Studies on Androgens and Propranolol Glucuronidation

Division of Pharmaceutical Chemistry Faculty of Pharmacy University of Helsinki Finland

Stereoselectivity of the Human UDP-glucuronosyltransferases (UGTs);

Studies on Androgens and Propranolol Glucuronidation

Taina Sten

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Pharmacy of the University of Helsinki, for public examination in Auditorium 1041, Viikki Biocenter 2 (Viikinkaari 5), on January 15th 2010, at 12 noon.

Helsinki 2010 Supervisor: Dr. Moshe Finel Centre for Drug Research (CDR) Faculty of Pharmacy University of Helsinki Finland

Reviewers: Professor John O. Miners Department of Clinical Pharmacology Flinders University Adelaide, Australia

Dr. Mikko Koskinen Translational Sciences Orion Corporation Espoo, Finland

Opponent: Professor Michael Court Department of Pharmacology & Experimental Therapeutics Tufts University School of Medicine Boston, MA, USA

ISBN 978-952-10-6005-2 (pbk.) ISBN 978-952-10-6006-9 (PDF) ISSN 1795-7079 http://ethesis.helsinki.fi/

Helsinki University Print Helsinki 2009 Contents

List of original publications 5

Abbreviations 6

Abstract 7

1 Introduction 8

1.1 Conjugation reactions in metabolism; UDP-glucuronosyltransferases 8

1.1.1 Structural aspects of the UGTs 9

1.1.2 Function and localization of the UGTs 11

1.1.2.1 Oligomerization and expression regulation 13

1.1.2.2 Polymorphism and metabolic interactions 13

1.2 Stereoisomers 15

1.2.1 Chiral compounds as drugs 15

1.2.3 Analysis of stereoisomers 17

1.3 Enzyme kinetic studies 18

1.4 Enzyme inhibition 19

1.5 Anabolic steroids 22

1.5.1 Anabolic steroids metabolism 22

1.5.1.1 Metabolism of testosterone 22

1.5.1.2 Glucuronidation of steroids 22

1.5.2 Implications for doping control 24

2. Aims of the study 26

3. Materials and Methods 27

3.1 Chemicals 27

3.2. General experimental conditions 28

3.2.1 Recombinant enzymes and other protein sources 28

3.2.2. Reaction conditions 29 3.2.3 Analytical methods 29

3.2.4. Data analysis 30

4. Results and Discussion 32

4.1. Propranolol glucuronidation (I) 32

4.1.1 Stereoselective glucuronidation of propranolol 32

4.1.2 Atypical kinetics in propranolol glucuronidation 34

4.2. Steroids glucuronidation (II, III) 35

4.2.1 Substrate selectivity of UGT2B7, UGT2B15 and UGT2B17 38

4.2.2 Correlation of in vitro results with in vivo findings 39

4.3 Inhibition of testosterone glucuronidation (II, IV) 40

5. Summary and Conclusions 43

6. Acknowledgements 44

7. References 46 List of original publications

This thesis is based on the following publications:

I Sten T, Qvisen S, Uutela P, Luukkanen L, Kostiainen R and Finel M (2006) Prominent but reverse stereoselectivity in propranolol glucuronidation by human UDP-glucuronosyltransferases 1A9 and 1A10. Drug Metab Dispos 34: 1488-1494.

II Sten T, Bichlmaier I, Kuuranne T, Leinonen A, Yli-Kauhaluoma J and Finel M (2009) UGT2B7 and UGT2B17 display converse specificity in testosterone and epitestosterone glucuronidation, whereas UGT2A1 conjugates both androgens similarly. Drug Metab Dispos 37: 417-423.

III Sten T, Kurkela M, Kuuranne T, Leinonen A and Finel M (2009) UDP- glucuronosyltransferases in conjugation of 5Į- and 5ȕ-androstane steroids. Drug Metab Dispos 37: 2221-2227.

IV Sten T, Finel M, Ask B, Rane A and Ekström L (2009) Non-steroidal anti- inflammatory drugs interact with testosterone glucuronidation. Steroids 74: 971-977.

The publications are referred to in the text by their roman numerals (I-IV).

The articles have been printed with permission of the copyright holders.

5 Abbreviations

CYP cytochrome P-450 DHEA dehydroepiandrosterone DHT dihydrotestosterone DMSO dimethyl sulfoxide E enzyme concentration ES enzyme-substrate complex ESI electrospray ionization HIM human intestine microsome HLM human liver microsome HPLC high pressure liquid chromatography I inhibitor concentration Ki inhibition constant Km Michaelis constant, substrate concentration resulting in 50% of Vmax LC liquid chromatography MS mass spectrometry MS/MS tandem mass spectrometry NSAID non-steroidal anti-inflammatory drug n Hill constant Q-TOF quadrupole time-of-flight R-pg R-propranolol glucuronide S substrate concentration SL saccharic acid lactone SN2 bimolecular nucleophilic substitution S-pg S-propranolol glucuronide S50 substrate concentration resulting in 50% of Vmax in Hill equation T/E testosterone to epitestosterone ratio UDP uridine 5’-diphosphate UDPGA uridine 5’-diphospho glucuronic acid UGT UDP-glucuronosyltransferase UGT UDP-glucuronosylransferase gene UPLC® Ultra performance liquid chromatograph Acquity, Waters Co. UV ultraviolet Vmax maximum reaction velocity 3Į-HSD 3Į-hydroxysteroid dehydrogenase 3ȕ-HSD 3ȕ-hydroxysteroid dehydrogenase 5Į-diol 5Į-androstane-3Į-,17ȕ-diol 5ȕ-diol 5ȕ-androstane-3Į-,17ȕ-diol 5Į-R 5Į-reductase 5ȕ-R 5ȕ-reductase 17ȕ-R 17ȕ-reductase

6 Abstract

Metabolic reactions are divided into functionalization and conjugation reactions. Uridine diphosphoglucuronosyltransferases (UGTs) are an important group of conjugative enzymes that catalyze the transfer of a sugar molecule to the substrate. In humans, 19 UGT enzymes are expressed in a tissue specific manner. Human UGTs are divided into subfamilies: UGT1A, UGT2A and UGT2B. Most UGTs have broad substrate selectivity and are involved in the metabolism of different endogenous and exogenous molecules. The steroids in the human circulation are mainly glucuronidated by UGT2B subfamily enzymes. Among steroids many stereoisomeric pairs are found. Stereoisomers differ from each other in the spatial orientation of the atoms. Mirror images are called enantiomers. In biological environments stereoisomers are differentiated. The current study is concerned with stereo- and regioselectivity of UGTs toward propranolol enantiomers and endogenous steroids. Stereoisomers offer a valuable tool in examination of UGT selectivity toward the substrates. Kinetic characterization of the reactions and inhibition studies can be used to further analyze the efficiency and type of the enzymatic reaction. The studies of this thesis were conducted with human recombinant UGTs incubated with the selected androgens and propranolol. Analysis was done by liquid chromatograph equipped with either ultraviolet or fluorescence detector, or a mass spectrometer. Propranolol was glucuronidated with opposite stereoselectively by UGT1A9 and UGT1A10, two closely related enzymes. The results also suggest the role of intestinal UGT1A10 in the stereoselective first pass metabolism of propranolol. Extrahepatic UGT2A1 was also active, showing low affinity and not discriminating propranolol enantiomers. Positive cooperativity was suggested in the reaction between propranolol enantiomers and UGT2A1. Endogenous C19-steroids were studied. Testosterone was mainly glucuronidated by UGT2B17, an enzyme that is absent in some individuals. Testosterone glucuronidation by UGT2B17 was inhibited with its diastereomeric pair epitestosterone, which is a high affinity substrate of UGT2B7, but not UGT2B17. Minor inhibition also occurred by a common non-steroidal anti-inflammatory drug (NSAID) diclofenac, but not ibuprofen. Both NSAIDs, however, inhibited UGT2B15. Other studied androgens were mostly glucuronidated by UGT2B7, 2B15, 2B17 and, often, UGT2A1. UGT2B15 was strictly regio and stereoselective toward 17ȕ-OH bearing steroids, whereas UGT2B17 accepted both 3Į- and 17ȕ-OH. If both were available, the 17ȕ-OH was favored over 3Į-OH. UGT2B7 accepted both 3Į- and 17Į-OH, but not 17ȕ-OH, which also significantly impaired the glucuronidation reaction when available in the molecule. For UGT2B7, structures with 17ȕ-OH seem to act as a competitive inhibitor for 3-O-glucuronidation if the Km is not very low. Interestingly, 17Į-OH as in epitestosterone served as a low Km substrate that was not inhibited by testosterone with 17ȕ-OH. The results shed new light on the selectivity of human UGTs toward endogenic C19 steroids.

7 1 Introduction

Glucuronide conjugation is catalyzed by uridine 5’-diphosphoglucuronosyltransferases, a group of microsomal enzymes expressed in many organs (Ohno and Nakajin, 2009; Tukey and Strassburg, 2000). UGTs catalyze the conjugation of the glucuronic acid moiety from UDP-glucuronic acid (UDPGA) to many endogenous and exogenous substrates, thus making it easier to excrete them into urine or bile. UGTs are divided into different subfamilies according to their sequence similarities (Mackenzie et al., 2005). The substrate specificity of UGTs is broad and partly overlapping. The present work focuses on the stereoselectivity of UGTs, structural aspects of the model substrates, and structure-activity relationships between the aglycones and the proteins. When authentic standards for glucuronides are not available, enzymatic synthesis of those metabolites is possible using biological catalyst, such as rat liver microsomes (I). Enzymes are biological catalysts with three-dimensional structures, and thus the interactions of drugs with drug-metabolizing enzymes are often stereoselective. It is of interest to investigate the interactions of chiral entities, propranolol enantiomers, by the use of chiral HPLC (I). The next line of the study was directed at steroid metabolism by recombinant UGTs using Ultra Performance LC with both UV and MS detection (II, III, IV). The studies on these steroids concentrated on both structure-function aspects (II, III), and inhibition of different UGTs and substrates (II, IV).

1.1 Conjugation reactions in metabolism; UDP- glucuronosyltransferases

Conjugation reactions, also referred to as phase II metabolic reactions, are primarily catalyzed by transferase enzymes. A molecular group from a cosubstrate needed for these reactions is conjugated with a functional group on the molecule. Typical functional groups able to undergo conjugation are hydroxyl-, carboxyl-, and amine groups that are often introduced or exposed during functionalization biotransformation reactions, also referred to as phase I metabolism. Conjugation biotransformation reactions include glucuronidation, sulfonation (more commonly called sulfation), acetylation, methylation, conjugation with glutathione (mercapturic acid synthesis), and conjugation with amino acids such as glycine, taurine, and glutamic acid. With the exception of methylation and acetylation, conjugation reactions usually result in a large increase in substrate hydrophilicity. The conjugation facilitates excretion of the compound into bile or urine through efflux transporters. UDP-glycosyltransferases (UGTs, EC 2.4.1.17) catalyze glucuronidation reactions, and are involved in about 35% of all drugs metabolized by conjugation (Evans and Relling, 1999). Glucuronic acid conjugation is one of the main metabolic pathways in detoxification of about 10% of the 200 most prescribed drugs (Williams et al., 2004).

