ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2014

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 186

Drug Metabolites Formed byCunninghamella Fungi

Mass Spectrometric Characterization andProduction for use in Doping Control

AXEL RYDEVIK

ISSN 1651-6192ISBN 978-91-554-8906-9urn:nbn:se:uu:diva-220906

Dissertation presented at Uppsala University to be publicly examined in B:41, BMC,Husargatan 3, Uppsala, Friday, 9 May 2014 at 09:15 for the degree of Doctor of Philosophy(Faculty of Pharmacy). The examination will be conducted in English. Faculty examiner:Professor David Cowan (King's College, Department of Forensic and Analytical Science).

AbstractRydevik, A. 2014. Drug Metabolites Formed by Cunninghamella Fungi. Mass SpectrometricCharacterization and Production for use in Doping Control. Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Pharmacy 186. 46 pp. Uppsala: ActaUniversitatis Upsaliensis. ISBN 978-91-554-8906-9.

This thesis describes the in vitro production of drug metabolites using fungi ofthe Cunninghamella species. The metabolites were characterized with mainly liquidchromatography-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 problemsassociated 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 wasinvestigated. The detected metabolites were compared with those formed in vitro and in vivo byhuman and horse and most phase I metabolites formed by mammals were also formed by thefungi. The higher levels of bupivacaine metabolites in the fungal samples allowed an extensivemass spectrometric structural characterization which shows that the fungi are relevant metabolicmodels.

Glucuronides are important drug metabolites but they are difficult to synthesize. Thediscovery that the fungus Cunninghamella elegans formed large amounts of glucosides ledto the idea that they could be used to form glucuronides. A new concept was developedwhere a fungal incubate containing a SARM S1 glucoside was mixed with the freeradical tetramethylpiperidinyl-1-oxy (TEMPO), sodium bromide and sodium hypochloritewhich produced a glucuronide. Isolation and characterization by nuclear magnetic resonancespectroscopy proved that the new method could produce glucuronides for use as referencematerial.

An investigation of reactive metabolite formation of the drugs paracetamol, mefenamic acidand diclofenac by the fungus Cunninghamella elegans was performed. It was demonstrated forthe 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 modelsformed identical metabolites.

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

Keywords: Cunninghamella blakesleeana, Cunninghamella elegans, Doping Control, DrugMetabolites, Glucuronide Production, Mass Spectrometry, Reactive Metabolites, ReferenceMaterial, Structural Characterization

Axel Rydevik, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry, Box574, Uppsala University, SE-75123 Uppsala, Sweden.

© Axel Rydevik 2014

ISSN 1651-6192ISBN 978-91-554-8906-9urn:nbn:se:uu:diva-220906 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-220906)

This thesis is dedicated to all thosewho have ever wanted a thesis

dedicated to them.

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Rydevik, A., Bondesson, U., Hedeland, M. (2012) 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. Rapid Com-munications in Mass Spectrometry, 26(11):1338–1346.

II Rydevik, A., Thevis, M., Krug, O., Bondesson, U., Hedeland, M. (2013) The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs). Xenobiotica, 43(5):409-420.

III Rydevik, A., Bondesson, U., Thevis, M., Hedeland, M. (2013) Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO. Journal of Pharmaceutical and Biomedical Analysis, 84:278-284.

IV Rydevik, A., Lagojda, A., Thevis, M., Bondesson, U., Hede-land, M. (2014) Isolation and characterization of a β-glucuronide of hydroxylated SARM S1 produced using a com-bination of biotransformation and chemical oxidation. In manu-script.

V Rydevik, A., Hansson, A., Hellqvist, A., Bondesson, U., Hede-land, M. (2014) A novel trapping system for the detection of re-active drug metabolites using the fungus Cunninghamella ele-gans and high resolution mass spectrometry. In manuscript.

Reprints were made with kind permission from the publishers.

Additional paper not included in this thesis

Guddat, S., Fußhöller, G., Beuck, S., Thomas, A., Geyer, H., Rydevik, A., Bondesson, U., Hedeland, M., Lagojda, A., Schänzer, W., Thevis, M. (2013) Synthesis, characterization, and detection of new oxandrolone metabolites as long-term markers in sports drug testing. Analytical and Bioanalytical Chemistry, 405(25):8285-8294.

Contents

Introduction ..................................................................................................... 9

Drug metabolism ........................................................................................... 11

Cunninghamella fungi .................................................................................. 13

Identification of drug metabolites ................................................................. 15 Analytical strategy for the identification of metabolites .......................... 15

Aims .............................................................................................................. 18

Results ........................................................................................................... 19

Discussion ..................................................................................................... 23 The fungi as a metabolism model ............................................................. 23 Drug metabolite production using fungi of the species Cunninghamella ....................................................................................... 28

Conclusions ................................................................................................... 33

Future studies ................................................................................................ 34

Svensk sammanfattning / Swedish summary ................................................ 35

Acknowledgements ....................................................................................... 38

References ..................................................................................................... 40

Abbreviations

C. blakesleeana Cunninghamella blakesleeana C. elegans Cunninghamella elegans CYP Cytochrome P450 DDA Data-Dependent Acquisition EMA European Medicines Agency FDA Food and Drug Administration HPLC High Performance Liquid Chromatography MS Mass Spectrometry MS/MS Tandem mass spectrometry MSn Multiple-stage mass spectrometry m/z Mass-to-charge ratio NMR Nuclear Magnetic Resonance Q-ToF Quadrupole-Time-of-Flight mass analyzer SARM Selective Androgen Receptor Modulator TEMPO Tetramethylpiperidinyl-1-oxy UPLC Ultra Performance Liquid Chromatography

9

Introduction

The Olympic Games in Athens in 2004 led to many more confirmed cases of doping than what was expected based on previous games [1]. In 2012 sam-ples from those Olympic Games were analyzed again using new methods, instruments and materials in an attempt to see if there were more doping cases that had not been discovered back in 2004. Thanks to the discovery that the fungus Cunninghamella elegans was able to produce two new long-term metabolites two medalists were caught for using the anabolic steroid oxandrolone and were subsequently stripped of their medals [2,3]. These cases would never have been discovered if it hadn’t been for metabolic stud-ies of drugs. Thus they make an excellent example of the importance of such studies.

The drugs being developed today are generally intended for therapeutic purposes; however some of them will be used for illicit purposes such as doping or drug abuse which means that there is a need to analyze them in doping control and forensic analysis. For all of these different areas of use it is important to know how the drugs are metabolized and how they can be detected by analyzing either the parent substance or its metabolites. This requires models that can be used to investigate how a drug is metabolized and production methods that can be used to create the metabolites for use as reference material.

In doping control and forensic analysis it is of interest to look at metabo-lites since a metabolite indicates that the substance has passed through the body and in many cases metabolites have a longer detection window after administration than the parent drug, e.g., the detection of metabolites of 19-norandrosterone and oxandrolone [2,4]. However one big problem is the lack of available reference material which is required for a verified result. Thus, new methods are needed which can be used to produce metabolites that can be difficult to synthesize with current methods, in quantities large enough for nuclear magnetic resonance (NMR) analysis and use as reference material.

In the development of a new drug the toxicity of the metabolites is readily investigated through the use of different in vitro models, both the European Medicines Agency (EMA) and the American Food and Drug Administration (FDA) have issued guidelines concerned with this [5–8]. Examples of in vitro models are human liver microsomes, sandwich-cultured hepatocytes from rat, mice and humans, hepatocellular carcinoma cells and more [9]. However most of these methods are expensive and they are performed in a

10

small scale which might make it difficult to isolate metabolites of interest for further analysis with mass spectrometry (MS) or NMR spectroscopy.

In this doctoral thesis fungi of the Cunninghamella species have been in-vestigated as a drug metabolism model that can also be used in the produc-tion of drug metabolites. It has shown the possibilities of these fungi, both as a metabolism model and as a way of producing metabolites, while also dem-onstrating a new important area of application for the Cunninghamella model.

11

Drug metabolism

Living organisms want to rid themselves of drugs that they are subjected to. They do this by making them more hydrophilic so that they can be more easily excreted [10]. The metabolism of a drug can be divided into two phases, phase I and phase II (see Figure 1 for an example), where phase I metabolism is the small additions or modifications of the drug creating func-tional groups while phase II metabolism is the conjugation of the functional groups of the drug with larger endogenous molecules such as glucuronic acid or glutathione [10,11].

Figure 1. An example of phase I and II metabolism of bupivacaine. In phase I an oxygen is added to the bupivacaine molecule and in phase II the phase I metabolite is conjugated with glucuronic acid.

The drugs functionalized in phase I can be excreted as they are or be sub-jected to further modifications. Some of the biotransformation processes involved in phase I metabolism are oxidation, hydrolysis dealkylation and reduction [11]. The most common of these processes is the oxidation [10] which can be seen in the large amount and variety of hydroxylated metabo-lites that are formed of drugs such as e.g., bupivacaine [12–14] which was studied in Paper I.

