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Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75651 Uppsala, Sweden.
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2018 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 1074-1075, p. 91-98Article in journal (Refereed) Published
Abstract [en]

LGD-4033 has been found in human doping control samples and has the potential for illicit use in racehorses as well. It belongs to the pharmacological class of selective androgen receptor modulators (SARMs) and can stimulate muscle growth, much like anabolic steroids. However, SARMs have shown superior side effect profiles compared to anabolic steroids, which arguably makes them attractive for use by individuals seeking an unfair advantage over their competitors. The purpose of this study was to investigate the metabolites formed from LGD-4033 in the horse in order to find suitable analytical targets for doping controls. LGD-4033 was administered to three horses after which plasma and urine samples were collected and analyzed for metabolites using ultra high performance liquid chromatography coupled to a high resolution mass spectrometer. In horse urine, eight metabolites, both phase I and phase II, were observed most of which had not been described in other metabolic systems. Six of these were also detected in plasma. The parent compound was detected in plasma, but not in non-hydrolyzed urine. The longest detection times were observed for unchanged LGD-4033 in plasma and in urine hydrolyzed with β-glucuronidase and is thus suggested as the analytical target for doping control in the horse. The metabolite profile determined in the horse samples was also compared to those of human urine and fungal incubate from Cunninghamella elegans. The main human metabolite, dihydroxylated LGD-4033, was detected in the horse samples and was also produced by the fungus. However, it was a not a major metabolite for horse and fungus, which highlights the importance of performing metabolism studies in the species of interest.

Place, publisher, year, edition, pages
2018. Vol. 1074-1075, p. 91-98
Keywords [en]
Doping, LGD-4033, Horse, Mass Spectrometry, Metabolite, SARM, Selective Androgen Receptor Modulator
National Category
Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-344303DOI: 10.1016/j.jchromb.2017.12.010ISI: 000425204900013PubMedID: 29334634OAI: oai:DiVA.org:uu-344303DiVA, id: diva2:1188025
Available from: 2018-03-06 Created: 2018-03-06 Last updated: 2018-05-07Bibliographically approved
In thesis
1. Structural Determination of Drug Metabolites from Doping Classed Compounds Using Mass Spectrometry
Open this publication in new window or tab >>Structural Determination of Drug Metabolites from Doping Classed Compounds Using Mass Spectrometry
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Doping control in equine sports is important for a fair competition, but also to ensure the integrity of the betting system, as well as for animal welfare reasons. To detect the use of illicit compounds, screening for the parent compound is common. However, by using a metabolite as the analytical target instead, the detection time can be prolonged. For some compounds, the use of a metabolite is a necessity since the parent drug may not be detected at all.

The metabolites of the selective androgen receptor modulators (SARM) S1, S4 and S22 were investigated in horse urine and plasma. The unchanged parent compounds had the longest detection time in plasma, but were not detected at all in urine. Instead, the longest detection time was measured for the metabolites 2-amino-5-nitro-4-(trifluoromethyl)phenyl hydrogen sulfate (SARMs S1 and S4) and 2-amino-5-cyano-4-(trifluoromethyl)phenyl hydrogen sulfate (SARM S22). These metabolites were thus suggested as analytical targets for doping control in urine while the parent compounds were suggested for plasma samples. 2-amino-5-nitro-4-(trifluoromethyl)phenyl hydrogen sulfate could also be produced in large quantities by the fungus Cunninghamella elegans to potentially be used as reference compound.

The horse metabolites of the SARM LGD-4033 were also studied in urine and plasma. The formate adduct of LGD-4033 had the longest detection time in plasma and in urine after hydrolysis with β-glucuronidase. In non-hydrolyzed urine, the glucuronidated LGD-4033 was detected instead.

Different in vitro models were used to predict in vivo metabolites of roxadustat, a hypoxia-inducible factor stabilizer. Cunninghamella elegans was successful in producing more metabolites compared to human and equine liver microsomes and human hepatocytes.

The metabolite detection and identification in all experiments were accomplished using a UHPLC-Q-TOF MS instrument, where the high-resolution MS data was vital in determining which metabolites were formed.

The thesis shows the benefits of investigating the metabolites of doping substances to allow for a successful doping control method in horse urine and plasma by prolonging the detection time. It also highlights the usefulness of Cunninghamella elegans as an alternative to the more commonly used in vitro models for both predicting and producing metabolites.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 251
Keywords
mass spectrometry, UHPLC-MS/MS, doping control, Cunninghamella elegans, selective androgen receptor modulator, SARM, andarine, ostarine, LGD-4033, roxadustat, HIF stabilizer
National Category
Medicinal Chemistry
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-344310 (URN)978-91-513-0276-8 (ISBN)
Public defence
2018-05-04, B:42, BMC, Husargatan 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2018-04-12 Created: 2018-03-14 Last updated: 2018-04-24

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Hansson, AnnelieBondesson, UlfHedeland, Mikael

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