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Structural Determination of Drug Metabolites from Doping Classed Compounds Using Mass Spectrometry
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
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 [en]
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: urn:nbn:se:uu:diva-344310ISBN: 978-91-513-0276-8 (print)OAI: oai:DiVA.org:uu-344310DiVA, id: diva2:1190549
Public defence
2018-05-04, B:42, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2018-04-12 Created: 2018-03-14 Last updated: 2018-04-24
List of papers
1. Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes
Open this publication in new window or tab >>Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes
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2015 (English)In: Drug Testing and Analysis, ISSN 1942-7603, E-ISSN 1942-7611, Vol. 7, no 8, p. 673-683Article in journal (Refereed) Published
Abstract [en]

Selective androgen receptor modulators, SARMs, constitute a class of compounds with anabolic properties but with few androgenic side-effects. This makes them possible substances of abuse and the World Anti-Doping Agency (WADA) has banned the entire class of substances. There have been several cases of illicit use of aryl propionamide SARMs in human sports and in 2013, 13 cases were reported. These substances have been found to be extensively metabolized in humans, making detection of metabolites necessary for doping control. SARMs are also of great interest to equine doping control, but the in vivo metabolite pattern and thus possible analytical targets have not been previously studied in this species. In this study, the urinary metabolites of the SARMs S1, S4, and S22 in horses were studied after intravenous injection, using ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS). Eight different metabolites were found for SARM S1, nine for SARM S4, and seven for SARM S22. The equine urinary metabolite profiles differed significantly from those of humans. The parent compounds were only detected for SARMs S4 and S22 and only at the first sampling time point at 3h post administration, making them unsuitable as target compounds. For all three SARMs tested, the metabolite yielding the highest response had undergone amide hydrolysis, hydroxylation and sulfonation. The resulting phase II metabolites (4-nitro-3-trifluoro-methyl-phenylamine sulfate for SARMs S1 and S4 and 4-cyano-3-trifluoro-methyl-phenylamine sulfate for SARM S22) are proposed as analytical targets for use in equine doping control.

Keywords
selective androgen receptor modulators, SARM, metabolite, equine, horse
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-261969 (URN)10.1002/dta.1768 (DOI)000359603700003 ()25560998 (PubMedID)
Available from: 2015-09-08 Created: 2015-09-07 Last updated: 2018-03-14Bibliographically approved
2. Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry
Open this publication in new window or tab >>Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry
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2016 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 30, no 7, p. 833-842Article in journal (Refereed) Published
Abstract [en]

RationaleSelective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma. MethodsEach SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrometric analysis was performed using negative electrospray, quadrupole time-of-flight mass spectrometry operated in MSE mode and triple-quadrupole mass spectrometry operated in selected reaction monitoring mode. For the quantification of SARM S1, a deuterated analogue was used as internal standard. ResultsThe numbers of observed metabolites were eight, nine and four for the SARMs S1, S4 and S22, respectively. The major metabolite was formed by the same metabolic reactions for all three SARMs, namely amide hydrolysis, hydroxylation and sulfonation. The values of the determined maximum plasma concentrations were in the range of 97-170 ng/mL for SARM S1, 95-115 ng/mL for SARM S4 and 92-147 ng/mL for SARM S22 and the compounds could be detected for 96 h, 12 h and 18 h, respectively. ConclusionsThe maximum plasma concentration of SARMs S1, S4 and S22 was measured in the first sample (5 min) after administration and they were eliminated fast from plasma. The proposed targets to be used in equine doping control are the parent compounds for all three SARMs, but with the metabolite yielding the highest response as a complementary target. 

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-286622 (URN)10.1002/rcm.7512 (DOI)000372508100006 ()26969924 (PubMedID)
Available from: 2016-04-28 Created: 2016-04-21 Last updated: 2018-03-14Bibliographically approved
3. Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites.
Open this publication in new window or tab >>Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites.
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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.

Keywords
Doping, LGD-4033, Horse, Mass Spectrometry, Metabolite, SARM, Selective Androgen Receptor Modulator
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-344303 (URN)10.1016/j.jchromb.2017.12.010 (DOI)000425204900013 ()29334634 (PubMedID)
Available from: 2018-03-06 Created: 2018-03-06 Last updated: 2018-05-07Bibliographically approved
4. 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
Open this publication in new window or tab >>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
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2017 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 134, p. 228-236Article in journal (Refereed) Published
Abstract [en]

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 MSE and 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.

Keywords
FG-4592, Drug metabolism, High resolution mass spectrometry
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-317588 (URN)10.1016/j.jpba.2016.11.041 (DOI)000392909900029 ()27918992 (PubMedID)
Available from: 2017-03-24 Created: 2017-03-24 Last updated: 2018-03-14Bibliographically approved

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