In phase II metabolism the unmodified drug or a phase I metabolite is conjugated with an endogenous molecule. In most cases the conjugation proceeds by an endogenous molecule replacing a hydrogen in a functional group, e.g., a hydroxyl group, in the drug or its metabolite [15]. The conju-gating molecules are hydrophilic and thus the resulting phase II metabolites are usually readily excreted in the urine [15]. In mammals a very common

CH3

CH3

NH

O

N

OH

CH3

CH3

NH

O

N

Parent molecule

Phase I metabolite

Phase II metabolite

O

OH

OHOH

OHO

O

CH3

CH3

NH

O

N

12

phase II conjugating agent is glucuronic acid which creates a sugar conjugate with the substrate. However, plants seem to prefer the use of glucose when creating sugar conjugates as in the case for diclofenac [16]. Other common phase II reactions are glutathione conjugation and sulphation.

In most cases the biotransformation of a drug results in metabolites with low or no pharmacological effect but in some cases the resulting metabolites have a higher pharmacological effect or they may even be toxic [17]. The increase in pharmacological effect can be utilized when a drug has a low bioavailability. By creating a so called pro-drug it is possible for the drug to reach the target area in the body and there gain the pharmacological effect when it undergoes a biotransformation process, an example of this is amitriptyline where it is its metabolite nortriptyline that has the pharmacol-ogical effect [18]. Drug toxicity can arise from the biotransformation when a metabolite is electrophilic and react with e.g., a protein. A classic example is paracetamol that can cause liver damage [19]. This damage is averted by conjugation of paracetamol’s reactive metabolite with glutathione [15,20] but when the reserves of glutathione run low serious liver damage can be the result.

The modifications in the metabolism process are enzyme driven and some groups of enzymes are very important. The enzymes that are responsible for the metabolism of a certain drug will affect the choice of which in vitro model to use in metabolic studies of the drug. The cytochrome P450 (CYP) enzyme superfamily is one of the most important groups of metabolizing enzymes responsible for metabolizing drugs [21]. It contains a very large number of enzymes and they can be found in plants, animals and fungi e.g., of the Cunninghamella family [22–24]. CYP enzymes are responsible for the oxidation and reduction of many different drugs e.g., propranolol, quinidine and fluoxetine [25]. Therefore models for predicting and understanding the CYP metabolism of drugs are very important for the pharmaceutical industry [26]. However CYP enzymes are only involved in phase I metabolism so it is also important to have models that possess conjugating enzymes that are involved in phase II metabolism. Important enzymes for phase II are UDP-glucuronosyltransferases, glutathione S-transferases and sulphotransferases [15].

13

Cunninghamella fungi

Research on the metabolism of drugs and chemicals by fungi of the species Cunninghamella has been performed at least since the late 1970s and early 1980s [24,27–29]. They have been shown to be possible to use as in vitro models of drug metabolism [30]. The fungi are so called filamentous fungi that can be found in soil and their appearance is similar to cotton (see Figure 2). Fungi are not the only type of microbes that are currently studied as metabolic models [31]. There are studies that have shown that fungi of the Cunninghamella species produce mammalian metabolites of different drugs such as meloxicam [32,33], naproxen [34], amitriptyline [35], omeprazole [36], clemastine [37] and more.

These fungi have metabolizing enzymes that can perform both phase I and phase II biotransformations [22–24,27,29] and their metabolism of a large number of substances have been investigated, as can be seen in a re-view from 2009 by Asha and Vidyavathi [30]. Apart from metabolism stud-ies the fungi have also been used to produce metabolites in amounts large enough for NMR characterization [38,39] while other studies have focused on optimizing the yield of metabolites through chemometrics [40] and modi-fying the media components [41].

The experimental procedure of cultivating the fungi and using them in biotransformations is simple, requires a low input of work and has a low cost. The protocol used in the studies presented in Papers I-V is described visually in Figure 2 and is as follows.

Figure 2. Schematic view of the experimental procedure for fungal incubations.

Agar plates are inoculated with fungus

Sample is used for analysis

Incubation at 26.5 °C

Fungal suspension used for incubations

Broth and fungal suspension are mixed

Drug is added to the fungus

The incubation is terminated with acetonitrile

Incubation at 26.5 °C

Incubation at 26.5 °C

Fungus is removed from the plate and mixed with saline solution

14

The fungi were grown on agar plates for 3-4 days in temperatures around 27 °C in an incubator after which the cultures were transferred to containers with saline solution (0.86 – 0.9 % in water) which were stored at 2 – 8 °C. Small aliquots of these were then grown in a broth in Erlenmeyer flasks. The broth can be prepared from premixed powders that are commercially avail-able or a specific mix can be created from scratch and they need to contain a carbon source and a nitrogen source. The Erlenmeyer flasks were incubated at approximately 27 °C for 2-4 days and the substrate was then added. After another period of incubation, that could go on for a week or more, the incu-bation was terminated through the addition of acetonitrile. Samples were then taken for pretreatment or direct analysis. In the review by Asha and Vidyavathi [30] more examples of the experimental protocol for Cunning-hamella fungi are presented.

15

Identification of drug metabolites

As has been described above there are a number of reasons for identifying drug metabolites, e.g., for use of the metabolites as analytical targets in dop-ing control. Several different analytical techniques have been used for the analysis of drug metabolites and today the main detectors used are mass spectrometers and NMR spectrometers [11]. Both these types of detectors give a lot of structural information which is critical for elucidating the struc-ture of a drug metabolite. An NMR instrument has the advantage that it can give the full structure of the metabolites describing the positions for different modifications however it lacks sensitivity compared to an MS instrument. The mass spectrometer on the other hand can give structural information even when the metabolite concentration is low but the exact positions of different substituents are often difficult to determine.

For more comprehensive information on the identification of both phase I and phase II metabolites using HPLC and MS there are two excellent re-views available [11,42].

Analytical strategy for the identification of metabolites The most important thing when analyzing drug metabolites using MS is to have a strategy in place for the search. Without a proper strategy even the most sophisticated instruments and programs are of little use. It is the au-thor’s experience that the process of looking for metabolites can be divided into four different steps with interpretation of data being part of steps two through four.

The first step is to create a list of the parent drug, the possible metabolites and their corresponding expected m/z values; this can be done manually or through the use of specialized software. When setting up the expected list of metabolites each expected biotransformation is listed along with its corre-sponding change in m/z. Each biotransformation is correlated to a nominal change in m/z and a change in accurate mass. The nominal change is mainly used when using a low resolution MS instrument which was the case in Pa-per I. If high resolution MS (HRMS) is used then the most important part in the m/z change is the change in the accurate mass since it is the accurate mass that contains the key to the elemental composition of the metabolite. Each element has a specific accurate mass, e.g., 1.0078 for hydrogen and not

16

1.0000, so every molecule will have an accurate mass that is dependent on its elemental composition. The higher the resolution and mass accuracy of the instrument the better is the information given by the mass defect.

The second step is to run a full scan experiment and then search for the expected m/z values. The detected analytes should then be investigated in a third step with tandem MS (MS/MS) so that characteristic fragments sup-porting their identities can be observed. Depending on the instrument being used steps two and three are either performed separately or as a combined step. The ion trap used in Paper I made it necessary to perform them as two separate steps while the quadrupole-time of flight (Q-ToF) instruments used in Papers II - V made it possible to combine these steps. Most modern high resolution instruments, such as the Q-ToF, can through the use of different techniques, e.g., data-dependent acquisition (DDA) and the scan technique MSE, collect MS/MS information on all metabolites of interest in one single run [43]. MSE is a technique where the instrument alternates between a low collision energy state and a high collision energy state and collects the data in two separate channels. These two traces then enable the software to pre-sent pseudo MS/MS data for each point in time throughout the whole chro-matographic run. When using these techniques the MS/MS information is obtained through data mining instead of running specific MS/MS experi-ments as is the case when running steps two and three as two different steps.

The fourth and final step is to search for unexpected phase I and II me-tabolites. This search can be performed by visual inspection of the chroma-tograms and by comparing the chromatograms of a sample and a control, e.g., a sample from an incubation with fungus but without the drug substrate. Any clear peaks observed in the sample or smaller peaks that are present in the sample but not in the control are then investigated. An example compari-son of the identification of metabolites can be seen in Figure 3. The search for unexpected metabolites can also be performed using neutral loss scan and precursor ion scan. Phase II metabolites tend to have characteristic neutral losses depending on the conjugating agent, sulphate conjugates show a neu-tral loss of 80 Da as an example, that can be scanned for. This is either done through experiments that only detect analytes exhibiting this characteristic loss (Paper I) or through data mining of the collected data (Papers II – V). It is also common that drug metabolites have fragments in common with the parent drug. A precursor ion scan can therefore be run to identify metabolites that exhibit these fragments.

