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Hansson, Annelie
Publications (9 of 9) Show all publications
Hansson, A., Knych, H., Stanley, S., Berndtson, E., Jackson, L., Bondesson, U., . . . Hedeland, M. (2018). Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites.. Journal of chromatography. B, 1074-1075, 91-98
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
Hansson, A. (2018). Structural Determination of Drug Metabolites from Doping Classed Compounds Using Mass Spectrometry. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
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)
Opponent
Supervisors
Available from: 2018-04-12 Created: 2018-03-14 Last updated: 2018-04-24
Garg, N., Hansson, A., Knych, H. K., Stanley, S. D., Thevis, M., Bondesson, U., . . . Globisch, D. (2018). Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control. Organic and biomolecular chemistry, 16(5), 698-702
Open this publication in new window or tab >>Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control
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2018 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 16, no 5, p. 698-702Article in journal (Refereed) Published
Abstract [en]

Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography- mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2018
National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-345712 (URN)10.1039/c7ob03030d (DOI)000423787600004 ()29319101 (PubMedID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-03-14 Created: 2018-03-14 Last updated: 2018-03-14Bibliographically approved
Hansson, A., Thevis, M., Cox, H., Miller, G., Eichner, D., Bondesson, U. & Hedeland, M. (2017). 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. Journal of Pharmaceutical and Biomedical Analysis, 134, 228-236
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
Hansson, A., Knych, H., Stanley, S., Thevis, M., Bondesson, U. & Hedeland, M. (2016). Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry. Rapid Communications in Mass Spectrometry, 30(7), 833-842
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
Rydevik, A., Hansson, A., Hellqvist, A., Bondesson, U. & Hedeland, M. (2015). A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry. Drug Testing and Analysis, 7(7), 626-633
Open this publication in new window or tab >>A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry
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2015 (English)In: Drug Testing and Analysis, ISSN 1942-7603, E-ISSN 1942-7611, Vol. 7, no 7, p. 626-633Article in journal (Refereed) Published
Abstract [en]

A new model is presented that can be used to screen for bioactivation of drugs. The evaluation of toxicity is an important step in the development of new drugs. One way to detect possible toxic metabolites is to use trapping agents such as glutathione. Often human liver microsomes are used as a metabolic model in initial studies. However, there is a need for alternatives that are easy to handle, cheap, and can produce large amounts of metabolites. In the presented study, paracetamol, mefenamic acid, and diclofenac, all known to form reactive metabolites in humans, were incubated with the fungus Cunninghamella elegans and the metabolites formed were characterized with ultra high performance liquid chromatography coupled to a quadrupole time of flight mass spectrometer. Interestingly, glutathione conjugates formed by the fungus were observed for all three drugs and their retention times and MS/MS spectra matched those obtained in a comparative experiment with human liver microsomes. These findings clearly demonstrated that the fungus is a suitable trapping model for toxic biotransformation products. Cysteine conjugates of all three test drugs were also observed with high signal intensities in the fungal incubates, giving the model a further indicator of drug bioactivation. To our knowledge, this is the first demonstration of the use of a fungal model for the formation and trapping of reactive drug metabolites. The investigated model is cheap, easy to handle, it does not involve experimental animals and it can be scaled up to produce large amounts of metabolites. Copyright (c) 2014 John Wiley & Sons, Ltd.

Keywords
paracetamol, mefenamic acid, diclofenac, Q-ToF, toxic metabolites, glutathione, trapping, cysteine
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-260866 (URN)10.1002/dta.1714 (DOI)000358611000009 ()25209992 (PubMedID)
Available from: 2015-08-26 Created: 2015-08-25 Last updated: 2018-01-11Bibliographically approved
Thevis, M., Lagojda, A., Kuehne, D., Thomas, A., Dib, J., Hansson, A., . . . Schaenzer, W. (2015). Characterization of a non-approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing. Rapid Communications in Mass Spectrometry, 29(11), 991-999
Open this publication in new window or tab >>Characterization of a non-approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing
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2015 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 29, no 11, p. 991-999Article in journal (Refereed) Published
Abstract [en]

RATIONALE: Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs). METHODS: In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl) pyrrolidin-1-yl)-2-(trifluoromethyl) benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations. RESULTS: By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono-and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl) pyrrolidin-1-yl)-2-(trifluoromethyl) benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies. CONCLUSIONS: The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-256112 (URN)10.1002/rcm.7189 (DOI)000354566900001 ()
Available from: 2015-06-23 Created: 2015-06-22 Last updated: 2017-12-04Bibliographically approved
Hansson, A., Knych, H., Stanley, S., Thevis, M., Bondesson, U. & Hedeland, M. (2015). Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes. Drug Testing and Analysis, 7(8), 673-683
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
Salomonsson, M. L., Hansson, A. & Bondesson, U. (2013). Development and in-house validation of a method for quantification of BMAA in mussels using dansyl chloride derivatization and ultra performance liquid chromatography tandem mass spectrometry. Analytical Methods, 5(18), 4865-4874
Open this publication in new window or tab >>Development and in-house validation of a method for quantification of BMAA in mussels using dansyl chloride derivatization and ultra performance liquid chromatography tandem mass spectrometry
2013 (English)In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 5, no 18, p. 4865-4874Article in journal (Refereed) Published
Abstract [en]

A new approach for the detection and quantification of beta-N-methylamino-L-alanine (BMAA) in mussels using chemical derivatization with dansyl chloride and ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented. The method, using dansyl chloride as the reagent, is simple, robust and cost efficient. Comparing the fragmentation patterns for derivatized BMAA and its isomer L-2,4-diaminobutyric acid derivatized a selective fragment for the derivatized BMAA was formed m/z 585 > m/z 71. To ensure an actual detection of BMAA, the ion ratio of the daughter ions m/z 71 and m/z 277 was calculated and compared with the calculated mean ion ratio. The method development resulted in a simplified sample preparation excluding solid phase extraction and, instead, performing filtration and dilution of the samples before the derivatization. Validation was performed and the limit of quantification was determined to be 0.15 mu g g(-1) wet mussel homogenate (33 fmol per injection) and the limit of detection was estimated to be 16 ng g(-1) (4 fmol per injection). The intra-and inter-day precisions were within the accepted criteria and the recovery was about 83%. The stability of BMAA in the stock solution was at least 3 months and the stability of derivatized extracts in vials for the quantification was good for 27 days. This is the first quantification method for BMAA in mussels with extensive validation data published and the method was also applied on real mussel samples collected on the west coast of Sweden. The concentrations of BMAA in the mussel samples were determined to be between 0.27 and 1.6 mu g g(-1) wet mussel homogenates.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-208177 (URN)10.1039/c3ay40657a (DOI)000323518200047 ()
Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2017-12-06Bibliographically approved
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