8 1.1.1 Structural aspects of the UGTs

Human UGTs belong to the GT1 family of the glycosyltransferase (GT) superfamily. Mammalian UDP-glycosyltransferases are divided into four families: UGT1, UGT2, UGT3, and UGT8 (Mackenzie et al., 2005; Meech and Mackenzie, 2009). UGT1 and UGT2 family members catalyse conjugation of glucuronic acid and are further discussed below. The abbreviation UGT in the text thus refers to UDP-glucuronosyltransferases. To date, 28 human UGT genes have been identified, including nine pseudogenes containing truncation mutations (Mackenzie et al., 1997; Mackenzie et al., 2005). UGTs are divided into subfamilies, UGT1A, UGT2A, and UGT2B, primarily according to gene sequence similarity (Mackenzie et al., 2005) (Fig. 1). Between the families of UGT1 and UGT2, there is at least 50% homology, and within each subfamily at least 60% homology in their DNA sequence (Guillemette, 2003). In humans, 19 UGTs are expressed in a tissue specific manner (Finel et al., 2005; Nakamura et al., 2008; Ohno and Nakajin, 2009; Tukey and Strassburg, 2000). Full-length crystal structure of any UGT is yet to be resolved. A crystal structure of the UDP-glucuronic acid binding domain of human UGT2B7 was recently published (Miley et al., 2007). The UGT1A family consists of nine functional proteins that are encoded by a single gene complex, located on chromosome 2q37 (Mackenzie et al., 1997; Owens and Ritter, 1995). The mRNA transcripts all share exons 2-5, but have a distinct exon 1. Thirteen exon 1 sequences in the gene locus encode nine functional enzymes. The four common exons are believed to yield the C-terminal part of the enzyme that includes the UDPGA binding domain (Mackenzie et al., 2003). The variable first exon yields the N-terminal domain of the protein, the substrate binding domain, explaining the different substrate selectivity between the enzymes despite the common C-terminal part. (King et al., 2000). The UGT enzymes exhibit distinct, although sometimes overlapping, substrate selectivities. It is not common that UGT-mediated metabolism would cause drug interaction, or a dysfunction in UGT metabolism would lead to a certain disease state. An important exception is UGT1A1, which is essential for bilirubin glucuronidation (Petit et al., 2008). The total lack of UGT1A1 activity in type I Crigler-Najjars syndrome leads to severe jaundice and kernicterus. The UGT2 enzymes are products of separate genes located on chromosome 4q13, and are further divided into two subfamilies, UGT2A and UGT2B (Fig. 1) (Mackenzie et al., 2005). Unlike the UGT1 family, the mRNA transcript in the UGT2B family is spliced from six exons. Even with the unique genes encoding the UGT2B family enzymes, they share very conserved protein sequences at the C-terminal portion, which both makes up the UDPGA binding domain and anchors the protein to the membrane (King et al., 2000; Tephly and Burchell, 1990). Analogously with the UGT1A family, the N-terminal part contributes to substrate selectivity (Lewis et al., 2007). UGT2A family enzymes are much less studied, and presumably of less importance in systemic metabolism. UGT2A1 is found mainly in olfactory epithelium, and with very low expression also in the brain and lungs (Jedlitschky et al., 1999; Somers et al., 2007). UGT2A2 was suggested to have a unique exon 1, and to share exons 2-6 that encode the C-terminal half of the enzyme, with UGT2A1 (Mackenzie et al., 2005). To date, UGT2A2 has been found in the liver and 9 small intestine (Tukey and Strassburg, 2001). A recent research paper, however reported UGT2A2 expression in nasal tissue but not in adult liver (Sneitz et al., 2009). UGT2A2 activity toward different substrates is broad but quite low, following substrate selectivity of UGT2A1 (Sneitz et al., 2009). UGT2A3 expression was recently published, but its activity was limited to bile acids (Court et al., 2008). UGT2A3 is mainly found in the small intestine, liver, and adipose tissue. Of the UGT2 family, mainly UGT2Bs play significant roles in drug and endogenic substances metabolism. They are extensively expressed in both liver and extrahepatic tissues, including reproductive organs (Nakamura et al., 2008; Ohno and Nakajin, 2009; Radominska-Pandya et al., 1998; Turgeon et al., 2001).

Figure 1. A phylogenetic tree of the 19 human UDP-glucuronosyltransferases (UGTs) (Patana, 2009).

10 1.1.2 Function and localization of the UGTs

Glucuronidation is a conjugation reaction, during which Į-D-glucuronic acid moiety is transferred from the co-substrate UDP-glucuronid acid (UDPGA) to the substrate, which in glycosylation prosesses is referred to as an aglycone, literally meaning the nonsugar component of a glycoside molecule (Fig. 2). UGTs are membrane bound enzymes, located in the endoplasmic reticulum and facing the lumenal side of the organelle (Tephly and Burchell, 1990; Tukey and Strassburg, 2000). The reaction is deduced to be similar to the bimolecular nucleophilic substitution reaction (SN2) producing only ȕ-D-glucuronides (Miley et al., 2007; Yin et al., 1994). The high energy bond between UDP and glucuronic acid holds the energy to form the new bond in the product. Glucuronic acid is very hydrophilic, thus increasing the substrates water solubility (Fisher et al., 2001; Wells et al., 2004). A study on UGT1A enzymes substrate binding order suggests that those enzymes exhibit a compulsory order mechanism, and that UDPGA is most likely the first binding substrate (Luukkanen et al., 2005). Recent studies support those findings, suggesting an ordered bi bi -mechanism, in which the aglycone may act as an inhibitor by binding to the enzyme-UDP complex (Patana et al., 2008).

O O OH O OH NH OH OH O O O O O N + R-OH + UDP P P O OH O O O OH O R OH OH OH OH OH OH

UDPGA

Figure 2. An example of formation of ether glucuronide. The reaction between co-substrate uridine-5-diphosphoglucuronic acid (UDPGA) and an aglycone bearing a hydroxyl group (R-OH). Note that after conjugation the configuration of the carbon is inverted (SN2).

Metabolic reactions occur mainly in the liver, but also in other tissues exposed to xenobiotics. Endogenous compounds, such as steroid hormones, are also metabolized by UGT enzymes. Ubiquitous UGTs are found in tissues such as the gastrointestinal tract (Finel et al., 2005; Radominska-Pandya et al., 1998; Tukey and Strassburg, 2000), lungs (Somers et al., 2007; Turgeon et al., 2001), and kidneys (Nakamura et al., 2008; Ohno and Nakajin, 2009), in a diverse expression pattern considering different UGTs. The UGT1A subfamily members UGT1A7-8 and UGT1A10 have a unique expression pattern as they are notably found only in extrahepatic tissues, particularly the gastrointestinal tract (Ohno and Nakajin, 2009; Radominska-Pandya et al., 1998; Tukey and Strassburg, 2000).

11 Glucuronidation is generally an end point reaction, and once formed, the glucuronic acid conjugates are not usually broken down. The chemical instability of conjugates must still be taken into consideration. Preanalytical stability of drugs and metabolites can be controlled with e.g. temperature, pH and light conditions (Shipkova et al., 2003; Skopp and Potsch, 2004). A reversible reaction to glucuronidation, namely dissociation of the glycosidic bond, is possible by ȕ-glucuronidase. It is a protein of either human or bacterial origin, present in e.g serum and urine (Ho, 1995; Lampe et al., 2002). Bacterial beta- glucuronidase is produced by certain intestinal bacterial strains, and thus mainly present in the gastrointestinal tract, but also in bile (Reddy et al., 1974; Skar et al., 1988). The activity of beta-glucuronidase compared to that of UGT enzymes is low, and only a limited amount of enzymatic degradation takes place (Bock and Kohle, 2009). Hence, the glucuronidated compound usually remains to be excreted. The organisms benefit from the glucuronidation reaction might also be due to neutralization of the pharmacological effect of substances, at least in most cases (Chouinard et al., 2008; Jedlitschky et al., 1999). Rare exceptions to this trend arise, such as morphine 6-glucuronide, which is a more potent analgetic than the parent compound (Osborne et al., 1990). In recent years, further metabolism of glucuronide conjugates has been detected especially for steroids. A repeated glucuronidation of steroid sex hormones, forming diglucuronides where two glucuronosyl groups are bound to a single hydroxyl group in tandem, was depicted in vitro (Murai et al., 2005). Dihydrotestosterone, for one, was shown to be metabolized in vitro via a previously unknown pathway involving sequential reactions of UGT2B17 and aldo- keto reductase (Jin et al., 2009). The clinical studies strongly suggest that this pathway would also be relevant in vivo. Different approaches have been developed to predict glucuronidation in vivo with help from in vitro data (IVIVC; in vitro in vivo –correlation), but even the simplest attempts, using substrates that do not undergo functionalization prior to glucuronidation, did not usually lead to good predictability (Miners et al., 2004; Miners et al., 2006). Typically, the in vitro resolved kinetic characteristics lead to underestimation of the in vivo clearance of UGT metabolized drugs. The reasons for these differences might lay in the incubation study conditions (Boase and Miners, 2002; Kilford et al., 2009; Soars et al., 2003). A noteworthy matter is the presence of long-chain fatty acids present in or released during incubations. Those fatty acids are inhibitory to certain UGT enzymes, at least UGT1A9 and UGT2B7, a reaction that can be overcome by the addition of bovine serum albumin to the incubation mixture as a free fatty acid sequestering agent (Rowland et al., 2007; Rowland et al., 2008). Addition of albumin to incubations provides physiologically relevant Km values. Recombinant UGTs are generally produced as membrane enzymes, which are not subject to other purification steps than differential centrifugation to concentrate the microsomal membrane fractions. The presence of a lipid bilayer membrane is thought to maintain the recombinant enzyme in its native membrane-bound conformation (Crespi and Miller, 1999). The luminal orientation of UGTs requires the action of different transporter proteins (Bock and Kohle, 2009). The systems in which the recombinant UGTs are produced, anyhow, do not generally express the transporter proteins that are present in human endoplasmic reticulum membranes. This can hinder both the availability of the substrates at the active site, and the release of the glucuronide 12 conjugates. The access through the membranes is especially critical in microsomal preparations, which can also be activated by using different mediators. The most common is probably the pore forming agent alamethicin (Al-Zoughool and Talaska, 2006; Cubitt et al., 2009). Saccharolactone (SL) is a ȕ-glucuronidase inhibitor that has often been used in glucuronidation studies with microsomes and recombinant UGTs. A recent study, however, suggests exclusion of SL on a routinely basis in the incubation mixtures, as it might cause inhibition of glucuronide formation in some cases (Oleson and Court, 2008).

1.1.2.1 Oligomerization and expression regulation

At least some, if not all, UGT enzymes are oligomeric, forming different quaternary structures, such as homo- and heterodimers (Finel and Kurkela, 2008; Kurkela et al., 2003; Kurkela et al., 2007; Radominska-Pandya et al., 2005). Although not always requisite, oligomerization might be necessary for enzymatic activity, and may alter the rates or even partially restore the glucuronidation capability in some cases (Fujiwara et al., 2007a; Kurkela et al., 2007; Olson et al., 2009; Ouzzine et al., 2003). Homo- and heterodimerization of certain UGTs was also recently shown by other researchers (Fujiwara et al., 2007a; Fujiwara et al., 2007b; Fujiwara et al., 2010; Nakajima et al., 2007; Operana and Tukey, 2007). Moreover, it was suggested that UGTs are acting together with other drug metabolizing enzymes such as cytochrome P-450 (CYP) via xenobiotic nuclear receptor coordinated regulation (Bock and Kohle, 2004). UGTs are inducible enzymes, and the induction is mediated by different receptors and transcription factors. Important nuclear receptors identified in UGTs regulation in human or animal models include the aryl hydrocarbon receptor, constitutive androstane receptor, and pregnane X receptor (Buckley and Klaassen, 2009; Court et al., 2008; Kohle and Bock, 2009). They can affect the expression of different UGT enzymes in a diverse manner.