17

Figure 3. A comparison of two base peak intensity chromatograms from a full scan analysis with UPLC-Q-ToF using a scan range of m/z 50 - 1200. The peaks visible in the fungal sample with the Selective Androgen Receptor Modulator (SARM) S1 (top) and not in the blank (bottom) should be investigated for the presence of me-tabolites.

Fungus + SARM S1

Only fungus

M11 M8

M7 + M7b

M3 + M13

M5 + M15 + M14

M6

M1b

M1cM2

M1a

S1

M10

M12

18

Aims

This thesis has been focused on the use of Cunninghamella fungi as a model for drug metabolism and a method to produce drug metabolites. The follow-ing aims have been pursued:

• To investigate the similarities in the metabolism of bupivacaine in

horse and the fungi Cunninghamella elegans and Cunninghamella blakesleeana (Paper I)

• To structurally characterize the produced metabolites of bupiva-caine through different mass spectrometric experiments (Paper I)

• To investigate the metabolism of the selective androgen receptor modulators S1, S4, and S24 by Cunninghamella elegans and compare it with mammalian metabolism (Paper II).

• To investigate the possibility of creating glucuronic acid conju-gates from glucose conjugates formed by the fungus Cunning-hamella elegans from the selective androgen receptor modulators S1, S4, S22 and S24 (Paper III)

• To produce, purify and NMR characterize the glucuronidated and hydroxylated selective androgen receptor modulator S1 (Paper IV) for possible use as reference material.

• To investigate the possibility of using Cunninghamella elegans as a model of reactive metabolite formation through detection of glu-tathione, glutathione ethyl-ester and cysteine conjugated phase II metabolites indicative of toxic metabolites (Paper V).

The overall aim of this thesis is to put the results from Papers I – V in a larger context. It also aims to clearly show the arguments for why fungi of the Cunninghamella species are useful as metabolism models and for the production of drug metabolites.

19

Results

The five different studies that have been performed can be divided into three separate areas. The results from the first study describe the metabolism of bupivacaine and the focus was on the structural characterization of the de-tected metabolites. The results from the second, third and fourth study are all connected where the results from the first two studies led to the fourth. Their combined focus is the production of drug metabolites that can be used as reference material. The focus of the final area is defined by the fifth study and it is the investigation of drug toxicity screening with the fungus Cun-ninghamella elegans. The structures of the different drugs that have been investigated are shown in Figure 4.

Figure 4. The chemical structures of the different drugs that have been investigated during the work with this thesis.

In Paper I a mass spectrometric investigation was performed on metabolites of the local anesthetic bupivacaine which is banned in horse racing due to animal health concerns. The investigation showed that the fungi C. elegans and C. blakesleeana formed two aromatically and five aliphatically hydroxy-

F3C NH

O

O

CH3 OH

F

NC

F3C NH

O

O

CH3 OH

NH

CH3

O

O2N

F3C NH

O

O

CH3 OH

F

O2N

F3C NH

O

O

CH3 OH

NC

CN

CH3

CH3

NH

O

N

NH

OHO

NH

Cl

Cl

OH

O

NH

OH

O

Bupivacaine(Paper I)

Diclofenac(Paper V)

SARM S22 (Paper III)

SARM S24 (Paper II and III)

SARM S1 (Paper II, III and IV)

SARM S4 (Paper II and III)

Mefenamic Acid(Paper V)

Paracetamol(Paper V)

20

lated metabolites of bupivacaine. The structures of the aliphatic metabolites were investigated using hydrogen/deuterium exchange and MSn experiments using an ion-trap mass spectrometer. Due to the high amount of sample and high metabolite abundance this resulted in a more detailed structural under-standing of the metabolites than what would be possible using in vivo sam-ples. It was also shown that the fungi formed the mammalian metabolites 3’-OH- and 4’-OH-bupivacaine. This means that fungal incubates can be used in an initial doping control screening.

In Paper II the metabolism of the SARMs S1, S4 and S24 by the fungus Cunninghamella elegans was investigated. Analysis with HRMS, using a Q-ToF instrument, showed the presence of both phase I and phase II metabo-lites. The MSE capability of the instrument gave an easier metabolite identi-fication thanks to the continuously collected pseudo MS/MS information. The most common modification was hydroxylation and in many cases it was combined with other changes e.g., conjugation with sulfate. The main results were that the fungus can produce mammalian metabolites of the SARMs and that it has a capability to perform a wide variety of metabolic reactions. It was also found that one of the main fungal metabolites of SARMs S1 and S24 was a hydroxylated and glucosidated phase II metabolite. It was this result that led to Paper III since the glucose conjugate of hydroxylated SARMs S1 and S24 had glucuronide analogues in mammals. Due to the existence of the glucuronide analogues the question was asked if the gluco-sides could be used in some way to produce the glucuronic acid conjugates.

In Paper III a study was performed with the aim to see if the glucose conjugates that were described in Paper II could be chemically oxidized to glucuronides. It was demonstrated for the first time that a reaction using the free radical tetramethylpiperidinyl-1-oxy (TEMPO) could turn the gluco-sides formed by the fungus C. elegans into glucuronic acid conjugates. This reaction could be performed directly on the fungal incubate and the glu-curonide could be detected when injected without any sample treatment. The reaction was successful for the SARMs S1, S22 and S24 and a comparison with human liver microsomes showed that the method produced the same metabolite as the microsomes (Figure 5).

21

Figure 5. A comparison of chromatograms and MS/MS spectra between the glu-curonides of SARMs S24 (top two), S22 (middle two) and S1 (bottom two) formed by human liver microsomes and the TEMPO oxidation of glucosides formed by Cunninghamella elegans in Paper III. The analysis was performed using a UPLC-Q-ToF instrument.

In Paper IV a proof of concept study was performed that showed that the method presented in Paper III could be used to produce glucuronic acid conjugates for use as reference material. A glucuronide of hydroxylated SARM S1 was produced using the TEMPO reaction and it was isolated and purified using solid phase extraction and preparative liquid chromatography. The isolated product was characterized with NMR and the structure was determined (See Figure 6). This proved that the method from Paper III could be used to produce glucuronides for use as reference material in e.g., doping control.

S24 liver microsome

S24 fungus

S22 liver microsome

S22 fungus

S1 livermicrosome

S1 fungus

22

Figure 6. An NMR spectrum of hydroxylated and glucuronidated SARM S1. This was one of the spectra leading to the determination of the structure of the glucuron-ide in Paper IV.

In Paper V a new area of application was investigated for the fungus C. ele-gans. Drugs known to form toxic metabolites were incubated with the fungus and trapping agents. A mass spectrometric study, using a Q-ToF mass spec-trometer, showed that C. elegans could form both glutathione and cysteine conjugates of the investigated drugs. The detection of these conjugates proves that the fungus can produce toxic metabolites of these drugs (See Figure 7). This important finding means that it would be possible to use the fungus to screen drugs for metabolites that are indicative of drug toxicity.

Figure 7. MS/MS spectra showing the fragments for glutathione (A), glutathione ethyl-ester (B), cysteine (C) and N-acetylcysteine (D) conjugates of hydroxylated mefenamic acid formed by the fungus Cunninghamella elegans in Paper V. The analysis was performed using a UPLC-Q-ToF instrument.

GSH

GSH-EE

Cysteine

N-acetylcysteine

A)

B)

C)

D)

23

Discussion

The fungi as a metabolism model As has been described above metabolism studies are essential for doping control analysis to find analytical targets. A detected drug metabolite in-creases the confidence of the positive result since a metabolite indicates that the drug has passed through the body. The detection window can also be significantly extended through detection of metabolites instead of the drug. The detection of the metabolite 18-nor-oxandrolone increased the detection window from three days for the intact drug oxandrolone to six days and by detecting a new metabolite the detection window was increased to eighteen days [2], a threefold increase, which shows the importance of metabolism studies to make it more difficult for cheaters in sports.

In the development of new drugs any metabolites identified in metabolic studies that are present above a threshold value (“10 % of total drug related exposure” [7,8]) needs to be characterized and investigated for toxicity and pharmacological effect. This investigation should be performed at an early stage in the development process before the initiation of clinical trials using both in vitro and in vivo models though unfortunately in some cases the tox-icity is not detected until the final trials or after the release of the drug.

Fungi from the species Cunninhamella have been investigated as an in vi-tro metabolism model for more than 30 years. In a review from 2009 by Asha and Vidyavathi [30] a summary of several studies on different com-pounds (both drugs and chemicals) have been put together as a table with many of the studies finding that mammals and fungi can produce the same metabolites. However, few studies have been performed that have been con-cerned with equine metabolites or that have focused on how the model can benefit doping control analysis. A small number of studies have described the formation of glucuronides but they are old and most of them only de-tected the UDP-glucuronidase enzymes [23,27,28]. Also, none of the studies has proven the fungal model’s capacity to predict drug toxicity which is an important area of use for drug metabolism models.

