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Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
2013 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 84, 278-284 p.Article in journal (Refereed) Published
Abstract [en]

A new concept for the production of drug glucuronides is presented and the products formed were characterized using ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Glucuronic acid conjugates are important phase II metabolites of a wide range of drugs. There is a lack of commercially available glucuronides and classic synthetic methods are tedious and expensive. Thus, new methods of glucuronide synthesis are needed. Selective androgen receptor modulators (SARMs) of the aryl propionamide class were used as model compounds and were incubated with the fungus Cunninghamella elegans which was previously known to conjugate drugs with glucose. The resulting glucoside metabolites were then oxidized with tetramethylpiperidinyl-1-oxy (TEMPO). UPLC-HRMS analysis showed that the peaks corresponding to the glucosides had disappeared after the reaction and were replaced by peaks with m/z consistent with the corresponding glucuronic acid conjugates. The MS/MS spectra of the reaction products were investigated and the observed fragment ion pattern corroborated the suggested structural change. A comparison in terms of retention times and product ion spectra between the glucuronides formed by the new method and those produced by liver microsomes indicated that the conjugates from the two different sources were identical, thus demonstrating the human relevance of the presented technique. Furthermore, the glucuronides formed by the presented method were readily hydrolyzed by β-glucuronidase which further gave evidence as to the fact that they were of β configuration. The investigated method was easy to perform, required a low input of work and had a low cost.

Place, publisher, year, edition, pages
2013. Vol. 84, 278-284 p.
Keyword [en]
High resolution mass spectrometry, UPLC, Glucuronides, SARM, TEMPO
National Category
Analytical Chemistry
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-205709DOI: 10.1016/j.jpba.2013.06.012ISI: 000322752800040PubMedID: 23867089OAI: oai:DiVA.org:uu-205709DiVA: diva2:642520
Available from: 2013-08-22 Created: 2013-08-22 Last updated: 2017-12-06Bibliographically 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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