1.1.2.2 Polymorphism and metabolic interactions

Some of the UGTs are polymorphic enzymes (Argikar et al., 2008; Evans and Relling, 1999; Lampe et al., 2000). Polymorphism has been identified for UGT1A1, UGT1A3-4, UGT1A6-9, UGT2A1, UGT2A3, UGT2B4, UGT2B7, UGT2B10, UGT2B15, UGT2B17 and UGT2B28 (Chen et al., 2007a; Court et al., 2008; Girard et al., 2006; Guillemette, 2003; Villeneuve et al., 2003; Wilson et al., 2004). Polymorphisms often show distinctive population differences (Bhasker et al., 2000; Jakobsson et al., 2006; Lampe et al., 1999; Thomas et al., 2006; Villeneuve et al., 2003). In the UGT1A subfamily the best known and most important polymorphism affects the UGT1A1 enzyme. Depending on the severity of the state, it leads to impairment or total lack of glucuronidation of bilirubin, a degradation product of heme bearing proteins such as CYPs, hemoglobin and myoglobin. The condition is known as either Crigler-Najjar or Gilberts syndrome (Petit et al., 2008; Strassburg, 2008). This is one of the rare cases when UGT-dependent metabolism leads to disease or severe impairment. The UGT-mediated clinical drug-drug interactions are not 13 typical either (Kiang et al., 2005). The high Km and general involvement of multiple UGTs in a definite pathway cause glucuronidation to be less susceptible to drug-drug interactions in comparison to CYP-dependent pathways (Williams et al., 2004). Situations in which UGT genotype is taken into consideration when using medication are also atypical. The only occasion so far is the UGT1A1*28 polymorphism (Gilbert’s syndrome), which affects the metabolism of a chemotherapeutic agent irinotecan. The UGT1A1 genotype is often tested in the case of irinotecan treatment, although its relevance has received criticism (Akiyama et al., 2008; Deeken et al., 2008; Perera et al., 2008). Recent evidence, however, associates an increased risk of certain diseases with polymorphism in UGT1A subfamily enzymes. The allelic variant UGT1A6*2 is associated with more rapid glucuronidation of salicylic acid, a major metabolite of a common NSAID acetylsalicylic acid (Chen et al., 2007b). The extrahepatic enzyme UGT1A7 is a key enzyme in catalyzing the glucuronidation of various carcinogens. For example, UGT1A7 haplotype is related to raised susceptibility of hepatocellular carcinoma in hepatitis B carriers (Kong et al., 2008). A UGT1A7 high acitivity genotype was more common in patients with laryngeal cancer, whereas low-activity genotype indicated risk for liver cirrhosis (Lacko et al., 2009; Tang et al., 2008). The expression level of UGT1A10, an estrogen conjugating UGT enzyme recently found in breast tissue, may be associated with breast cancer (Starlard-Davenport et al., 2008). Among the UGT2B family there are many polymorphic enzymes. Polymorphs UGT2B4(D458E), UGT2B7(H268Y), and UGT2B15(D85Y) were found by Lampe et al. (Lampe et al., 2000). Also, UGT2B17 and UGT2B28 polymorphisms exist, but they are copy number variations (McCarroll et al., 2006; Wilson et al., 2004). UGT2B7H268Y polymorphism is common, showing distinctive interethnic difference between Caucasian and Japanese populations (Bhasker et al., 2000; Coffman et al., 1998). It may be of clinical importance e.g. with interaction of lorazepam and valproic acid (Chung et al., 2008) but not with epitestosterone glucuronide formation and androgen disposition, nor in the metabolism of menthol and morphine (Bhasker et al., 2000; Schulze et al., 2008a). Despite a minor difference in polymorphs of UGT2B7 in androsterone glucuronidation was shown in vitro, it was not evident in vivo (Bhasker et al., 2000; Swanson et al., 2007a). The genetic susceptibility to diclofenac-induced hepatotoxicity in correlation to UGT2B7 genotype was recently reported (Daly et al., 2007). UGT2B15 is encoded by a polymorphic gene, yielding polymorphs UGT2B15(D85Y), (T352I), and (K523T) (Court et al., 2004). The isolation and characterization of UGT2B15(D85Y) was first reported by (Levesque et al., 1997) and is of some clinical importance in S-oxazepam glucuronidation (Court et al., 2004). (Swanson et al., 2007b) showed that UGT2B15(D85Y) and 2B17 deletion polymorphisms also predict the glucuronidation pattern of androgens and fat mass in men Deletion polymorphism of the UGT2B17 gene was first reported by Wilson et al. (2004), and it was shown to be genetically linked to UGT2B15. Later studies suggest that lack of the UGT2B17 enzyme may be compensated for by increased UGT2B15 transcription (Schulze et al., 2008a). Prominent interracial differences in UGT2B17 copy- number occur, the influence of which is also observed in certain endogenous compound glucuronidation, such as testosterone (Jakobsson et al., 2006; Schulze et al., 2008c; Xue et 14 al., 2008). Deletion of UGT2B17 leads to decreased level of urinary testosterone, which may lead to misinterpretation of doping control assays. UGT2B17 gene is associated with an increased risk for prostate cancer and correlates to gene expression in the prostate (Karypidis et al., 2008). Moreover, the gene is a strong determinant of androgen excretion in healthy pubertal boys, and associated with the pathogenesis of osteoporosis (Juul et al., 2009; Yang et al., 2008). Studies on the impact of single polymorphic UGTs on a disease state or as a risk factor, however, are probably insufficient. The coexistence of the polymorphisms may have more or less profound effects on hormonal metabolism (Menard et al., 2009). Thus, the haplotype analyses are needed for further evaluation of the importance of the UGTs polymorphism.

1.2 Stereoisomers

Molecules that have identical atomic constitution and bonding, but different three- dimensional arrangement of the atoms are called stereoisomers, a general term for all isomers that differ only in the spatial orientation of the atoms. Within the group of stereoisomers, different subcatecories can be distinguished. Mirror images that differ only by one chiral center are called enantiomers. Isomers of compounds with more than one chiral center, that are not mirror images of one another, are referred to as diastereomers. Also geometric (cis/trans) isomers are included under the definition of stereoisomers. Diastereoisomers and geometric isomers are chemically diverse molecules, and possess different pharmacological effects (unless they are interconverted in vivo). In the pharmaceutical industry and pharmacotherapy, enantiomers are probably the most interesting stereoisomers. They essentially have identical physical (except for optical rotatory) and chemical properties. In a chiral environment, however, enantiomers are distinguished. This is of interest clinically, as living organisms represent chiral environment and, hence, in biological systems enantiomers are discriminated. They may have different pharmacokinetic properties (absorption, distribution, biotransformation and excretion) and quantitatively or qualitatively different pharmacologic or toxicologic effects. Another relevant point is the complication in analyses of the enantiomers, as they are only separated in chiral environment and thus require specific chiral separation techniques.

1.2.1 Chiral compounds as drugs

As discussed above, chiral substances are discriminated in a homochiral environment (Fig. 3). Thus, it is of considerable interest to separately examine and evaluate the pharmacokinetic and pharmacodynamic characteristics of each individual stereoisomer. This is partly because of the U.S. Food and Drug Administrations (FDA) guidelines for

15 the development of new stereoisomeric drugs in 1992 (www.fda.gov). It was recently estimated, that of marketed drugs today, approximately one half are optically active and of those approximately 50 % were used as a racemate (Campo et al., 2009). Among the new drugs approved by FDA, in 2005-2008, however, one half are still optically active, but only 8% were racemates. Many of those racemic drugs’ therapeutic action is due to the eutomer, and the distomer can be less active or inactive as in the cases of citalopram and ibuprofen (Hao et al., 2005; Hyttel et al., 1992), or cause a totally different pharmacological effect such as propranolol (Zipper et al., 1983). A safer pharmacological profile, as in the case of levobupivacain, can also be a reason to choose either of the two active enantiomers (Burlacu and Buggy, 2008). Some drug molecules, such as the anti- inflammatory agent ibuprofen, undergo chiral inversion to the other enantiomer in vivo (Hao et al., 2005). The study of the enantiomers’ metabolism in the human body is of considerable interest. Examples of differences in biological action and metabolism of drug enantiomers are numerous. Racemization of the once pure enantiomer can happen, however, during storage and in vitro studies, as well as in the body (Ali et al., 2007). This is particularly important if the opposite enantiomer was toxic.

B B

A C C A

A’ C’ A’ C’ B’ B’

Figure 3. The molecule on the left with contact points A, B and C matches the corresponding receptor sites A', B' and C'. The mirror-imaged enantiomeric form of the molecule (right) does not match this receptor, thereby enabling chiral discrimination. According to (Kuhnle et al., 2002).

Conjugation of chiral drugs is a research field, which has recently gained increasing attention. The vast amount of optically active substances on the market, together with the prevalence of UGT contribution to drug metabolism, makes this study field an ever growing theme. Substances are not always directly conjugated, and this metabolic reaction may take place via a functionalization reaction. Thus, it is also possible that initially a non-optically active substance, bearing a prochiral group such as ketone, is metabolized in vivo into two stereomers capable of undergoing conjugation reactions (Chiarotto and Wainer, 1995). Chiral inversion from one enantiomer to another in vivo is also possible (Wsol et al., 2004), the metabolism in a living organism is a sum of multiple processes and is hard to evaluate in vitro.

16 Propranolol, [1-isopropylamino-3-(1-naphtoxy)-2-propanol] is an example of a widely used chiral drug. It is a non-selective ȕ-adrenergic blocking agent that is used as a racemic mixture in the treatment of hypertension, cardiac arrhythmias and angina pectoris. Propranolol is metabolized through different pathways such as conjugation, side-chain oxidation and ring oxidation. It is glucuronidated to two diastereomeric O-glucuronides in human, as well as in dog and rat (Bai and Walle, 1984; Thompson et al., 1981). The role of presystemic glucuronidation has been evaluated, but the results have been somewhat contradictory. Extensive presystemic glucuronidation of propranolol in human was suggested following oral propranolol administration (Midha et al., 1983). The major site of propranolol glucuronidation, anyhow, is thought to be the liver rather than intestine (Buch and Barr, 1998). Approximately 17% of the dose is metabolized through stereoselective glucuronidation in human (Silber et al., 1982; Walle et al., 1985). When racemic propranolol was administered to man, the concentration of S-propranolol glucuronide (S-pg) was higher than R-propranolol glucuronide (R-pg) in both plasma and urine (Luan et al., 2005; Silber et al., 1982). It was previously reported that at least UGT1A9, UGT2B4 and UGT2B7 are involved in propranolol glucuronidation in human (Coffman et al., 1998; Hanioka et al., 2008; Li et al., 2001). UGT2B7 did not exhibit a clear preference to either R- or S-propranolol, whereas UGT1A9 prefers catalyzing S-enantiomer to R-enantiomer (Coffman et al., 1998; Yu et al., 2009).

1.2.3 Analysis of stereoisomers

In analytical perspective, enantiomers can be directly separated by chiral chromatography, or analyzed after derivatization with a homochiral derivative either on- line or offline, and separating the resulting diastereomers by conventional, achiral chromatographic methods (Bobbitt and Linder, 2001; Haginaka, 2002). Analytically, conjugates of drug enantiomers are not usually too challenging, since they are chemically diastereomers and can be considered the same as derivatized chiral compounds, as with glucuronides (Laethem et al., 1996). On the other hand, if there is a need for separation of unconjugated substrates or other possible chiral metabolites, as often in clinical studies, chiral separation is usually needed. Metabolite standards are also needed, not only for clinical studies, but also therapeutic and legal drug monitoring. Different metabolites of a drug or an endogenous compound can also mirror certain disease states or biopharmacologically important genotypes. The drugs are often readily metabolized, and the parent drug can be detected in blood or urine only for a limited time period. The conjugated metabolites can usually be detected for a longer period of time, which is not only used in therapeutic drug monitoring but also doping analysis (Peng et al., 2000; Pozo et al., 2008). The need to use non-invasive methods, such as urine collection, also fortifies the importance of the hydrophilic metabolites as a research focus. The metabolite standards are limitedly available, thus decreasing the possibility to use these as biological markers. Several attempts to synthesize glucuronides have been taken place. Chemical synthesis (Alonen et al., 2009; Lucas et al., 2009; Oatis et al., 1983) and enzymatic 17 approaches (Alonen et al., 2005; Jäntti et al., 2007; Luukkanen et al., 1999; Soars et al., 2002; Stevenson and Hubl, 1999) have been generated. An alternative way of acquiring metabolites standards is to extract them from the urine of either humans or test animals treated with the parent drug (Bai and Walle, 1984). The advantage of different enzymatic or in vivo approaches is that only the biologically relevant ȕ-anomers are formed, but the purification can be difficult and the yields are generally low. This methodology can also raise discussion about study ethics. The chemical syntheses often give better yields, but several impurities, including the non-biological Į-glucuronide anomers can be formed, thus leading to additional purification steps.

1.3 Enzyme kinetic studies

Enzyme kinetics is a study of enzyme catalyzed chemical reactions, which aim at resolving the catalytic mechanism of the enzyme, its role in the metabolism, and controlling the activity. Enzyme kinetic studies focus on how compounds are metabolized, on the reaction rates, substrate affinity, and how do the substances affect a certain enzyme. The interaction between enzyme and substrate is often described by the Michaelis Menten equation (Equation 1).