This thesis includes three papers where metabolic studies have been per-formed. The results in Paper I and Paper II are interesting from a doping control perspective and show the similarities in drug metabolism of bupiva-caine and the SARMs S1, S4 and S24 between fungi and mammals. From a toxicological perspective the results in Paper V are more interesting since

24

they show that the fungus Cunninghamella elegans can produce phase II conjugates indicative of reactive metabolites. These results add to the knowledge base of the metabolic capabilities of the fungi and introduce a new area of use.

It is important to point out the potential areas of use for drug metabolism models. It has been observed that the fungi tend to produce a relatively rich pattern of hydroxylated metabolites. This has been observed in both Paper I and Paper II and it has been shown in papers by other authors as well [35,37,44,45]. Based on this one area of use for the Cunninghamella fungi are as a hydroxylation model. In addition to this Papers I, II and V show that the fungi exhibit a rich pattern of different metabolic reactions. There-fore another area of use is investigations of the possible biotransformation pathways that a newly developed drug might undergo. The results in Paper V that show for the first time that the fungus Cunninghamella elegans can produce glutathione and cysteine conjugates of drugs with known toxicity clearly show that a new area of use could be as a predictive model for drug toxicity.

Paper I is a good example of the prominent oxidation processes in the Cunninghamella fungi. The results in Paper I, that investigated the metabo-lism of bupivacaine in the fungi C. elegans and C. blakesleeana, showed several different hydroxylated metabolites in the incubates. The different bupivacaine metabolites that were observed can be seen in Figure 8. In addi-tion to the main metabolites also found in horse, 3’-OH-bupivacaine [46], and in man, 4’-OH-bupivacaine [47], several other hydroxylated metabolites were also detected some of which were also observed in horse urine. All the phase I metabolites detected in horse were also detected in the fungi. It is important that the detected aromatically hydroxylated metabolites also have been found in horse [46], human [12], monkey [14] and rat [14,48] since this shows the relevance of the fungal model from a mammalian perspective.

25

Figure 8. A metabolic map, from Paper I, showing the metabolites formed by Cun-ninghamella fungi and horse. Metabolites with their name in blue were found in both fungi and horse while those marked in red were only found in the horse. Me-tabolites with their names in black were only found in the fungal samples.

Paper II also presents results that show the oxidative capabilities of the fun-gus Cunninghamella elegans. The analysis of fungal incubates for the SARMs S1 and S24 showed that the main phase I metabolism reaction was hydroxylation. The hydroxylated metabolites constituted more than 50 % of the detected metabolites for each of the two SARMs. More importantly the findings also demonstrated the relevance of the fungus as a metabolic model for the substance class arylpropionamide SARMs. This was observed through comparisons of samples of the SARMs S4 and S24 formed from either incubation with human and equine liver microsomes or through incu-bation with the fungus C. elegans. This comparison showed that all the im-portant isomers formed by human and equine liver microsomes were also formed by the fungus and that the isomeric pattern for SARM S24 was the same for both human and fungus (see Figure 9). This match in the isomer pattern between human and fungus clearly show the potential that the fungus has as a predictive drug metabolism model.

NH

O

N1'

2'3'

4'

5'

6'

12

3

4

5

6

NH

O

N

OH

NH

O

N

HOC

NH

O

N

HOOC

NH2

NH

O

N

OH

NH

O

N

OH

NH

O

N

OH

NH

O

NO

NH

O

N

O

Gluc

NH

O

N

OH

NH

O

N

OH

OGluc

A1

Aldehyde

B4

4’-OH-bupivacaine

B5

B1, B2, B3

3’-OH-bupivacaine4’-OH-bupivacaine glucuronide

3’-OH-bupivacaine glucuronide

Dihydroxybupivacaine

Sulfated hydroxybupivacaine

N-desbutylbupivacaine

Carboxylic acid2,6-dimethylaniline

Dihydroxy glucuronide (2 isomers)

NH

O

N

OH

OH

NH

O

NHOSO3

NH

O

N

OGluc

OH

NH

O

NH

Bupivacaine

26

Figure 9. Comparison of the isomeric pattern for hydroxylated SARM S24 metabo-lites in equine and human liver microsomes and the fungus Cunninghamella ele-gans. The figure is from Paper II.

A study by Amadio et al. [45] where the biotransformation of flurbiprofen by various Cunninghamella strains was investigated is another good example of the oxidative capabilities of the fungi. Seven metabolites formed by the fungus C. elegans were detected that all had been hydroxylated and the main metabolite was also formed in humans [49]. Their study shows, just like the results in Paper I and II does, that these fungi can produce several different hydroxylated isomers.

One important aspect of the results in Paper I is the difference in metabo-lite formation between different Cunninhamella strains. This difference was most notable for the sulphate conjugates of hydroxylated bupivacaine where C. blakesleeana formed two isomers in approximately similar amounts while C. elegans favored the formation of one of the isomers and produced it in much larger amounts than the other (see Figure 10). This difference could be due to different formation of the hydroxylated phase I metabolites that were then conjugated. However, the difference in their formation, although pre-sent, was not as obvious. These results lead to the conclusion that not every Cunninghamella fungi is a suitable model for every substance. Instead inves-tigations should be performed on several strains in a parallel screening proc-ess. This is in line with one of the benefits of microbial models listed by Pervaiz et al. [31].

RT: 0.00 - 15.00

0 2 4 6 8 10 12 14

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Rel

ativ

e A

bun

danc

e

0

20

40

60

80

1007.49

6.91 8.116.15 8.57

6.95

7.55

8.16

6.97

7.55

8.172.06 8.61 11.06

Equine

C. elegans

Human

27

Figure 10. A comparison showing the differences in formation of sulphate conju-gates of hydroxylated bupivacaine by the fungi Cunninghamella elegans and Cun-ninghamella blakesleeana. The figure is part of a larger figure presented in Paper I.

In the author’s opinion, the results in Paper V are the most important for the argument that fungi of the Cunninghamella species can be used as an impor-tant metabolic model for humans. Drug toxicity is one of the major reasons for the failure in phase III trials for new drugs being developed [50] making the studies of drug toxicity very important to pharmaceutical companies. Thus it is very important that drugs that can form toxic metabolites are iden-tified as early as possible during their development process in order to re-duce costs as well as to minimize the potential harm to humans and animals. In Paper V it is demonstrated for the first time that the fungus C. elegans can form both glutathione and cysteine conjugates of paracetamol, me-fenamic acid and diclofenac which are all known to form toxic metabolites (see Figure 11 for an example).

0 2 4 6 8 100

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

Fungal incubate, C. elegans

Fungal incubate, C. blakesleeana

Time (min)

28

Figure 11. A comparison of mass spectra for a glutathione conjugate of paracetamol formed by the fungus Cunninghamella elegans and human liver microsomes. The figure is from Paper V. The analysis was performed using a UPLC-Q-ToF instru-ment.

The detected conjugates are indicative of these toxic metabolites and it is this kind of indicative model that is needed to discover previously unknown toxic metabolites. It has been shown previously that Cunninghamella ech-inulata can form the toxic metabolite responsible for the toxicity of paracetamol [51]. However, the detection of previously known toxic metabo-lites does not show that the model can be used to identify unknown toxic metabolites. Since conjugation with glutathione is a metabolic way to neu-tralize toxic metabolites [15,20] the detection of such conjugates is clear evidence that the model can be used in the search for unknown toxic metabo-lites. This is the most important conclusion in Paper V. One of the main advantages of using the fungal model compared to human liver microsomes is that a larger volume of sample is obtained. This allows for the isolation of larger amounts from each sample.

Together the Papers I, II and V show that fungi of the Cunninghamella species have a role to play as a metabolic model. They show that the fungi can form metabolites that are relevant for both human and equine doping control analysis and in Paper V it is demonstrated that the model can be used in the very important assessment of drug toxicity.

Drug metabolite production using fungi of the species Cunninghamella There are several areas of analysis that are concerned with drugs and drug metabolites in biological samples. In doping control blood and urine samples

Cunninghamella Elegans

Human liver microsomes

29

are analyzed in order to catch the users of banned substances [4,52,53]. In forensic analysis many types of biological samples are analyzed for a multi-tude of reasons e.g., detecting drug abuse [54–56]. The pharmaceutical in-dustry does a lot of investigations into drug metabolism to ascertain that a drug is safe for use before it is released to the market [57,58].

All these different areas have a common need for characterized reference material of the drugs and drug metabolites that they are investigating. For a positive result in doping control to be accepted as verified a comparison has to be made with a positive control [59,60] while the pharmaceutical industry has a need for larger amounts of characterized drugs and drug metabolites in order to be able to assess their safety. Unfortunately the commercial avail-ability of drug metabolites, especially phase II metabolites, is low and those that are available are expensive. This means that there is a need for new methods that can be used to produce needed metabolites and this thesis will show that Cunninghamella fungi can be one of those methods.

Paper IV included in this thesis and another paper not included in the thesis by Guddat et al. [2], describe the production of NMR characterized metabolites that could be used or were used as reference materials. In Paper IV a glucuronide of hydroxylated SARM S1 was produced, purified and characterized by NMR while Guddat et al. 2013 describes the production of a metabolite of oxandrolone that was used successfully as an indirect refer-ence material in doping control analysis on samples from athletes participat-ing in the Olympic Games in Athens 2004.

