create profiles for the elimination of IOX4 from the system.
- As a result of this method, the detection times of total IOX4 in both urine and plasma samples were effectively stretched to at least 312 hours post-administration.
Cite This Article
APA
Ishii H, Shibuya M, So YM, Wong JKY, Ho ENM, Kusano K, Sone Y, Kamiya T, Wakuno A, Ito H, Miyata K, Yamada M, Leung GN.
(2021).
Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high-resolution mass spectrometer for the purpose of doping control.
Drug Test Anal, 14(2), 233-251.
https://doi.org/10.1002/dta.3172 Publication
Researcher Affiliations
- Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
- Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Miyagi, Japan.
- Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong.
- Veterinarian Section, Equine Department, JRA, Minato, Tokyo, Japan.
- Veterinarian Section, Equine Department, JRA, Minato, Tokyo, Japan.
- Equine Veterinary Clinic, Horse Racing School, Japan Racing Association, Shiroi, Chiba, Japan.
- Equine Veterinary Clinic, Horse Racing School, Japan Racing Association, Shiroi, Chiba, Japan.
- Equine Veterinary Clinic, Horse Racing School, Japan Racing Association, Shiroi, Chiba, Japan.
- JRA Equestrian Park Utsunomiya Office, Utsunomiya, Tochigi, Japan.
- Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
- Drug Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.
MeSH Terms
- Animals
- Chromatography, Liquid / methods
- Doping in Sports / prevention & control
- Glucuronides
- Horses
- Plasma
- Spectrometry, Mass, Electrospray Ionization / methods
Grant Funding
- Japan Racing Association
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Citations
This article has been cited 3 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. Generic approach for the discovery of drug metabolites in horses based on data-dependent acquisition by liquid chromatography high-resolution mass spectrometry and its applications to pharmacokinetic study of daprodustat. Anal Bioanal Chem 2022 Nov;414(28):8125-8142.
- 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.
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