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Drug Metabolites Formed by Cunninghamella Fungi: Mass Spectrometric Characterization and Production for use in Doping Control
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
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 [en]
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: urn:nbn:se:uu:diva-220906ISBN: 978-91-554-8906-9 (print)OAI: oai:DiVA.org:uu-220906DiVA: diva2:706895
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
List of papers
1. Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry
Open this publication in new window or tab >>Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry
2012 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 26, no 11, 1338-1346 p.Article in journal (Refereed) Published
Abstract [en]

RATIONALE: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial.

METHODS: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d9. Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MSn and hydrogen/deuterium exchange.

RESULTS: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana.

CONCLUSIONS: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials.

National Category
Medicinal Chemistry Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-173545 (URN)10.1002/rcm.6225 (DOI)000303597100009 ()
Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2017-12-07Bibliographically approved
2. The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs)
Open this publication in new window or tab >>The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs)
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2013 (English)In: Xenobiotica, ISSN 0049-8254, E-ISSN 1366-5928, Vol. 43, no 5, 409-420 p.Article in journal (Refereed) Published
Abstract [en]

1. Selective androgen receptor modulators (SARMs) are a group of substances that have potential to be used as doping agents in sports. Being a relatively new group not available on the open market means that no reference materials are commercially available for the main metabolites. In the presented study, the in vitro metabolism of SARMs by the fungus Cunninghamella elegans has been investigated with the purpose of finding out if it can produce relevant human and equine metabolites.

2. Three different SARMs, S1, S4 and S24, were incubated for 5 days with C. elegans. The samples were analysed both with and without sample pretreatment using ultra performance liquid chromatography coupled to high resolution mass spectrometry.

3. All the important phase I and some phase II metabolites from human and horse were formed by the fungus. They were formed through reactions such as hydroxylation, deacetylation, O-dephenylation, nitro-reduction, acetylation and sulfonation.

4. The study showed that the fungus produced relevant metabolites of the SARMs and thus can be used to mimic mammalian metabolism. Furthermore, it has the potential to be used for future production of reference material.

National Category
Pharmaceutical Sciences Medicinal Chemistry Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-196318 (URN)10.3109/00498254.2012.729102 (DOI)000316952100002 ()
Note

MEDLINE AN 2013135005(Journal; Article; (JOURNAL ARTICLE))

Available from: 2013-03-07 Created: 2013-03-07 Last updated: 2017-12-06Bibliographically approved
3. Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO
Open this publication in new window or tab >>Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO
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.

Keyword
High resolution mass spectrometry, UPLC, Glucuronides, SARM, TEMPO
National Category
Analytical Chemistry
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-205709 (URN)10.1016/j.jpba.2013.06.012 (DOI)000322752800040 ()23867089 (PubMedID)
Available from: 2013-08-22 Created: 2013-08-22 Last updated: 2017-12-06Bibliographically approved
4. Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation
Open this publication in new window or tab >>Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation
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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.

National Category
Medicinal Chemistry Analytical Chemistry
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-220769 (URN)10.1016/j.jpba.2014.05.001 (DOI)000339859800005 ()
Available from: 2014-03-20 Created: 2014-03-20 Last updated: 2017-12-05Bibliographically approved
5. A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry
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
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Medicinal Chemistry Pharmaceutical Sciences Analytical Chemistry
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-220770 (URN)
Available from: 2014-03-20 Created: 2014-03-20 Last updated: 2014-04-29

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