One of the most classical methods of producing drugs and drug metabo-lites is through chemical synthesis. It is an important method for the pharma-ceutical industries and it has the possibility to produce large amounts of drugs and metabolites [61]. This can be an expensive and complicated pro-cedure so new methods are needed that can simplify the process. A number of different studies have investigated new methods that can be used to pro-duce drugs and/or their metabolites utilizing both chemical [62–66] and bio-technical solutions [36,38,67–71]. However, there is still a need for new methods.

Perhaps one of the more promising ways is to create new methods using both chemical synthesis in combination with biotransformation processes. Zöllner et al. [72] used such a combined chemical and biotransformation approach when they used recombinant yeast strains to produce a long term metabolite of methandienone that could be used as a reference material in doping control. In 2012 two medalists from the 2004 Olympic Games in Athens were stripped of their medals due to methandienone use, this was due to a long term metabolite characterized in 2006 [73] and synthesized with the method developed by Zöllner et al.

Using a combined approach with both biotransformation and chemical synthesis also make it easier to create metabolites of the correct stereochem-istry. It can be difficult to synthesize the correct isomer but biological sys-

30

tems tend to form specific isomers thanks to the specificity of the enzymes involved in the metabolism. In the paper written together with Guddat el al. [2] (not included in the thesis) the difficulties with synthesizing the correct isomers of the metabolite 17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one and its isomer 17α-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one led to the investigation of whether the fungus Cunninghamella elegans could be used to produce them instead (the biotransformation process is described in Figure 12).

Figure 12. The structural modification performed by the fungus Cunninghamella elegans in the production of the new long term oxandrolone metabolites [2].

The results showed that the fungus could form the desired metabolite if it was incubated with a precursor and the metabolites were then isolated and characterized by NMR. The success of this investigation resulted in two medalists being caught for oxandrolone use and lost their medals eight years after the Olympic Games in Athens.

Glucuronic acid conjugates tend to be especially difficult to synthesize [63] so their availability is low but they are very important both in doping control [59,60] and in the safety assessment of drugs [63] so efficient meth-ods of preparing them is needed. One of the ways to produce glucuronides that have been investigated is the selective oxidation of glucosides into glu-curonides using the free radical TEMPO [62,64,66]. It has been shown pre-viously, in a synthetic chemistry setting, that mixing a glucoside with TEMPO in the presence of NaBr and NaOCl will oxidize the glucoside to a glucuronide with high efficiency. However it has not been previously shown that the reaction will work when used with complex samples instead of pure solutions.

Paper III and Paper IV showed that the TEMPO reaction can be used in combination with biotransformation processes. Together the papers demon-strated, for the first time, that the combination of biotransformation and se-

O

OH

O

OH

OH

O

OH

OH

31

lective oxidation was a possible way for doping control laboratories to pro-duce glucuronides that can be used as e.g., reference material (See Figure 13).

Figure 13. An NMR spectrum of hydroxylated and glucuronidated SARM S1. This was one of the spectra leading to the determination of the structure of the glucuron-ide in Paper IV.

The developed method from Papers III and IV can fill an important func-tion for these laboratories since the experimental protocol for the fungal incubations is simple. It is also quite cheap to set up since the total cost of the starting material needed (fungi, broth, agar plates and saline solution) amount to approximately 600-700 euro. This will then be enough for, at the very minimum, 200-300 incubations before new broth has to be purchased or new fungal cultures have to be grown on agar plates. Each of these incuba-tions will be enough for the production and isolation of glucuronides at the microgram scale.

While micrograms of metabolites are enough amounts for use as reference material other areas need higher amounts. The TEMPO reaction, for exam-ple, would be more easily controlled if the substrate measured in milligrams instead of micrograms. Unfortunately in some cases the yield of drug me-tabolites is low. Thus, it would be beneficial if it was possible to affect the biotransformation or scale it up so that metabolites could be isolated in mil-ligrams or more. Experiments were made where different additives were mixed with the fungi and broth during the fungal incubations. Some caused the fungus to obtain a larger biomass than without them (See Figure 14).

32

Others changed the metabolic profile of the fungus. Unfortunately the varia-tion between replicates was too large for any clear conclusions to be drawn.

Figure 14. The fungus on the left was grown with St. John’s wort as an additive while the fungus on the right was grown in ordinary broth (control). As can be seen the additive caused the fungi to grow more than the control.

In a study performed by Pearce and Lushnikova [36] a scaled up biotrans-formation process was used to incubate 40 mg of omeprazole with the fun-gus. This enabled the isolation and purification of three metabolites that were characterized NMR analysis. The same approach used by Pearce et al. [36] should be applicable to the results of Paper V that showed that large amounts of glutathione, cysteine and N-acetylcysteine conjugates could be found in incubates with paracetamol, mefenamic acid and diclofenac. Simply by scaling up the incubation from the 1 mg that was used to 40 mg of sub-strate used it should be possible to obtain these conjugates in amounts large enough for NMR analysis and further safety assessment. This would further prove that the fungus can be used to investigate the possible toxicity of drugs. Not only by identifying the types of conjugates (e.g., glutathione and cysteine conjugates) indicative of toxicity but also by structural characteriza-tion of the metabolites giving an enhanced understanding of the toxicity mechanism.

The results from Paper IV and the other described results all clearly sup-port the fact that fungi of the Cunninghamella species have a role to play in the production of drug metabolites that can be used as characterized refer-ence material in doping control and other areas.

33

Conclusions

In this thesis it has been shown that fungi of the Cunninghamella species are valuable drug metabolism models that can also be used for the production of drug metabolites.

It has been demonstrated in Paper I and Paper II that Cunninghamella fungi can form mammalian metabolites of bupivacaine and the SARMs S1, S4 and S24. A new method was introduced in Paper III and a proof-of-concept study on it was performed in Paper IV. The method can be used to produce glucuronides of different drugs without any difficult or expensive synthetic procedures. In Paper V it was demonstrated for the first time that the fungus C. elegans can form phase II metabolites indicative of toxic drug metabolites. The detection of glutathione, glutathione ethyl-ester and cys-teine conjugates mean that the fungus can be used in the drug development process to identify possible drug toxicity at an early stage.

The discussion above has shown that the results in Papers I – V are part of a larger context. Papers I and II are two more pieces of evidence that show the potential of the fungal model Cunninghamella as a metabolism model. Together with results published by other authors they make a con-vincing argument that this model should be more extensively used in the future. In particular these two papers show the potential of the model within the area of doping control. Paper V also builds onto the argument that this fungal model is a good drug metabolism model. Even more important is that as the discussion shows there is a need for drug metabolism models that can be used in the assessment of drug toxicity and that the fungus C. elegans can fill this need.

It was discussed that there is a need for new ways to produce drug me-tabolites. There are published papers that show that it is possible to produce drug metabolites through incubations with fungi and other microorganisms. However, the results in Paper III and IV show for the first time that it is possible to produce glucuronides using C. elegans. The developed method, as the discussion shows, can help fill the need for new glucuronide produc-tion methods.

To summarize, this thesis have introduced two new areas of application for the fungal model Cunninghamella. It has also added to our understanding of the metabolic capabilities of the model something which is necessary for its future use.

34

Future studies

The need for in vitro and in vivo drug metabolism models and metabolite production methods will always exist. It is the author’s firm belief that fungi of the species Cunninghamella have a role to play as one of these models. However there are more research and development that needs to be per-formed to increase the utility of the model.

The method that has been developed to produce and isolate glucuronide conjugates using the fungi needs to be further investigated so that it can be better understood and improved to increase the yield. One approach that should be tested is to scale up the incubation so that the glucoside substrate for the oxidation can be isolated and measured in milligrams before oxida-tion. Another possible route of interest is to use immobilized TEMPO which could lead to better control of the oxidation process and make the method more environmentally friendly.

Further studies on the metabolism of drugs with known toxicity issues should be undertaken. This would create a better knowledge base from which the model could be introduced as a drug toxicity predictor that would also have the capability of producing potentially toxic metabolites in large amounts.

A very interesting method of increasing the yield of metabolites that has been investigated [74] is the immobilization of the fungi to create bioreactors that can be used over and over and that would have an increased contact surface compared to fungi growing on the surface of the broth. Further stud-ies should be performed to see if the immobilization can also increase the yield of metabolites from drugs other than the already tested. However, it is a promising method that could help further the use of Cunninghamella fungi as a metabolite production method.

From a wider perspective it is the author’s hope that research would go into genetic modification of these fungi to further enhance their oxidative and conjugative capabilities. If their enzymatic expression can be changed to be more directed towards specific types of metabolites then that is where their true potential lies. In their current state they are perfect for small labs that want a model that can give a big range of different products. However, if they are to be used in a larger setting then they have to be able to give higher yields of a smaller number of metabolites.