Equation 1.

k1 k2 E + S ES E + P k-1

The Michaelis-Menten model is somewhat simple for many purposes, but it is the most used enzyme kinetic model. The Michaelis constant, Km, is defined as (k2 +k-1)/k1. It should be noted that Km is not a binding constant that measures the strength of binding between the enzyme and substrate. Often Km is called affinity, but actually its value includes the affinity of substrate for enzyme, and also the rate at which the substrate bound to the enzyme is converted into product. Only if k2 is much smaller than k-1 will Km equal a binding affinity. Vmax is the maximum reaction velocity, and the ratio of Vmax/Km can be used as an indication of the catalytic efficiency. Enzyme reaction is commonly visualized as a plot of rate of product formation (velocity) versus substrate concentration (Fig. 4). Metabolism of a compound is classically described kinetically with the Michaelis-Menten equation, which yields a hyperbolic rate profile (Fig 4A). Not all enzyme and substrate reactions are best described by this equation, and different models and equations are also used, two examples of which are presented in the Fig. 4. The equations concerning these models are introduced in chapter 4.2.4.

18 y t i A B C c o l e v n o i t

c

V V V a e Reaction velocity R Reaction velocity V/S V/S V/S Substrate concentration Substrate concentration Substrate concentration

Figure 4. Illustrative kinetic plots showing Michaelis-Menten hyperbolic kinetic profile (A), substrate inhibition profile (B) and sigmoidal autoactivation profile (C). In the insets corresponding Eadie-Hofstee plots are shown.

Formerly, linear transformations of Michaelis-Menten equation were widely used, since they are easily exploited with simple conventional tools (Table 1). These linear plots were used for rapid identification of kinetic parameters like Km and Vmax.

Table 1. Linear transformations of Michelis-Menten equation.

Transformation On horizontal (x) axis On vertical (y) axis Eadie-Hofstee Y/X No change Hanes-Woolf No change X/Y Lineweaver-Burk 1/X 1/Y Scatchard Y Y/X

As nonlinear regression methods, however, are easy to use with modern computer software, and significantly more accurate, the linearization in parameter calculation is not longer generally used. Nevertheless, these methods offer a valuable way to easily visualize the properties of enzyme kinetics. For example in the case of atypical kinetics, the Eadie- Hofstee plot is characteristically changed from a straight line to a certain parabola (Fig 4. insets).

1.4 Enzyme inhibition

Many drugs used today exert their pharmacological effect via enzyme inhibition. They either decrease the enzymes ability to bind substrates, or lower the enzymes catalytic activity, or both. Two main classes exist, irreversible and reversible inhibitors, differing by their affinity to the enzyme. Reversible inhibitors bind to and dissociate from the protein in an equilibrium process. Irreversible enzymes bind tightly to the enzyme to form 19 an essentially permanent complex. Drugs that irreversibly inhibit proteins, and are not dissociated once bound, can lead to toxicity that could not be reversed in poisoning cases. The time course of the irreversible inhibition is determined by the kinetics of enzyme synthesis, activation, and elimination. Thus, this type of enzyme inhibitor can only be used for certain target proteins. Finasteride is used for treatment of prostate hyperplacia and hair loss. It irreversibly inhibits 5Į-reductase type II enzyme in the prostate and hair follicles (Span et al., 1999). Another example contains the gastric acid lowering agent omeprazole whose target protein is H+,K+-ATP-ase, and acetylsalicylic acid that inhibits cyclo-oxygenase I -enzyme. Irreversible inhibitors bind to and inactivate enzymes, thus lowering the available enzyme concentration [E], leading to decreased Vmax. But then, Km would be unchanged because it is an intrinsic constant of the enzyme, not dependent on the [E]. Reversible inhibitors can be classified as competitive, noncompetitive, and uncompetitive (Copeland, 2005). If the detailed mechanism of inhibition is known, classification into these groups can be done by relation to type of interference. This means the place on the enzyme that the inhibitor binds to, and the order in which the inhibitor binds relative to substrate. One mechanism is blocking the substrate binding, namely a competition for the binding site. As the inhibitor competes with a substrate for an enzymes active site, it lowers the enzymes likelihood of binding the substrate and reduces the reaction velocity, but leaves the maximal activity at saturating substrate concentration unchanged. Determination of simple kinetic parameters can often be used to identify the type of inhibition. Typical steady-state kinetic experiments can be performed, in which the reaction velocity is measured in the presence of varying substrate concentrations. If the inhibitor is then added, and the data shows an increase in Km, yet the Vmax is unaffected, it is the sign of a competitive inhibitor (Fig. 5). Linearizations, such as double reciprocal Lineweaver-Burk plot, have widely been used before the development of efficient computer programs. The linearized plots still serve an easily visualizated plot, which also can clarify the inhibition type (Cortes et al., 2001). Uncompetitive inhibitors bind exclusively to the enzyme-substrate (ES) complex. In contrast to the competitive type, the inhibition is more pronounced when the substrate concentration is higher, as more ES complexes are available. A noncompetitive inhibitor can bind to both the unoccupied enzyme and ES complex. All inhibition types show distinctive patterns in direct Michaelis-Menten plot, but the inhibition type is easier visualized using Lineweaver-Burk plot. (Fig. 5). Dixon plot is a graphical method for determination of the inhibition constant (Ki) and the type of enzyme inhibition (Dixon, 1953). The effect on the reaction velocity (v) is determined at two or more substrate concentrations [S], and over a range of inhibitor concentrations [I]. Keeping [S] constant, 1/v is plotted against [I] and a straight line will be obtained. Data for each [S] will cut one another at I=-Ki and 1/v=1/Vmax (competitive inhibition), or at the abscissa (1/v=0) and I=- Ki (non-competitive inhibition. For uncompetitive inhibition the lines are parallel. If the binding affinity to E and ES is equal, the inhibitor is a true noncompetitive type.

20 Michaelis-Menten Lineweaver-Burk A Vmax

V 1/V 1/2 Vmax

1/Vmax

[S] 1/[S] KM KM 1/KM 1/KM Michaelis-Menten B Lineweaver-Burk Vmax Vmax V 1/V 1/2 Vmax 1/Vmax

[S] 1/[S] KM 1/KM Michaelis-Menten C Lineweaver-Burk

Vmax

V 1/V Vmax 1/2 Vmax 1/Vmax 1/2 Vmax

[S] 1/[S] KM KM 1/KM 1/KM

Figure 5. A; Competitive inhibition, leading to increased Km but unaffected Vmax. B; Non- competitive inhibition; Vmax is decreased, Km is unaffected.C; uncompetitive inhibition, both Vmax and Km are decreased. The kinetic parameters changed due to inhibition are written in italics.

A mixed inhibitor binds to a site on the enzyme, and interferes with both the parent substrates affinity and catalytic turnover (Cornish-Bowden et al., 1983). Mixed inhibitors do not bind directly to the active site. Therefore, they do not block substrate binding, but instead bind to another site that can be proximal or distal to the active site. Mixed inhibitors can bind to free enzyme prior to substrate, causing the active sites conformation to change to an unoptimal form for the substrate binding and for catalysis. The enzymatic turnover rate is slowed. Mixed inhibitors can also bind after substrate to the ES complex, decreasing the apparent substrate binding. Mixed inhibitors can either increase or decrease Km and decrease Vmax. 21 1.5 Anabolic steroids

Human male sex steroids consist of a 19 carbon structure (Fig. 6). Male sex steroids that possess androgenic potential are also called androgens. To include all structurally related compounds regardless the androgenic activity, steroids discussed here are referred to as C19-steroids, according to their structure.

18 19 19 11 H 13 12 17 2 10 D 1 9 C H 1 10 A B 8 14 15 16 4 6 6 3 5 H 7 H 2 5 H 3 4 5a -androstane 5b -androstane

Figure 6. Androstane structure of theC19-steroids. The numbering of the carbons as well as nomenclature of the ring structure is depicted. Note the major conformational difference in the steroid backbone between 5Į- and 5ȕ-androstane.

1.5.1 Anabolic steroids metabolism

1.5.1.1 Metabolism of testosterone

Testosterone is the main male hormone, secreted by the testes and adrenal gland. It is metabolized to various metabolites, and both testosterone and many of the metabolites are available for conjugation reactions, such as sulfonation and glucuronidation (Fig. 7).

1.5.1.2 Glucuronidation of steroids

Glucuronidation of endogenous steroids and their metabolites is an important metabolic pathway, also of interest in the fight against illegal steroid abuse in sports. The most important UGTs with respect to androgen steroid metabolism are UGT2B family members, 2B4, 2B7, 2B15 and 2B17 (Belanger et al., 2003; Turgeon et al., 2001). Testosterone and its active metabolite dihydrotestosterone (DHT) are glucuronidated mainly by UGT2B17 (Turgeon et al., 2001). Androsterone glucuronide is the major androgen metabolite found in the circulation (Belanger et al., 1991). Androsterone is glucuronidated by UGTs 2B4, 2B7 and 2B17 (Turgeon et al., 2001).

22 O OH 12 17 11 13 H 16 H 1 9 14 2 10 8 15 17-b HSD H H H H 3 5 7

OH 4 6 OH DHEA androst-5-ene-3bb , 17 -diol

3b-HSD OH 3b-HSD H

H H O O Testosterone H 17-b HSD

H H O androst-4-ene 3, 17-dione

5a-R 5a-R 5-b R 5-b R

OH O O OH

H H H H

H H H H H H H H O O O O H H H H 5a -DHT 5a -androstanedione 5b -androstanedione 5b-DHT 3a-HSD OH 3a-HSD O 3a-HSD O 3a-HSD OH

H H H H

H H H H H H H H OH OH OH OH H 17b -HSD H H 17b -HSD H

5a -diol androsterone etiocholanolone 5b -diol

Figure 7. Metabolism of testosterone. Functionalization metabolism of androgen hormones and enzyme that are involved are presented. DHEA, dihydroepiandrosterone; DHT, dihydrotetosterone.3Į- and 3ȕ-HSD, 3Į- and 3ȕ-hydroxysteroid dehydrogenase, respectively; 17ȕ-HSD, 17ȕ-hydroxysteroid dehydrogenase; 5Į- and 5ȕ-R, 5Į- and ȕ-reductase, respecttively. The numbering of the carbons 1-19 is explained in the DHEA structure. (Dufort et al., 2001; Labrie et al., 2003; Schänzer, 1996).

The Km values in androsterone glucuronidation by UGT2B7 and UGT2B17 in previous studies were 9.7 µM and 0.4 µM, respectively (Beaulieu et al., 1996; Coffman et al., 1998). Interestingly, even though UGT2B17 seems to be of great importance in vitro, its deletion did not effect the androsterone glucuronide serum concentration (Swanson et al., 2007b). Most, if not all, steroids are conjugated to some degree by UGT2B7, which is abundantly expressed in the liver, intestine, kidney, and bladder (Nakamura et al., 2008; Turgeon et al., 2001). Etiocholanolone is glucuronidated by UGTs 2B7 and 2B17 (Turgeon et al., 2001). Although it seems to be preferentially glucuronidated by UGT2B17, when Vmax is considered, (relative Vmax 46 pmol/mg protein/min compared to that of 19.5 pmol/mg protein/min for UGT2B7), the total absence of UGT2B17 does not

23 have a significant effect on etiocholanolone glucuronide excretion in urine (Jakobsson et al., 2006; Turgeon et al., 2001). The etiocholanolone Km for UGT2B17 is lower than for UGT2B7, 1.3 and 4.8 µM, respectively and thus its glucuronidation efficiency is about 20 fold compared to UGT2B7. As endogenous concentrations of etiocholanolone in serum are at the sub-nanomolar range, (Kancheva et al., 2007), the high affinity to the enzyme is important, and the kinetic parameters can not directly explain why the conjugation in vivo is not disturbed by the deficiency of UGT2B17. For the urinary excretion of etiocholanolone glucuronides, like other steroid glucuronides, the conjugation in the kidneys and bladder can be important. It may be significant that in both organs UGT2B7, but not 2B17, is extensively expressed (Nakamura et al., 2008). The construction of the in vitro study may also offer an explanation. As discussed above, UGT2B7 is inhibited by long chain fatty acids present or released during incubation (Rowland et al., 2007). Including fatty acid sequestering agent, such as albumin, in the incubation mixture would most likely lead to lower estimated Km value for UGT2B7. Another androgen metabolite, 5Į-diol (Fig. 7), is glucuronidated by UGT2B7, 2B15 and 2B17 with high affinity (Km 0.5, 2.2 and 1.0 µM, respectively) (Beaulieu et al., 1996; Levesque et al., 1997; Turgeon et al., 2001). There is, however, a limitation in comparing literature kinetic values on the glucuronidation of 5Į-diol and 5ȕ-diol. These diols are glucuronidated by different UGTs at variable regioselectivity, while the published results often contain both glucuronide regioisomers. Formerly it has been assumed that of 5Į-diol, only 17-O-glucuronide exists in the circulation (Rao et al., 1987). Both glucuronides were, however, detected in serum (Chouinard et al., 2008; Swanson et al., 2007b).

