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Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
Bayer CropScience AG.
Institute of Biochemistry and Center for Preventive Doping Research, German Sport University, Cologne.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
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2014 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 98, 36-39 p.Article in journal (Refereed) Published
Abstract [en]

In this study, using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, it has been confirmed that biotransformation with the fungus Cunninghamella elegans combined with chemical oxidation with the free radical tetramethylpiperidinyl-1-oxy (TEMPO) can produce drug glucuronides of beta-configuration. Glucuronic acid conjugates are a common type of metabolites formed by the human body. The detection of such conjugates in doping control and other kinds of forensic analysis would be beneficial owing to a decrease in analysis time as hydrolysis can be omitted. However the commercial availability of reference standards for drug glucuronides is poor. The selective androgen receptor modulator (SARM) SARM Si was incubated with the fungus C elegans. The sample was treated with the free radical TEMPO oxidizing agent and was thereafter purified by SPE. A glucuronic acid conjugate was isolated using a fraction collector connected to an ultra high performance liquid chromatographic (UHPLC) system. The isolated compound was characterized by NMR spectroscopy and mass spectrometry and its structure was confirmed as a glucuronic acid beta-conjugate of hydroxylated SARM Si bearing the glucuronide moiety on carbon C-10.

Place, publisher, year, edition, pages
2014. Vol. 98, 36-39 p.
National Category
Medicinal Chemistry Analytical Chemistry
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-220769DOI: 10.1016/j.jpba.2014.05.001ISI: 000339859800005OAI: oai:DiVA.org:uu-220769DiVA: diva2:706438
Available from: 2014-03-20 Created: 2014-03-20 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Drug Metabolites Formed by Cunninghamella Fungi: Mass Spectrometric Characterization and Production for use in Doping Control
Open this publication in new window or tab >>Drug Metabolites Formed by Cunninghamella Fungi: Mass Spectrometric Characterization and Production for use in Doping Control
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the in vitro production of drug metabolites using fungi of the Cunninghamella species. The metabolites were characterized with mainly liquid chromatography-mass spectrometry using ion-trap and quadrupole-time-of-flight instruments. A fungal in vitro model has several advantages e.g., it is easily up-scaled and ethical problems associated with animal-based models are avoided.

The metabolism of bupivacaine and the selective androgen receptor modulators (SARMs) S1, S4 and S24 by the fungi Cunninghamella elegans and Cunninghamella blakesleeana was investigated. The detected metabolites were compared with those formed in vitro and in vivo by human and horse and most phase I metabolites formed by mammals were also formed by the fungi. The higher levels of bupivacaine metabolites in the fungal samples allowed an extensive mass spectrometric structural characterization which shows that the fungi are relevant metabolic models.

Glucuronides are important drug metabolites but they are difficult to synthesize. The discovery that the fungus Cunninghamella elegans formed large amounts of glucosides led to the idea that they could be used to form glucuronides. A new concept was developed where a fungal incubate containing a SARM S1 glucoside was mixed with the free radical tetramethylpiperidinyl-1-oxy (TEMPO), sodium bromide and sodium hypochlorite which produced a glucuronide. Isolation and characterization by nuclear magnetic resonance spectroscopy proved that the new method could produce glucuronides for use as reference material.

An investigation of reactive metabolite formation of the drugs paracetamol, mefenamic acid and diclofenac by the fungus Cunninghamella elegans was performed. It was demonstrated for the first time that the fungus could produce glutathione, glutathione ethyl-ester, cysteine and N-acetylcysteine conjugates that are indicative of a preceding formation of reactive intermediates. A comparison with conjugates formed by human liver microsomes showed that both models formed identical metabolites.

The presented investigations prove that Cunninghamella fungi are relevant drug metabolism models. They show that the fungi to a large extent forms the same metabolites as mammals and that they can produce metabolites for use as reference material in, e.g. doping control. It was also demonstrated that the fungal model can be used in the important assessment of drug toxicity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 46 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 186
Keyword
Cunninghamella blakesleeana, Cunninghamella elegans, Doping Control, Drug Metabolites, Glucuronide Production, Mass Spectrometry, Reactive Metabolites, Reference Material, Structural Characterization
National Category
Medicinal Chemistry Pharmaceutical Sciences
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-220906 (URN)978-91-554-8906-9 (ISBN)
Public defence
2014-05-09, B:41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-04-17 Created: 2014-03-23 Last updated: 2014-04-29

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Rydevik, AxelBondesson, UlfHedeland, Mikael

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