35

Svensk sammanfattning / Swedish summary

Sommaren 2012 skedde en ny analys av åtta år gamla prover från OS i Aten 2004. Syftet var att med modernare metoder se om det fanns några fler fall av doping som inte hittades vid den ursprungliga analysen. Tack vare en nedbrytningsprodukt för den anabola steroiden oxandrolon som framställts med hjälp av trådsvampen Cunninghamella elegans så kunde två nya fall avslöjas som ledde till att två medaljörer blev av med sina medaljer.

Oavsett hur vi får i oss läkemedel så kommer de att brytas ner i kroppen. Denna process, kallad metabolism, syftar till att förändra substanserna så att de blir mer vattenlösliga och därmed lättare kan utsöndras från kroppen.

Metabolismen gör att det kan vara svårt att detektera användningen av olika läkemedel genom analys av t.ex. blod- och urinprover. Nedbrytningen gör att det inte är säkert att den substans som togs är den substans som ut-söndras utan det kan istället vara en så kallad metabolit (nedbrytningspro-dukt) som utsöndras istället. För att därför säkert kunna detektera t.ex. an-vändningen av en dopingsklassad substans så måste man titta efter en eller flera metaboliter. Fördelen med att detektera en metabolit är att en metabolit visar att substansen måste ha gått igenom kroppen vilket stärker rättssäker-heten i analysen då ett förorenat prov inte borde innehålla några metaboliter. I flera fall så går det också att detektera metaboliter långt efter det att den tagna substansen passerat igenom kroppen. När en substans detekteras i ett dopingprov så genomförs en jämförande analys tillsammans med ett refe-rensmaterial för att substansens identitet ska bekräftas. Ett problem är dock att det i många fall saknas rena metaboliter som kan användas som refe-rensmaterial vid analyserna.

Läkemedelsmetabolismen är många gånger en positiv process som hjälper kroppen att ha kontroll över vilka ämnen som den har i sig men ibland så leder den till att skadliga metaboliter bildas. Ett exempel är nedbrytningen av paracetamol. När paracetamol bryts ner i kroppen så bildas bl.a. en metabolit som kan binda till proteinerna i levern och på så sätt ge leverskador. Det är därför väldigt viktigt vid utvecklingen av läkemedel att metabolismen kart-läggs grundligt så att läkemedel med skadliga metaboliter kan stoppas innan de testas på djur eller människor.

På grund av anledningarna ovan så är det viktigt med studier av olika lä-kemedels metabolism så att måltavlor för dopingkontroll kan hittas och för att skadliga läkemedel ska stoppas tidigt i läkemedelsutvecklingen. Dessa studier kan utföras med olika typer av modeller som antingen kan vara cel-

36

ler, mikroorganismer, djur eller människor. Trådsvamparna av släktet Cun-ninghamella är en av de modeller som undersöks och som har visat sig vara användbar för metabolismstudier.

I den här avhandlingen så har ett antal delarbeten presenterats som har ut-forskat svampmodellen Cunninghamella för att hitta sätt att använda den inom dopingkontroll. Tillsammans så har dessa delarbeten utökat kunskapen kring hur dessa svampar bryter ner läkemedel och Delarbete V har visat att de kan användas i den viktiga granskningen av ett läkemedels toxicitet. De har också visat att vi kan tillverka metaboliter som är viktiga för dopingkon-trollen.

I Delarbete I undersöktes hur svamparna bröt ner bupivakain som an-vänds som lokalbedövning. Inom hästsporten så förekommer det att bupiva-kain och andra lokalbedövande ämnen används för att få skadade hästar att träna eller tävla trots skadan. För att skydda hästarna så är lokalbedövande substanser såsom bupivakain förbjudna inom hästsport vilket gör att det är viktigt med studier av deras metabolism. Studien kom fram till att svampar-na C. elegans och C. blakesleeana bildade flera olika metaboliter av bupiva-kain. Många av dessa metaboliter bildas också av däggdjur och en av dessa metaboliter används som måltavla inom dopingkontrollen. Vidare så fann studien att de höga halter av metaboliter som återfanns gjorde det lättare att göra en strukturell bestämning av metaboliterna än vad det hade varit om bara urinprov från hästar hade använts.

Delarbete II presenterade en undersökning av hur de anabola ämnena SARM S1, S4 och S24 bröts ner i svampen C. elegans. De funna metaboli-terna jämfördes med de som bildas av hästar och människor. Studien fann att svampen bildade ett rikt mönster av metaboliter som också återfanns i le-vermikrosomprover från människor och hästar. Slutsatsen blev att det fanns viktiga likheter i metabolism mellan svamp, människa och häst.

Utvecklandet av en ny metod för att producera glukuronider (en typ av metaboliter) av SARMar presenterades i Delarbete III. Metoden gick ut på att svampen C. elegans först bröt ner SARMarna S1, S4, S22 och S24 till en metabolit som sedan omvandlades i en kemisk reaktion till den önskade glu-kuroniden. Den utvecklade metoden fungerade för SARMarna S1, S22 och S24 och var enkel i sitt utförande. Den kan därmed användas för att fylla det behov av glukuronider som finns inom bl.a. dopingkontroll och läkemedels-utveckling.

I Delarbete IV användes metoden från Delarbete III för att framställa en glukuronid av SARM S1 i sådan utsträckning att den kan användas som referensmaterial inom dopingkontrollen. Glukuroniden producerades med den tidigare utvecklade metoden och renades därefter fram innan den analy-serades med analystekniken NMR. Analysen fastställde med god säkerhet

37

metabolitens struktur. Detta innebar att substansen skulle kunna användas som referensmaterial.

Studien som presenteras i Delarbete V gick ut på att undersöka om svampen C. elegans kunde användas för att förutsäga om ett läkemedel ris-kera att brytas ner till skadliga metaboliter. Genom att detektera en typ av metaboliter som bildas från skadliga nedbrytningsprodukter går det att förut-säga om ett läkemedel riskerar att vara toxiskt eller ej. Studien visade att svampen kan användas för att undersöka bildandet av skadliga metaboliter, något som inte visats tidigare.

Sammanfattningsvis så har denna avhanding och arbetet med den visat att Cunninghamella-svampar har en roll att spela inom kartläggningen av olika läkemedels nedbrytning i kroppen hos däggdjur. Det har visats att svamp-modellen underlättar strukturbestämningen av metaboliter och att den kan användas för produktion av metaboliter som behövs inom dopingkontrollen. Slutligen så har det också visats att den kan fylla en roll inom den viktiga säkerhetsundersökningen av nya läkemedel.

Det är författarens förhoppning att den här modellen kommer användas i ökande utsträckning och att den bidrar till att fylla några av alla de luckor som finns inom utvecklandet och användningen av läkemedel.

38

Acknowledgements

The work in this thesis has been carried out at the Division of Analytical Pharmaceutical Chemistry at Uppsala University and at the Department of Chemistry, Environment and Feed Hygiene at the National Veterinary Insti-tute (SVA). There are several people that have helped me in my work and that I would like to thank.

My main supervisor, Visiting Prof. Mikael Hedeland. Thank you for your never ending enthusiasm and dedicated guidance. I know that I still have some ways to go but you have had a big impact on how I promote myself and my results. I couldn’t have asked for a better supervisor.

Prof. Ulf Bondesson, my assistant supervisor. Thank you for all the fun meetings and great ideas that you have contributed with. I’m really grateful to have had you as a supervisor.

Prof. Mario Thevis. You have contributed a lot to my work and I wish to thank you for all the great collaborations that we’ve had. All the other col-laborators and co-authors also deserve a thank you for everything.

I would also like to thank the people at the Division of Analytical Pharma-ceutical Chemistry. Prof. Curt Pettersson, thank you for all the answers to my questions. Dr. Ylva Hedeland, I really appreciate all the help you have given me during my time at the division and with this thesis. Dr. Olle Gyllenhaal, I wish to thank you for taking the time to look at this thesis and for helping making it better. To all the other current and former colleagues at the division, thank you for making the experience fun and interesting and thank you for all the help with everything.

The people at the Department of Chemistry, Environment and Feed Hygiene also deserve a big thank you for putting up with me during these four years. I especially want to thank Gabor Sellei for all the help with the instruments and for the great company at lunch. I shouldn’t forget to thank Elisabeth Fredriksson as well for all the fun conversations and for all the rhubarb. Another thank you goes out to Lena Ekman and Lena Grandin for all the help with practical issues during these four years. Annica Tevell Åberg and

39

Matilda Lampinen Salomonsson are two more from SVA that I want to thank for putting up with my questions and for all the help.

I also feel that I want to thank my former MSc students, Anna Hellqvist, Nina Klasson and Johanna Wedin. Your work has been important for the progression of this project and I have personally grown from working to-gether with you.

I want to thank APS stiftelse för vetenskaplig forskning and Anna Maria Lundins stipendienämnd for their financial support that allowed me to par-ticipate in scientific conferences.