1.5.2 Implications for doping control

Doping in sports is a problem. The list of prohibited substances in sports is published yearly by the World Antidoping Agency (WADA) (www.wada-ama.org). For over 50 years some athletes have used exogenous testosterone supplements (Bowers et al., 2009). Testosterone induces growth of muscle size and strength (Bhasin et al., 1996). Doping control became possible in the late 60s, along with the development of suitable methods, such as mass spectroscopy, capillary gas chromatography, and radioimmunoassay (Shackleton, 2009). The use of anabolic steroids was first prohibited by the International Olympic Committee at the Montreal Olympic Games in 1976. Since 1983 the method of choice for detecting exogenous testosterone misuse has been based on the urinary testosterone to epitestosterone ratio (T/E) (Donike et al., 1983). Testosterone and epitestosterone exist in approximately eaqual amounts in urine. Presently, T/E >4.0 is considered suspicious and requires further examination The parent steroids are extensively metabolized, and can only be detected in the urine for a short time after use. Generally the analytes are excreted as glucuronides and sulphates, and steroids are analyzed after hydrolysis of the conjugates (Schänzer and Donike, 1993; Schänzer, 1996). Glucuronidation of endogenous steroids and their metabolites is thus also of interest in illegal steroid abuse. Both synthetic and naturally occurring steroids (e.g. testosterone, 24 DHT, and 4-androstenedione) are abused in sports (Cawley et al., 2008; Dehennin and Matsumoto, 1993; Donike et al., 1995). Endogenous steroids are metabolized to several androgens such as testosterone, DHT, DHEA, androstenedione, androsterone and etiocholanolone (Fig. 7). The problem with endogenous steroid determination is that since they are naturally present in the samples, a simple qualitative approach is not adequate. Screening for the misuse of naturally occurring anabolic steroids is based on not only monitoring the absolute amounts in urine, but also the ratios of certain endogenous steroids, the most important of which has been the testosterone to epitestosterone ratio (T/E) (Donike et al., 1983). Establishment of proper threshold levels is a consistent issue. The levels are based on population statistics, and many studies have shown significant population differences in the average basal levels of the studied metabolites (Bowers, 2008; Jakobsson et al., 2006; Schulze et al., 2008c; Sottas et al., 2008; Strahm et al., 2009). Current research is mostly concentrated on a UGT2B17 deletion polymorphism, as it reduces testosterone glucuronidation to negligible levels. This has led to alternative suggestions for steroid abuse detection, such as population or genotype based T/E cut-off levels, or individual longitudinal steroid profile follow up (Bowers, 2008; Schulze et al., 2008b; Schulze et al., 2008c; Sottas et al., 2008; Strahm et al., 2009). Samples with adverse analytical findings are submitted to isotope ratio mass spectroscopy analysis to verify the origin of the steroid, as the percentage of C13 in endogenous molecules is different from that of synthetic origin. Other suspicious findings include higher than threshold concentration of epitestosterone, testosterone, androsterone, etiocholanolone, and DHEA (all equivalent to the glucuronide) (WADA technical report 2004).

25 2. Aims of the study

This work was done to study the reactions of UDP-glucuronosyltransferases with stereoisomeric molecules. The more detailed aims of the included publications were:

· Biosynthesis of R- and S-propranolol glucuronides for reference compounds (I)

· Examination of the enantioselective glucuronidation of R- and S-propranolol by human UGTs along with microsomes from human liver and intestine (I).

· To study the glucuronidation of several structurally related androgens by recombinant UGTs, for determining the regio- and stereoselectivity in such reactions (II, III).

· To study the inhibition on testosterone glucuronidation by UGT2B15 (IV) and UGT2B17 (II, IV).

26 3. Materials and Methods

3.1 Chemicals

A B

O N O N H H OH H H OH

OH C OH D

O O

E OH F OH

H H

OH OH H H

O G O H

OH OH H H I J

Cl O

NH OH OH O Cl

Figure 8. Structures of the studied compounds: A - R-propranolol; B - S-propranolol; C - testosterone; D - epitestosterone; E - 5Į-diol; F - 5ȕ-diol; G - androsterone; H - etiocholanolone; I - diclofenac; J - ibuprofen.

27 Stereoisomeric pairs of compounds were studied, the structures of which are shown in figure 8. In propranolol [1-isopropylamino-3-(1-naphtoxy)-2-propanol] enantiomers, the conjugative OH-group is at the chiral center (I). A similar difference occurred between the diastereomers testosterone (4-androsten-17ȕ-ol-3-one) and epitestosterone (4-androsten- 17Į-ol-3-one) (II). Androsterone (5Į-androstan-3Į-ol-17-one), etiocholanolone (5ȕ-androstan-3Į-ol-17-one), 5Į-diol (5Į-androstan-3Į-,17ȕ-diol), and 5ȕ-diol (5ȕ-androstan-3Į-17ȕ-diol) have more extensive differences at C5 that do not result in simple configurative changes right at the stereocenter, but also in the steroid backbone (III). Additionally, the four latter compounds let us study the regioselectivity of the UGTs with respect to the 3-OH and 17-OH groups of those androgens. The inhibition of testosterone glucuronidation by common NSAIDs, ibuprofen and diclofenac was studied using human liver microsomes and recombinant UGTs (IV). Detailed descriptions of the chemicals used can be found in the original publications I-IV. All reagents were of analytical or chromatographic grade.

3.2. General experimental conditions

Experimental methods used in the studies included in this thesis are described in detail in the original publications I-IV or references therein. The general methods are outlined briefly below.

3.2.1 Recombinant enzymes and other protein sources

Human recombinant UGTs were cloned and expressed in baculovirus-infected insect cells in our laboratory (Finel et al., 2005; Kurkela et al., 2003; Kuuranne et al., 2003; Sneitz et al., 2009). The recombinant UGTs from our laboratory carried a short C-terminal fusion peptide ending with 6 His residues, a His-tag. The relative expression level of each recombinant UGT was determined by dot-blot analyses using tetra-His antibodies (Qiagen, Hilden, Germany), as detailed elsewhere (Kurkela et al., 2004; Kurkela et al., 2007). Protein concentrations were determined by the BCA method (Pierce Biotechnology Inc., Rockford, IL, USA). Due to observed poor activity of the UGT2B15 produced in our laboratory, recombinant human UGT2B15 of commercial origin (BD SupersomesTM Gentest, BD Biosciences, MA, USA) was included in certain studies (II, III, IV). This enzyme, like our recombinant UGTs, is expressed in baculovirus-infected insect cells, but does not carry a His-tag. Hence, its expression level could not be compared to the other UGTs. Human liver and intestinal microsomes (HLM and HIM, respectively) were purchased from Gentest (BD Biosciences, MA, USA) (I).

28 3.2.2. Reaction conditions

The incubation reactions are described in detail in the original publications I-IV. The studies with recombinant UGTs and liver microsomes were carried out with 2.5-5 mM co- substrate UDPGA in 50 mM phosphate buffer (pH 7.4) containing 5 mM MgCl2. The substrates were dissolved in DMSO (I-IV) or ethanol (IV) and the organic solvent concentration in the reactions was 1-5 %. When saccharic acid lactone (a ȕ-glucuronidase inhibitor) was used, its concentration was 5 mM (I-III). The reactions were started by the addition of UDPGA, incubated in a heat bath at +37°C and terminated by the addition of cooled methanol (I) or 4 M perchloric acid (II-IV), chosen according to the analytes and chromatographic conditions. The control incubations were carried out in the presence of UDPGA, but in the absence of substrate, and vice versa. The glucuronides were identified by HPLC/MS/MS analyses. For kinetic analyses, 2-3 protein concentrations were first incubated with 2-3 different substrate concentrations for varying time spans, to specify the conditions when product formation is linear with respect to protein concentration and time. The determination of linear conditions was done individually for each pair of recombinant protein and substrate.

3.2.3 Analytical methods

The chromatographic devices used for the studies are listed below (Tables 2 and 3). When the analyses were done in another laboratory, namely GC/MS analyses (III) and HPLC- UV analysis of HLM samples (IV), the equipment is not listed here.

Table 2. Instrumentation used in the studies I-IV.

Equipment Supplier Study Chromatoghaphs Detection Note and columns Acquity UPLC® UV-Vis Empower Waters Corp., Millford, MA, USA II, IV Ȝ 246 nm software Acquity BEH Shield Waters Corp., Millford, MA, USA RP18 column (100x 1mm i.d. with 1.7- µm particle size Acquity UPLC® Mass spetrometry MassLynx 4.0 Waters Corp., Millford, MA, USA III software Acquity BEH Shield Waters Corp., Millford, MA, USA RP18 column (100x 1mm i.d. with 1.7- µm particle size Agilent HPLC 1100 Fluorescence ChemStation Agilent Technologies Inc., Palo I 227 (ex) 336 (em) software Alto, CA, USA Chirobiotic T (250 x nm 4.6 mm i.d. with 5- Advanced Separation Technologies µm particle size Inc., Whippany, NJ, USA

29 Table 3. Instrumentation used in the studies I-IV.

Equipment Supplier Study Mass Ion source Analysis Spectrometers Perkin Elmer Sciex ESI LC-MS/MS Sciex, Concord, Canada I API 3000 Waters Micromass ESI LC-MS and Waters Corp., Millford, MA, USA III Q-TOF LC-MS/MS

3.2.4. Data analysis

Computer analyses of the enzyme kinetic data were done using GraphPad Prism software (version 4.02 for Windows; GraphPad Software, San Diego, CA, USA). In addition to computer analyses, data transformation and visualization in Eadie-Hofstee (II) and Dixon plots (IV) was done. The equations used are as follow, with symbol explanations below.

1. Michaelis-Menten equation I-III

´ SV v = max m + SK

2. Substrate inhibition: I, III

V v = max 1 (Km /S)(++ S/Ksi )

3. Hill equation: I-III

´ SV n v = max n n 50 + SS

4. Competitive inhibition model equation: II

´ SV v = max æ1+ I ö ç ÷ K m ç ÷ + S è K i ø

5. Modified sigmoidal dose response equation IV

Top + (Bottom - Top) Y = 1 10^((X -+ LogIC50)´ HillSlope) 30 v = velocity Vmax = maximum velocity S = substrate concentration Km = Michaelis-Menten constant (substrate concentration at 0.5 Vmax) Ki = inhibition constant Ksi = the constant describing the substrate inhibition interaction S50 = the substrate concentration resulting in 50% of Vmax n = Hill coefficient I = inhibitor concentration IC50 = half maximal inhibitory concentration X = logarithm of the inhibitor concentration Y = degree of inhibition, expressed as % of remaining activity while the uninhibited activity is taken as 100%. Y starts at the Top and goes to the Bottom with a sigmoid shape.

31 4. Results and Discussion

The results of this study are mainly presented in the attached articles (I-IV). Additional data about propranolol glucuronidation by UGT2A-family enzymes, and alternative data processing of the inhibitory study (IV) are presented here. The parts of the studies (II, IV) that were carried out by the researchers other than the writer of the present study are excluded from this dissection.

4.1. Propranolol glucuronidation (I)

4.1.1 Stereoselective glucuronidation of propranolol

Glucuronidation of propranolol enantiomers by HLM and HIM as well as human recombinant UGTs was studied and analyzed by HPLC with UV-detection. Racemization of the propranolol enantiomers did not happen during the incubation. Glucuronidation of propranolol enantiomers was stereoselective in HLM and HIM (Table 4). The contribution of certain UGT enzymes to this selectivity was established during incubation of propranolol enantiomers with these enzymes, both in the presence and absence of the opposite enantiomer. The concentration of an individual enantiomer was kept constant.