Thank you, mom and dad, for all the encouragement during the years. I wouldn’t have done this if it hadn’t been for you.

A big thank you to my family and all my friends for all the fun during the years. It was the fun that made me have the energy to get this done.

Till sist, tack Johanna för att du gör mitt liv lite intressantare och mycket härligare.

40

References

[1] International Olympic Committee Factsheet - The fight agains doping and promotion of athletes’ health http://www.olympic.org/documents/reference_documents_factsheets/fight_against_doping.pdf (accessed Mar 17, 2014).

[2] Guddat, S., Fußhöller, G., Beuck, S., Thomas, A., Geyer, H., Rydevik, A., Bondesson, U., Hedeland, M., Lagojda, A., Schänzer, W. and Thevis, M. Synthesis, characterization, and detection of new oxandrolone metabolites as long-term markers in sports drug testing. Anal. Bioanal. Chem., 2013, 405(25), 8285–94.

[3] Olympic.org IOC disqualifies four medallists from Athens 2004 following further analysis of stored samples http://www.olympic.org/news/ioc-disqualifies-four-medallists-from-athens-2004-following-further-analysis-of-stored-samples/184931 (accessed Mar 12, 2014).

[4] Strahm, E., Baume, N., Mangin, P., Saugy, M., Ayotte, C. and Saudan, C. Profiling of 19-norandrosterone sulfate and glucuronide in human urine: implications in athlete’s drug testing. Steroids, 2009, 74(3), 359–64.

[5] European Medicines Agency Guideline on the Investigation of Drug Interactions - CPMP/EWP/560/95 http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf (accessed Mar 12, 2014).

[6] Food and Drug Administration Guidance for Industry Guidance for Industry Drug Interaction Studies — Study Design , Data Analysis , Implications for Dosing, and Labeling Recommendations http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm292362.pdf (accessed Mar 12, 2014).

[7] European Medicines Agency ICH guideline M3 ( R2 ) on non-clinical safety studies for the conduct of human clinical trials and marketing authorisation for pharmaceuticals http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002720.pdf (accessed Mar 12, 2014).

[8] Food and Drug Administration Guidance for Industry Safety Testing of Drug http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079266.pdf (accessed Mar 12, 2014).

41

[9] Alqahtani, S., Mohamed, L. A. and Kaddoumi, A. Experimental models for predicting drug absorption and metabolism. Expert Opin. Drug Metab. Toxicol., 2013, 9(10), 1241–54.

[10] Ionescu, C. and Caira, M. Pathways of Biotransformation - Phase I Reactions. In Drug Metabolism; Ionescu, C. and Caira, M., Eds.; Springer Netherlands: Dordrecht, 2005; pp. 41–128.

[11] Levsen, K., Schiebel, H.M., Behnke, B., Dötzer, R., Dreher, W., Elend, M. and Thiele, H. Structure elucidation of phase II metabolites by tandem mass spectrometry: an overview. J. Chromatogr. A, 2005, 1067(1-2), 55–72.

[12] Ledger, R. Nonaromatic hydroxylation of bupivacaine during continuous epidural infusion in man. J. Biochem. Biophys. Methods, 2003, 57(2), 105–114.

[13] Krisko, R. M., Schieferecke, M. A., Williams, T. D. and Lunte, C. E. Determination of bupivacaine and metabolites in rat urine using capillary electrophoresis with mass spectrometric detection. Electrophoresis, 2003, 24(14), 2340–7.

[14] Goehl, T. J., Davenport, J. B. and Stanley, M. J. Distribution, biotransformation and excretion of bupivacaine in the rat and the monkey. Xenobiotica., 1973, 3(12), 761–72.

[15] Ionescu, C. and Caira, M. Pathways of Biotransformation — Phase II Reactions. In Drug Metabolism; Ionescu, C. and Caira, M., Eds.; Springer Netherlands: Dordrecht, 2005; pp. 129–170.

[16] Huber, C., Bartha, B. and Schröder, P. Metabolism of diclofenac in plants--hydroxylation is followed by glucose conjugation. J. Hazard. Mater., 2012, 243, 250–6.

[17] Kalgutkar, A. S. and Soglia, J. R. Minimising the potential for metabolic activation in drug discovery. Expert Opin. Drug Metab. Toxicol., 2005, 1(1), 91–142.

[18] Claesson, A., Danielsson, B. and Svensson, U. Läkemedelskemi; Apotekarsocieteten: Stockholm, 2005.

[19] Prescott, L. F. Hepatotoxicity of mild analgesics. Br. J. Clin. Pharmacol., 1980, 10 Suppl 2, 373S–379S.

[20] Van Bladeren, P. J. Glutathione conjugation as a bioactivation reaction. Chem. Biol. Interact., 2000, 129(1-2), 61–76.

[21] Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C. and Nebert, D. W. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics, 1996, 6(1), 1–42.

[22] Anzenbacher, P. and Anzenbacherová, E. Cytochromes P450 and metabolism of xenobiotics. Cell. Mol. Life Sci., 2001, 58(5-6), 737–47.

42

[23] Zhang, D., Yang, Y., Leakey, J. E. and Cerniglia, C. E. Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics. FEMS Microbiol. Lett., 1996, 138(2-3), 221–226.

[24] Ferris, J. P., MacDonald, L. H., Patrie, M. A. and Martin, M. A. Aryl hydrocarbon hydroxylase activity in the fungus Cunninghamella bainieri: Evidence for the presence of cytochrome P-450. Arch. Biochem. Biophys., 1976, 175(2), 443–452.

[25] Smith, D. A. and Jones, B. C. Speculations on the substrate structure-activity relationship (SSAR) of cytochrome P450 enzymes. Biochem. Pharmacol., 1992, 44(11), 2089–98.

[26] De Groot, M. J. Designing better drugs: predicting cytochrome P450 metabolism. Drug Discov. Today, 2006, 11(13-14), 601–6.

[27] Cerniglia, C. E., Freeman, J. P. and Mitchum, R. K. Glucuronide and sulfate conjugation in the fungal metabolism of aromatic hydrocarbons. Appl. Environ. Microbiol., 1982, 43(5), 1070–5.

[28] Cerniglia, C. E., Miller, D. W., Yang, S. K. and Freeman, J. P. Effects of a fluoro substituent on the fungal metabolism of 1-fluoronaphthalene. Appl. Environ. Microb., 1984, 48(2), 294–300.

[29] Wackett, L. P. and Gibson, D. T. Metabolism of xenobiotic compounds by enzymes in cell extracts of the fungus Cunninghamella elegans. Biochem. J., 1982, 205(1), 117–22.

[30] Asha, S. and Vidyavathi, M. Cunninghamella--a microbial model for drug metabolism studies--a review. Biotechnol. Adv., 2009, 27(1), 16–29.

[31] Pervaiz, I., Ahmad, S., Madni, M. A., Ahmad, H. and Khaliq, F. H. Microbial biotransformation: a tool for drug designing. Appl. Biochem. Microbiol., 2013, 49(5), 437–450.

[32] Shyam Prasad, G. Studies on Microbial Transformation of Meloxicam by Fungi. J. Microbiol. Biotechnol., 2009, 19(9), 922–931.

[33] Tevell Åberg, A., Olsson, C., Bondesson, U. and Hedeland, M. A mass spectrometric study on meloxicam metabolism in horses and the fungus Cunninghamella elegans, and the relevance of this microbial system as a model of drug metabolism in the horse. J. Mass Spectrom., 2009, 44(7), 1026–37.

[34] Zhong, D.F., Sun, L., Liu, L. and Huang, H.H. Microbial transformation of naproxen by Cunninghamella species. Acta Pharmacol. Sin., 2003, 24(5), 442–7.

[35] Zhang, D., Evans, F. E., Freeman, J. P., Duhart, B. and Cerniglia, C. E. Biotransformation of amitriptyline by Cunninghamella elegans. Drug Metab. Dispos., 1995, 23(12), 1417–25.

[36] Pearce, C. M. and Lushnikova, M. V Microbiological production of omeprazole metabolites by Cunninghamella elegans. J. Mol. Catal. B Enzym., 2006, 41(3-4), 87–91.

43

[37] Tevell Åberg, A., Löfgren, H., Bondesson, U. and Hedeland, M. Structural elucidation of N-oxidized clemastine metabolites by liquid chromatography/tandem mass spectrometry and the use of Cunninghamella elegans to facilitate drug metabolite identification. Rapid Commun. Mass Spectrom., 2010, 24(10), 1447–56.

[38] Amadio, J. and Murphy, C. D. Production of human metabolites of the anti-cancer drug flutamide via biotransformation in Cunninghamella species. Biotechnol. Lett., 2011, 33(2), 321–6.

[39] El Sayed, K. A., Halim, A. F., Zaghloul, A. M., Dunbar, D. C. and McChesney, J. D. Transformation of jervine by Cunninghamella elegans ATCC 9245. Phytochemistry, 2000, 55(1), 19–22.

[40] Milanova, R., Stoynov, N. and Moore, M. The optimization of triptoquinone production by Cunninghamella elegans using factorial design. Enzyme Microb. Technol., 1996, 19(2), 86–93.

[41] Swathi, D., Vidyavathi, M., Padmavathi, S. and Viswa, M. Optimization of media components for hesperidine metabolism by Cunninghamella elegans. Pharmacologyonline, 2011, 365(1), 356–365.

[42] Holcapek, M., Kolárová, L. and Nobilis, M. High-performance liquid chromatography-tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites. Anal. Bioanal. Chem., 2008, 391(1), 59–78.

[43] Zhu, M., Zhang, H. and Humphreys, W. G. Drug metabolite profiling and identification by high-resolution mass spectrometry. J. Biol. Chem., 2011, 286(29), 25419–25.

[44] Kang, S.I., Kang, S.Y. and Hur, H.G. Identification of fungal metabolites of anticonvulsant drug carbamazepine. Appl. Microbiol. Biotechnol., 2008, 79(4), 663–669.

[45] Amadio, J., Gordon, K. and Murphy, C. D. Biotransformation of flurbiprofen by Cunninghamella species. Appl. Environ. Microbiol., 2010, 76(18), 6299–303.

[46] Harkins, J. D., Lehner, A., Karpiesiuk, W., Woods, W. E., Dirikolu, L., Boyles, J., Carter, W. G. and Tobin, T. Bupivacaine in the horse: relationship of local anaesthetic responses and urinary concentrations of 3-hydroxybupivacaine. J. Vet. Pharmacol. Ther., 1999, 22(3), 181–195.

[47] Fawcett, J. P., Kennedy, J., Kumar, A., Ledger, R. and Zacharias, M. Stereoselective urinary excretion of bupivacaine and its metabolites during epidural infusion. Chirality, 1999, 11(1), 50–5.

[48] Caldwell, J., Notarianni, L. J., Smith, R. L. and Snedden, W. The metabolism of bupivacaine in the rat [proceedings]. Br. J. Pharmacol., 1977, 61(1), 135–136.

[49] Risdall, P. C., Adams, S. S., Crampton, E. L. and Marchant, B. The Disposition and Metabolism of Flurbiprofen in Several Species Including Man. Xenobiotica, 1978, 8(11), 691–703.

44

[50] Arrowsmith, J. Trial watch: phase III and submission failures: 2007-2010. Nat. Rev. Drug Discov., 2011, 10(2), 87.

[51] Ratna, K. T., Vidyavathi, M., Asha, S. and Prasad, K. V. S. R. G. Biotransformation of paracetamol by Cunninghamella echinulata. J. Pharm. Res., 2009, 8(1), 1–5.

[52] Musenga, A. and Cowan, D. A. Use of ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry for fast screening in high throughput doping control. J. Chromatogr. A, 2013, 1288, 82–95.

[53] Eibye, K., Elers, J., Pedersen, L., Henninge, J., Hemmersbach, P., Dalhoff, K. and Backer, V. Formoterol concentrations in blood and urine: the World Anti-Doping Agency 2012 regulations. Med. Sci. Sports Exerc., 2013, 45(1), 16–22.

[54] Chericoni, S., Stefanelli, F., Iannella, V. and Giusiani, M. Simultaneous determination of morphine, codeine and 6-acetyl morphine in human urine and blood samples using direct aqueous derivatisation: validation and application to real cases. J. Chromatogr. B, 2013, 949-950, 127–132.

[55] Park, M., Kim, J., Park, Y., In, S., Kim, E. and Park, Y. Quantitative determination of 11-nor-9-carboxy-tetrahydrocannabinol in hair by column switching LC-ESI-MS(3.). J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 2014, 947-948C, 179–185.

[56] Scheidweiler, K. B. and Huestis, M. A. Simultaneous quantification of 20 synthetic cannabinoids and 21 metabolites, and semi-quantification of 12 alkyl hydroxy metabolites in human urine by liquid chromatography-tandem mass spectrometry. J. Chromatogr. A, 2013, 1327, 105–117.

[57] Nakayama, S., Takakusa, H., Watanabe, A., Miyaji, Y., Suzuki, W., Sugiyama, D., Shiosakai, K., Honda, K., Okudaira, N., Izumi, T. and Okazaki, O. Combination of GSH trapping and time-dependent inhibition assays as a predictive method of drugs generating highly reactive metabolites. Drug Metab. Dispos., 2011, 39(7), 1247–54.

[58] Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A. and Baillie, T. A. Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol., 2004, 17(1), 3–16.

[59] Badoud, F., Guillarme, D., Boccard, J., Grata, E., Saugy, M., Rudaz, S. and Veuthey, J.-L. Analytical aspects in doping control: challenges and perspectives. Forensic Sci. Int., 2011, 213(1-3), 49–61.

[60] Wong, J. K. Y. and Wan, T. S. M. Doping control analyses in horseracing: A clinician’s guide. Vet. J., 2014, (January), 1–9.

[61] Cusack, K. P., Koolman, H. F., Lange, U. E. W., Peltier, H. M., Piel, I. and Vasudevan, A. Emerging technologies for metabolite generation and structural diversification. Bioorg. Med. Chem. Lett., 2013, 23(20), 5471–83.

45

[62] Melvin, F., McNeill, A., Henderson, P. J. and Herbert, R. B. The improved synthesis of β-D-glucuronides using TEMPO and t-butyl hypochlorite. Tetrahedron Lett., 1999, 40(6), 1201–1202.

[63] Stachulski, A. V and Jenkins, G. N. The synthesis of O-glucuronides. Nat. Prod. Rep., 1998, 15(2), 173–86.

[64] Bouktaib, M., Atmani, A. and Rolando, C. Regio- and stereoselective synthesis of the major metabolite of quercetin, quercetin-3-O-β-d-glucuronide. Tetrahedron Lett., 2002, 43(35), 6263–6266.

[65] Yang, J., Morton, M. D., Hill, D. W. and Grant, D. F. NMR and HPLC-MS/MS analysis of synthetically prepared linoleic acid diol glucuronides. Chem. Phys. Lipids, 2006, 140(1-2), 75–87.

[66] Ho, K., Murphy, M. B., Wan, Y., Fong, B. M.W., Tam, S., Giesy, J. P., Leung, K. S.Y. and Lam, M. H.W. Synthesis and characterization of bromophenol glucuronide and sulfate conjugates for their direct LC-MS/MS quantification in human urine as potential exposure markers for polybrominated diphenyl ethers. Anal. Chem., 2012, 84(22), 9881–8.

[67] Caron, P., Trottier, J., Verreault, M., Bélanger, J., Kaeding, J. and Barbier, O. Enzymatic production of bile acid glucuronides used as analytical standards for liquid chromatography-mass spectrometry analyses. Mol. Pharm., 2006, 3(3), 293–302.

[68] Drăgan, C.-A., Buchheit, D., Bischoff, D., Ebner, T. and Bureik, M. Glucuronide production by whole-cell biotransformation using genetically engineered fission yeast Schizosaccharomyces pombe. Drug Metab. Dispos., 2010, 38(3), 509–15.

[69] Geier, M., Braun, A., Emmerstorfer, A., Pichler, H. and Glieder, A. Production of human cytochrome P450 2D6 drug metabolites with recombinant microbes--a comparative study. Biotechnol. J., 2012, 7(11), 1346–58.

[70] Kang, J.Y., Ryu, S. H., Park, S.H., Cha, G. S., Kim, D.H., Kim, K.H., Hong, A. W., Ahn, T., Pan, J.G., Joung, Y. H., Kang, H.S. and Yun, C.-H. Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnol. Bioeng., 2014, 9999(xxx), 1–10.

[71] Wollenberg, L. A., Kabulski, J. L., Powell, M. J., Chen, J., Flora, D. R., Tracy, T. S. and Gannett, P. M. The Use of Immobilized Cytochrome P4502C9 in PMMA-Based Plug Flow Bioreactors for the Production of Drug Metabolites. Appl. Biochem. Biotechnol., 2013, .

[72] Zöllner, A., Parr, M. K., Drăgan, C.-A., Dräs, S., Schlörer, N., Peters, F. T., Maurer, H. H., Schänzer, W. and Bureik, M. CYP21-catalyzed production of the long-term urinary metandienone metabolite 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one: a contribution to the fight against doping. Biol. Chem., 2010, 391(1), 119–27.

46

[73] Geyer, H., Fußho, G., Halatcheva, N., Scha, W., Kohler, M., Parr, M., Guddat, S., Thomas, A. and Thevis, M. Mass spectrometric identification and characterization of a new long-term metabolite of metandienone in human urine. 2006, , 2252–2258.

[74] Amadio, J., Casey, E. and Murphy, C. D. Filamentous fungal biofilm for production of human drug metabolites. Appl. Microbiol. Biotechnol., 2013, 97(13), 5955–63.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 186

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofPharmacy. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-220906

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2014