Table 4. Glucuronidation rates of propranolol enantiomers and their dependence on the presence or absence of the opposite enantiomer.

Formation of S-propranolol glucuronide (S-pg) and R-propranolol glucuronide (R-pg) was determined by incubations in the presence of 0.5 mM concentration of the tested enantiomer (in both presence and absence of the other enantiomer). Values are ± S.D. n=3. The limit of quantification corresponds to reaction velocity of 0.3 pmol/mg/min Racemic mixture Individual enantiomers reaction velocity ± SD reaction velocity ± SD ( pmol/mg/min) ( pmol/mg/min) S-pg R-pg S-pg R-pg UGT1A1 0.7 ± 0.0 detectable 0.6 ± 0.1 0.3 ± 0.0 UGT1A9 8.3 ± 0.3 1.1 ± 0.1 21.6 ± 0.6 2.0 ± 0.4 UGT1A10 5.5 ± 0.8 26.8 ± 4.2 8.7 ± 1.3 36.7 ± 1.7 UGT2B4 4.9 ± 0.2 2.8 ± 0.1 5.8 ± 0.1 4.1 ± 0.5 UGT2B7 19.5 ± 0.5 25.2 ± 0.6 32.9 ± 3.3 41.3 ± 2.0 UGT2B17 1.1 ± 0.2 detectable 1.4 ± 0.3 0.4 ± 0.0 UGT2A1 1.9 ± 0.0 1.7 ± 0.0 1.4 ± 0.1 1.2 ± 0.3 UGT2A2 0.7 ± 0.0 detectable 0.6 ± 0.0 detectable HLM 70.7 ± 4.9 50.4 ± 3.2 74.2 ± 8.9 47 ± 1.8 HIM 7.3 ± 0.7 13.1 ± 1.2 10.0 ± 2.8 21.8 ± 1.8

32 The most active enzymes were the UGTs 1A9, 1A10, 2B4, and 2B7. The most prominent stereoselectivity was observed for UGT1A9 and UGT1A10, the first favoring S- propranolol, and the latter R-enantiomer (Table 4). These results might suggest the enantioselective first pass glucuronidation of propranolol, as seen with HIM. Generally, the presence of the other enantiomer diminished the glucuronide formation, as in the case of UGT1A9. An exception to this was found with UGT2A1, since the glucuronide formation was somewhat stimulated by the presence of both enantiomers. According to the kinetic data, even the most active UGTs were low to medium capacity enzymes in both propranolol enantiomers glucuronidation (Table 5). Sub- millimolar Km values of UGTs 1A9-10 and UGT2B7 indicated rather high affinity, whereas UGT2B4 and UGT2A1 were low affinity enzymes. According to the present knowledge, however, some of these results could be questioned. At least UGT1A9 and UGT2B7 are shown to be inhibited by long chain fatty acids present in the incubation media, a phenomenon that can be overcome with the addition of albumin in the reaction mixture (Rowland et al., 2007; Rowland et al., 2008). Another matter worth to consider in the studies is the use of SL in the reaction (Oleson and Court, 2008). It changes the pH of the incubation mixture, and the 50 mM phosphate buffer may not have had sufficient buffering capacity. Organic solvents also variably affect UGT enzyme activity, which may have obscured contributions of some UGT enzymes (Uchaipichat et al., 2004). Considering the data processing, application of multi-site kinetic models might have offered further insights into the kinetic studies.

Table 5. Kinetic constants (± SE) for S- and R-propranolol aglucuronidation by recombinant UGTs 1A9-10, 2B4, 2B7, and 2A1.

The initial rate data were fitted to eqs. 1 to 3 and the kinetic constants were derived from the best fit.

S-propranolol R-propranolol

Vmax Km or S50 Vmax Km or S50 (pmol/mg/min) (mM) (pmol/mg/min) (mM) UGT1A9 35.3 ± 3.7 0.24a ± 0.04 2.0 ± 0.2 0.12 ± 0.04 UGT1A10 11.4 ± 0.7 0.29 ± 0.06 81.6 ± 4.8 0.50 ± 0.1 UGT2B4b 25.6 ± 2.4 2.6 ± 0.5 17.9 ± 0.7 1.4c ± 0.1 UGT2B7 73.4 ± 4.4 0.60 ± 0.1 N.D. UGT 2A1 74.1 ± 3.1 5.0d ± 0.3 77.1 ± 4.6 4.4d ± 0.3

a) Substrate inhibition. Ksi = 1.2 ± 0.2 b) Saturating concentration not reached; experimental data up to 5 mM fitted to the equations. c) Hill equation. n = 1.9 ± 0.2 d) Hill equation. n (S-p)= 1.8 ± 0.1; n (R-p)=2.5 ± 0.3 N.D., not determined

The most interesting result in catalytic activity point of view was the prominent, but opposite, stereoselectivity of the closely related enzymes UGT1A9 and UGT1A10. This 33 inverse preference for the propranolol enantiomer was a significant finding, especially considering the highly similar primary structures of UGT1A9 and UGT1A10. In the total of 505 residues in these mature proteins, 32 are nonidentical, denoting 93% similarity in the amino acid sequence. It is also worth mentioning that UGT1A7 and UGT1A8 were practically inactive in propranolol glucuronidation despite the considerable homology that they share with UGTs 1A9 and 1A10. In UGT1A9, 14 amino acids are unique compared to UGT1A7-8 and 1A10, and in a recent study Arg42 and Asn152 were suggested to contribute largely to the dissimilarity in the substrate specificity between UGT1A8 and UGT1A9 (Fujiwara et al., 2009). The critical amino acids affecting the substrate selectivity of UGT1A9 and UGT1A10 still remain to be studied.

4.1.2 Atypical kinetics in propranolol glucuronidation

Atypical kinetics refers to kinetics diverged from the Michaelis-Menten type. In the case of propranolol glucuronidation, both substrate inhibition and cooperativity were evidenced (Fig. 9). Those types were also clearly illustrated by the Eadie-Hofstee plots for S- propranolol glucuronidation by UGT1A9 and UGT2A1 (Fig. 9).

25 A B

20 60

15 25 80 40 V 10 V 20 5 0 0

V, pmol/min/mg protein pmol/min/mg V, 0 10 0 150 protein pmol/min/mg V, V/S V/S 0 0 0 1 2 3 0 2 4 6 8 10 [S], mM [S], mM

Figure 9. Atypical kinetics of S -propranolol glucuronidation by UGT1A9 (A) and UGT2A1 (B). Substrate inhibition with UGT1A9 and autoactivation kinetics by UGT2A1 are illustrated with Eadie-Hofstee plots in the insets.

The mechanism of substrate inhibition is not fully known. Substrate inhibition may arise from multiple mechanisms, although all arise from the binding of two or more substrate molecules. One possible explanation of observed inhibition with certain UGTs might be the product inhibition caused by the released UDP (Fujiwara et al., 2008). When one of the enantiomers has higher glucuronidation turnover with the enzyme, the other might hinder the conjugation via non-productive binding as a competitive inhibitor. In both enantiomers conjugation by UGT1A9, the reaction rate was diminished when the opposite enantiomer was also present (Table 4). The effect that the opposite enantiomers presence induces on the kinetic values of propranolol glucuronidation by UGT1A9 has 34 recently been elucidated (Yu et al., 2009). The Vmax of either enantiomer was significantly lower when the substrate was used as a racemic compound. The assay done with the racemic substrate most likely represents the situation in vivo better, if the compound is used as a racemate. The determination of the true kinetic values and possible inhibition constant of the opposite enantiomer, however, is unreliable if neither of the enantiomers concentration can be held steady. When an enzyme does not discriminate between the two enantiomers, the activity toward both is similar. Such was the case with the propranolol enantiomers glucuronidation by UGT2A1 (Table 4). This finding was further studied by conducting the kinetic experiments of propranolol glucuronidation by UGT2A1. Both propranolol enantiomers were glucuronidated by UGT2A1 with very high S50 values, suggesting low affinity to the enzyme (Table 5). Furthermore, the data illustrated positive autoactivation kinetics and suggested multiple binding sites. The sigmoidal kinetics is depicted in figure 9. Positive cooperativity explains the positive influence in the glucuronidation rate of either of the propranolol enantiomers by the opposite enantiomer, particularly at a concentration range significantly below the S50 (Table 4). The study could continue with full kinetic characterization of propranolol enantiomers in the presence of the opposite enantiomer to further ascertain the positive cooperativity in action. The study of propranolol glucuronides has yielded new information on the stereoselectivity of UGTs, particularly for the closely related enzymes UGT1A9 and UGT1A10. The studies have been repeated and extended by other authors with a kinetic study of racemic propranolol, and stereoselectivity of propranolol glucuronidation in cynomolgous monkey liver microsomes (Hanioka et al., 2008; Yu et al., 2009). The results also suggest that the use of chiral drugs as probe substrates in studies of structurally related enzymes, such as UGT1A9 and UGT1A10, may be very valuable for elucidating substrate selectivity differences. For example, stereoselective glucuronidation of oxazepam was previously used as a probe of induced forms of UGTs in rabbits (Yost and Finley, 1985). In humans, S-oxazepam is conjugated mainly by UGT2B15, that is inactive in R-oxazepam glucuronidation, whereas R-oxazepam is glucuronidated by UGT1A9 and UGT2B7 (Court et al., 2002).

4.2. Steroids glucuronidation (II, III)

The glucuronidation of six structurally related endogenic C19-steroids was screened with 19 recombinant human UGTs (Table 6).

35 Table 6. Glucuronidation rates of androsterone, etiocholanolone, and 5Į- and 5ȕ-diol glucuronidation by recombinant UGTs. Det., glucuronide was detected but below quantification level. n.d., no glucuronide was detected. UGT2B15* was of commercial origin (BD Biosciences, USA). Glucuronide formation, pmol/mg protein/min UGT Testosterone Epitestosterone Androsterone Etiocholanolone Į-diola ȕ-diola 17-glucuronide 17-glucuronide 3-glucuronide 3-glucuronide 3-glucuronide 17-glucuronide 3-glucuronide 17-glucuronide 1A1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1A3 4.6 ± 0.3 n.d. Det. Det. Det Det n.d. n.d. 1A4 5.5 ± 0.3 4.9 ± 0.8 11.7 ± 0.0 Det. 5.2 77.4 Det Det 1A5 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1A6 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1A7 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1A8 2.7 ± 0.1 n.d. n.d. n.d. n.d. 4.9 n.d. n.d. 1A9 10.4 ± 0.5 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1A10 7.9 ± 0.8 n.d. n.d. n.d. n.d. 11.1 n.d. n.d.

2A1 253.0 ± 5.4 172.0 ± 0.9 25.0 ± 1.3 565.4 ± 18.5 n.d. 97.6 52.0 n.d. 2A2 13.5 ± 0.5 69.7 ± 5.4 15.0 ± 1.7 26.1 ± 1.0 12.0 38.4 60.6 n.d. 2A3 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

2B4 n.d. 8.1 ± 0.6 Det. Det. 10.5 n.d. 8.1 n.d. 2B7 4.6 ± 0.1 312.5 ± 20 687.0 ± 47.2 192.0 ± 39.8 264.6 n.d. 63.9 n.d. 2B10 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2B11 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2B15 7.9 ± 0.1 n.d. n.d. n.d. n.d. 70.7 n.d. 4.1 2B15* 63.0 ± 2.9 n.d. n.d. n.d. n.d. 570.3 n.d. 30.4 2B17 508.9 ± 19.6 n.d. 365.0 ± 8.3 786.0 ± 5.4 12.8 745.9 27.9 361.2 2B28 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

The protein concentration was 0.5-1.25 mg/ml, substrate concentration 50 µM and incubation time 120 min, n=3. On protein concentration of 0.5 mg/ml, LOQ corresponds to reaction velocity of 4.6 pmol/mg/min (testosterone and epitestosterone) and 2.8 pmol/mg/min (androsterone and etiocholanolone). a The diol glucuronides are quantified based on androsterone glucuronide standard curve.

36 Both stereo- and regioselectivity was evident in steroid glucuronidation (Table 6). Formation of steroid diglucuronides was not evident. Kinetic characterization of the most active UGTs in androgens glucuronidation revealed generally high affinity (low Km) of 2B7 and 2B17 (Table 7). Recent study (Bowalgaha et al., 2007) determined a somewhat lower Km in testosterone glucuronidation, 3.8 µM versus our 10 µM (II). The substrate concentrations we used for studying kinetic constants should have been lower for better Km value estimation. UGT2A1 was capable of glucuronidation of all studied steroids, but with variable activity and regioselectivity (Table 6). In a bent steroid skeleton, such as etiocholanolone and 5ȕ-diol, 3-O-glucuronide was formed abundantly (etiocholanolone), but 17-O- glucuronide formation was negligible (5ȕ-diol) (Fig. 7). Apparently, non-productive binding of 5ȕ-diol in direction, when the D-ring comes close to the active site, reduced otherwise abundant 17-O-glucuronidation. In contrast, planar structure of androsterone and 5Į-diol was only insignificantly conjugated at the 3-position, but was a rather good substrate for 17-O-glucuronidation, which was also evident in the case of testosterone and epitestosterone (Table 6). The glucuronidation efficiency of UGT2A1 toward the substrates, however, was rather poor (Table 7). The Km in etiocholanolone glucuronidation, 250 µM, was exceptionally high. Regardless of the high capacity, UGT2A1 is not presumed to play a major role in etiocholanolone glucuronidation, as a consequence of both low affinity and the local expression pattern of UGT2A1. The results, nonetheless, might be misguiding due to the fact that UGT2B7 is shown to be inhibited by long chain fatty acids (Rowland et al., 2007). Thus the actual Kms are supposed to be even lower than the reported ones. The effect of the fatty acids in UGT2A1 mediated glucuronidation is to be investigated.

Table 7. Kinetic parameters of testosterone, epitestosterone, androsterone, and etiocholanolone glucuronidation by UGTs 2A1, 2B7 and 2B17. Values are mean ± S.E.

UGT2A1 UGT2B7 UGT2B17

Vmax Km Vmax Km Vmax Km pmol/mg/min µM pmol/mg/min µM pmol/mg/min µM

Testosterone 427 ± 9.5 38.7 ± 6.7 n.d. n.d. 1002 ± 16.7 10.0 ± 0.8 Epitestosterone 271 ± 6.7 11.6 ± 1.2 337 ± 4.8 1.7 ± 0.2 n.d. n.d. Androsterone n.d. n.d. 713 ± 41a 4.3 ± 0.6 280 ± 4.9 0.9 ± 0.1 Etiocholanolone 4900±700 250 ± 50 189 ± 4.7 5.4 ± 0.5 814 ± 4.3b 1.3 ± 0.3 a Data fitted to substrate inhibition equation Ksi 658 ± 333 µM b Data fitted to substrate inhibition equation, Ksi 494 ± 211 µM n.d. stands for “not detected”

37 4.2.1 Substrate selectivity of UGT2B7, UGT2B15 and UGT2B17

Prominent difference appeared between UGT2B7 and UGT2B17 stereoselectivity in glucuronide conjugation at the C17 position of the steroids (Table 6). UGT2B17 was strictly stereoselective in 17ȕ-OH glucuronidation (testosterone), whereas UGT2B7 strongly favored 17Į-configuration (epitestosterone) (II). This was consistent with other findings on steroids glucuronidation (Itäaho et al., 2008). UGT2B17 is also active in 3-O- glucuronidation of androsterone, etiocholanolone and dehydroepiandrosterone (III) (Turgeon et al., 2001). The aglycones have two possible alignments in the conjugating enzymes, either the A- or D-ring toward the catalytic site of the enzyme. For conjugation with glucuronic acid by UGT2B17, the configuration of the carbon that is bearing the hydroxyl undergoing the conjugation has to be in either the 17ȕ- or 3Į-orientation (Fig. 10). Although no structure bearing the hydroxyl in a 3ȕ-orientation was included in this study, it has been shown by others (Beaulieu et al., 1996) that epiandrosterone, an androsterone stereoisomer with 3ȕ-OH instead of 3Į-OH, was not a substrate for UGT2B17. Additional information on the substrate selectivity of UGT2B17 still has to be acquired, but our study adds both new insights and a proof of concept to the current knowledge on UGT2B17 substrate selectivity in steroids glucuronidation. A possible explanation for UGT2B7 stereoselectivity could be that methyl groups cause hindrance for glucuronidation if they are positioned in cis-configuration with respect to the hydroxyl group, considering the plane of steroid skeleton. This would explain why androsterone, etiocholanolone, epitestosterone, and 5Į-diol-3-O-glucuronide, but not testosterone glucuronide, are formed by UGT2B7. If it is assumed that a steroid structure can occupy the enzyme active site in two possible directions, then both 17Į-OH and 3Į- OH bearing steroids would be conjugated by this enzyme. In fact, the earlier results on androgens glucuronidation by UGT2B enzymes also indicate that 3ȕ- as well as 17ȕ- hydroxylated androgens are rather poor substrates to UGT2B7, while 3Į-hydroxylated serve as good aglycones (Turgeon et al., 2001). The metabolite formation is dependent on both binding affinity and catalytic turnover. Epitestosterone serves as a particularly good substrate for UGT2B7, as it has not only a high Vmax but also a low Km, yielding high efficiency (Table 7). The notable difference in affinity became evident since testosterone was not inhibiting epitestosterone glucuronidation by UGT2B7, although the opposite was true in UGT2B17-catalyzed testosterone glucuronidation (II). The diols glucuronidation at 3-OH was diminished when an additional OH-group was available at C17 (5Į-diol) (Table 6). The diol may exhibit non-productive binding on the C17-direction, decreasing the production of 3-O- glucuronides. UGT2B15 is strictly selective for 17ȕ-O-glucuronidation, according to both our studies (II, III) and previous results (Turgeon et al., 2001). Possibly due to the direction of the hydrophobic methyl groups, which in the case of UGT2B15 would prevent reaction when directing the opposite (trans) side of the steroid plane.

38 Conjugated OH

H H H OH 3a O H Androsterone H H H OH 3a OH H 5a-diol

H OH

17b U G 7 OH H H

B H T

2 5a-diol 2

T

B 1

G 7 U H OH O 17b H H Testosterone H O OH H H 17a Epitestosterone

H OH H O 3a H H

Etiocholanolone

Figure 10. Selectivity of UGT2B7 and UGT2B17 toward 3Į- and 17Įȕ-OH groups in C19 steroids.

4.2.2 Correlation of in vitro results with in vivo findings

The results on steroid glucuronidation also have clinical implications. Most, if not all, steroids are conjugated by UGT2B7, which is abundantly expressed in liver, intestines, and kidneys (Nakamura et al., 2008; Ohno and Nakajin, 2009; Turgeon et al., 2001). The 39 UGT2B subfamily enzymes are all present in liver and it has recently been reported that UGT2B4 expression is particularly high in this organ (Izukawa et al., 2009; Ohno and Nakajin, 2009). Hence, UGT2B4 might have a prominent contribution to metabolism in vivo, despite its relatively low activity in vitro. Although not only testosterone, but also androsterone and etiocholanolone are preferentially glucuronidated in vitro by UGT2B17 (Table 6), the deletion of UGT2B17 does not seem to have a significant effect on these steroid glucuronide excretion in urine (Jakobsson et al., 2006; Juul et al., 2009). Since endogenous concentrations of steroids are low (unconjugated etiocholanolone in plasma 1.4 nM) (Sato and Ohsawa, 1977) the high affinity to the enzyme is important, and therefore the kinetic parameters can not directly explain why the conjugation in vivo is not disturbed by the deficiency of UGT2B17. The explanation might lay in the relatively low expression level of UGT2B17 in the liver, and its near absence from the kidney (Nakamura et al., 2008; Ohno and Nakajin, 2009). Since 5ȕ-diol-17-O-glucuronide is only produced by UGTs 2B15 and 2B17, its concentration could be expected to be related to UGT2B17 deletion polymorphism, as seen with testosterone (Jakobsson et al., 2006). The total 5ȕ-diol glucuronide amount in urine is reduced among samples with a UGT2B17 deletion polymorphism (Jakobsson et al., 2006), suggesting that in vivo a considerable amount is glucuronidated at the 17-OH by UGT2B17. On the other hand, the effect of UGT2B17 deletion was not seen or was minor in 5Į-diol glucuronidation (Jakobsson et al., 2006; Juul et al., 2009). Our results can explain these findings, as especially 5ȕ-diol-17-glucuronide formation is almost exclusively due to UGT2B17 activity, whereas UGT2B15 can largely compensate the lack of UGT2B17 in the case of 5Į-diol (Table 6). Even though the study by Juul et al. discussed all conjugated metabolites of the studied androgens, and the diol glucuronides concentrations consisted of both 3-O- and 17-O-glucuronide, the difference was highly significant, suggesting a central role of UGT2B17 in 5ȕ-diol glucuronidation in vivo. UGT2A1 was active in all androgens glucuronidation, at least to some extent, but generally the Km in every substrates glucuronidation was quite high (Table 6-7). The contribution of UGT2A1 to steroids systemic metabolism is presumed negligible, as it is only abundantly expressed in the nasal cavity, although reports about low level expression of UGT2A1 mRNA in the lung and trachea have been published (Jedlitschky et al., 1999; Nishimura and Naito, 2006; Somers et al., 2007). The UGT2A1, however, should not be ignored in prospective studies. Nasal delivery in brain targeting has gained increasing interest (Costantino et al., 2008; Hanson and Frey, 2008), and even testosterone administration to the brain of a mouse, using the intranasal route, was recently described (Banks et al., 2009).

4.3 Inhibition of testosterone glucuronidation (II, IV)

We studied inhibition of testosterone glucuronidation by different compounds using recombinant UGT2B17 (II, IV) and UGT2B15 (IV). Inhibitory effects of diclofenac and ibuprofen on testosterone glucuronidation by HLM, UGT2B15, and UGT2B17 were investigated at fixed substrate concentrations and variable inhibitor concentrations (IV). 40 Competitive inhibition was evident in most cases with the exception of ibuprofen inhibition of UGT2B17-catalyzed testosterone glucuronidation. Weak inhibition was seen in this case, but no clear conclusion of the inhibition type could be drawn based on the Dixon plots (IV). The study of inhibition potency of epitestosterone on testosterone glucuronidation by UGT2B17 (II) was conducted by full kinetic characterization in presence of varying inhibitor concentrations. As computer programs for calculating global values for kinetic and inhibition parameters offer better estimation of true values, such a study design is advisable (Kakkar et al., 1999). This study design also allows the use of Lineweaver-Burk plots in visualizing the inhibition type. Fairly good estimations with only two substrate concentrations could be acquired when our data (IV) was analyzed with the above mentioned techniques (Fig. 11). Lineweaver-Burk plots strongly suggest competitive type inhibition by diclofenac and epitestosterone, but uncompetitive type by ibuprofen (Fig. 11).

1000 800 1000

800 A B 800 C 600

600 600 400 400 400

200 200 200 V (pmol/mg protein/min) V (pmol/mg V (pmol/mg protein/min) protein/min) V (pmol/mg

0 0 0 0 10 20 30 40 50 0 10 20 30 40 50 0 25 50 75 100 125 [S] µM [S] µM [S] µM

0.020 0.005 0.006

D E 0.004 F 0.015 0.004 0.003 0.010 1/V 1/V 1/V 0.002 0.002 0.005 0.001

-0.025 0.000 0.025 0.050 0.075 0.100 -0.10 -0.05 0.00 0.05 0.10 -0.10 -0.05 0.00 0.05 0.10 1/[S] 1/[S] 1/[S]

Figure 11. Testosterone glucuronidation by UGT2B17 and inhibition by diclofenac (A), ibuprofen (B) and epitestosterone (C) represented in direct plot (A-C) and semireciprocal Lineweaver-Burk plot (D-F, respectively). Data was fit in competitive inhibition (A,C) or uncompetitive inhibition equation (B) with GraphPad Prism 5.0.

UGT2B17 is the most active enzyme in testosterone glucuronidation, both in vitro and in vivo (II, IV) (Jakobsson et al., 2006; Turgeon et al., 2001). If UGT2B17 is inhibited in vivo, it could have a minor contribution to homeostasis of certain substances, such as testosterone. The inhibitors, still, should be far more effective than the tested ones. The Ki of diclofenac inhibition, calculated from the above mentioned data, was 56 µM (± S.E. 4.8 µM), and the Ki for ibuprofen was 605 µM (± S.E. 78 µM). The plasma concentration observed for diclofenac varies between 3.6 and 6.1 µM (Cryer and Feldman, 1998; Vietri et al., 2000). The therapeutic plasma concentrations of ibuprofen have been reported to be between 39 and 110 µM (Cryer and Feldman, 1998) depending on the dose. This suggests that, considering UGT2B17 metabolism in vivo, these NSAIDS are not expected to significantly affect its conjugative activity. UGT2B15 was inhibited by both diclofenac 41 and ibuprofen with Kis of 13 (±0.8) µM and 40 (±3.3) µM, respectively (IV). This indicates that ibuprofen, despite having a lower potential inhibitory effect, might modulate the testosterone glucuronidation more in vivo, especially in the case of UGT2B17 del/del genotypes, in which UGT2B15 is probably the main enzyme responsible in testosterone glucuronidation (II). However, both diclofenac and ibuprofen are very highly protein bound in blood, and on that basis, inhibition of testosterone glucuronidation may not be expected.

42 5. Summary and Conclusions

Stereoisomers offer useful probes for investigating UGT substrate specificity and structural aspects influencing substrate selectivity and affinity. Propranolol was glucuronidated by UGT1A9 and UGT1A10, two very similar enzymes, with opposite stereoselectivity. The results suggest a role of intestinal UGT1A10 in the first pass metabolism of propranolol. Extrahepatic UGT2A1 was also active, even though both propranolol enantiomers were poor low affinity substrates. Allosteric activation was suggested in the reaction between propranolol enantiomers and UGT2A1. Studies on androgens glucuronidation by human recombinant UGTs mostly offered a proof of concept to the previous studies, but also some novel findings. Testosterone glucuronidation by UGT2B17 was inhibited by its diastereomeric pair epitestosterone, a high affinity substrate of UGT2B7. NSAIDs, diclofenac and ibuprofen, were not considerably inhibiting in testosterone glucuronidation by UGT2B17, whereas UGT2B15 was inhibited by both. The androgens were mostly glucuronidated by UGT2B7, UGT2B15, UGT2B17, and often UGT2A1. In both regio- and stereoselectivity, UGT2B15 was strictly selective. It only conjugated the androgens at the 17-OH position in the ȕ-configuration, whereas UGT2B17 accepted both 3Į- and 17ȕ-OH as the acceptor group. If both of those were available, the 17ȕ-OH was favored over the 3Į-OH. The third active UGT in steroid glucuronidation, UGT2B7, accepted both 3Į- and 17Į-OH, but not 17ȕ-OH, which also significantly impaired the glucuronidation reaction when available in the molecule. For UGT2B7, a structure with a 17ȕ-OH, seems to act as a competitive inhibitor for 3-O-glucuronidation unless the Km of 3-O-glucuronidation is very low. Interestingly, epitestosterone with a 17-OH in Į-configuration served as a low Km substrate that was not inhibited by testosterone with 17ȕ-OH. The results shed new light on the selectivity of human UGTs toward endogenic C19 steroids.

43 6. Acknowledgements

This study was carried out at the Division of Pharmaceutical Chemistry and Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, during the years 2002-2009. During that time span I attained valuable skills in the laboratory, and on university courses and congresses, gave birth to a beautiful child, learned to enjoy my life at home, purchased two homes, lost relatives and friends, made new friends for life, suffered from depression but gained pride and confidence again and drove around 150 000 km in my red car. There are numerous people and elements I need to express my gratitude to. The person to begin with is my respectable supervisor, mentor and friend, Dr. Moshe Finel. Things just began clicking into places after your intervention. I also need to thank my other supervisors, Pharm.Lic Leena Luukkanen and professor Risto Kostiainen, for their efforts in my research. I acknowledge World Antidoping Agency, Karolinska Institutet, the Swedish National Centre for Research in Sports, as well as Emil Aaltonen foundation for funding the work. Waters Corp., Finland is thanked for giving us the privilege to use their UPLC® device. I want to express my greatest thankfulness to all my co-workers in the lab and other people contributing to the research done. My best friend Mika Kurkela, for his endless patience, trust and respect. My student, Saana Qvisen, for her hardworking and trustworthy graft in the lab. My collague and friend, Päivi Uutela, for introducing me to the analytical equipment, productively criticizing my work, results and conclusions about them, and allowing me to consume her secret hidden candies. For the scientific expertise lent to my use, and respective collaboratorion I am truly thankful to the other co-authors Lena Ekström, Birgitta Ask, Anders Rane, Ingo Bichlmaier, Jari Yli-Kauhaluoma, Tiia Kuuranne and Antti Leinonen. Professor John Miners and Dr. Mikko Koskinen are acknowledged for carefully reviewing the manuscript and providing valuable comments. The contribution of certain people to my work in the laboratory was crucial, and the exceptional laboratory and support work from my dear friends Johanna Mosorin, Sanna Sistonen, Inkeri Huttunen, Irene Kylänlahti and Ismo Väisänen are acknowledged. The working period in the Division of Pharmaceutical Chemistry was hard but rewarding. There are numerous more people to thank about tutoring and teaching me, and also for warm friendship. Katariina and Tuomo, thank you for making me a part of your lives. Very special thanks to Nina S, for everything! Wonderful people; Anna, Elisa, Hannele, Johanna I, Sirkku, Katriina, Kati H, Kirsi, Liisa, Piia, Nenad, Hongbo, Tapio, and Teemu, you all invigorated me during these years. Life wasn’t worth living if it didn’t happen predominantly outside the laboratory. I think that I have the best family and friends anyone could ask for. My significant other Marko; thanks for taking care of both the house and the garden. Dear children, Valtteri, Ella and Leea. I could have hoped for some more help in the household projects, but not much more love, trust, challenge and encouraging intellectual company than what you did offer. My brothers, sister and father as well as my parents-in-law deserve many thanks for helping us in the house renovation, child care and economics. Although I have been away for years, there is still a home to go back to, whenever it is needed.

44 Despite my occasional low or unsettled moods, foolish behavior and aggressions, I do have some lovely friends for which I am most grateful. Many fruitful discussions, relaxing coffee breaks, meals, hikes, expeditions, bacchanalia and evenings in sauna have taken place with Ellu, Epe, Hanna, Jaana, Leena, Seija, Suvi, Elysee, Hessu, Jansu, Jukka, Jyri, Pate, Ville and numerous other lively people. I was born on 1975, the very year that John Cornforth and Vladimir Prelog were honored with the Nobel Prize in Chemistry for “his work on the stereochemistry of enzyme-catalyzed reactions” and for "his research into the stereochemistry of organic molecules and reactions". It probably did not guide me into the field, but still gives a special bond to this theme of research. I began this work the same year my dear mother passed away, in the middle of her own licentiate studies. I dedicate this thesis to my mother, Aila. You would have been proud of me once again.

Tuusula

Taina Sten

45 7. References

Akiyama Y, Fujita K, Nagashima F, Yamamoto W, Endo H, Sunakawa Y, Yamashita K, Ishida H, Mizuno K, Araki K, Ichikawa W, Miya T, Narabayashi M, Kawara K, Sugiyama M, Hirose T, Ando Y, Sasaki Y. (2008) Genetic testing for UGT1A1*28 and *6 in japanese patients who receive irinotecan chemotherapy. Ann Oncol 19:2089-2090. Ali I, Gupta VK, Aboul-Enein HY, Singh P, Sharma B. (2007) Role of racemization in optically active drugs development (abstract). Chirality 19:453-463. Alonen A, Aitio O, Hakala K, Luukkanen L, Finel M, Kostiainen R. (2005) Biosynthesis of dobutamine monoglucuronides and glucuronidation of dobutamine by recombinant human UDP-glucuronosyltransferases. Drug Metab Dispos 33:657-663. Alonen A, Gartman M, Aitio O, Finel M, Yli-Kauhaluoma J, Kostiainen R. (2009) Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides. Eur J Pharm Sci 37:581-587. Al-Zoughool M, Talaska G. (2006) 4-aminobiphenyl N-glucuronidation by liver microsomes: Optimization of the reaction conditions and characterization of the UDP- glucuronosyltransferase isoforms. J Appl Toxicol 26:524-532. Argikar UA, Iwuchukwu OF, Nagar S. (2008) Update on tools for evaluation of uridine diphosphoglucuronosyltransferase polymorphisms. Expert Opin Drug Metab Toxicol 4:879- 894. Bai SA, Walle T. (1984) Isolation, purification, and structure identification of glucuronic acid conjugates of propranolol and alprenolol and their ring-hydroxylated metabolites. Drug Metab Dispos 12:749-754. Banks WA, Morley JE, Niehoff ML, Mattern C. (2009) Delivery of testosterone to the brain by intranasal administration: Comparison to intravenous testosterone. J Drug Target 17:91-97. Beaulieu M, Levesque E, Hum DW, Belanger A. (1996) Isolation and characterization of a novel cDNA encoding a human UDP-glucuronosyltransferase active on C19 steroids. J Biol Chem 271:22855-22862. Belanger A, Brochu M, Lacoste D, Noel C, Labrie F, Dupont A, Cusan L, Caron S, Couture J. (1991) Steroid glucuronides: Human circulatory levels and formation by LNCaP cells. J Steroid Biochem Mol Biol 40:593-598. Belanger A, Pelletier G, Labrie F, Barbier O, Chouinard S. (2003) Inactivation of androgens by UDP-glucuronosyltransferase enzymes in humans. Trends Endocrinol Metab 14:473-479. Bhasin S, Storer TW, Berman N, Callegari C, Clevenger B, Phillips J, Bunnell TJ, Tricker R, Shirazi A, Casaburi R. (1996) The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. N Engl J Med 335:1-7. Bhasker CR, McKinnon W, Stone A, Lo AC, Kubota T, Ishizaki T, Miners JO. (2000) Genetic polymorphism of UDP-glucuronosyltransferase 2B7 (UGT2B7) at amino acid 268: Ethnic diversity of alleles and potential clinical significance. Pharmacogenetics 10:679-685. Boase S, Miners JO. (2002) In vitro-in vivo correlations for drugs eliminated by glucuronidation: Investigations with the model substrate zidovudine. Br J Clin Pharmacol 54:493-503. Bobbitt DR, Linder SW. (2001) Recent advances in chiral detection for high performance liquid chromatography. Trends in Analytical Chemistry 20:111-123.

46 Bock KW, Kohle C. (2004) Coordinate regulation of drug metabolism by xenobiotic nuclear receptors: UGTs acting together with CYPs and glucuronide transporters. Drug Metab Rev 36:595-615. Bock KW, Kohle C. (2009) Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: Advances and open questions. Biochem Pharmacol 77:1458-1465. Bowalgaha K, Elliot DJ, Mackenzie PI, Knights KM, Miners JO. (2007) The glucuronidation of Delta4-3-keto C19- and C21-hydroxysteroids by human liver microsomal and recombinant UDP-glucuronosyltransferases (UGTs): 6alpha- and 21-hydroxyprogesterone are selective substrates for UGT2B7. Drug Metab Dispos 35:363-370. Bowers LD. (2008) Testosterone doping: Dealing with genetic differences in metabolism and excretion. J Clin Endocrinol Metab 93:2469-2471. Bowers LD, Clark RV, Shackleton CH. (2009) A half-century of anabolic steroids in sport. Steroids 74:285-287. Buch A, Barr WH. (1998) Absorption of propranolol in humans following oral, jejunal, and ileal administration. Pharm Res 15:953-957. Buckley DB, Klaassen CD. (2009) Induction of mouse UDP-glucuronosyltransferase mRNA expression in liver and intestine by activators of AhR, CAR, PXR, PPAR{alpha}, and Nrf2. Drug Metab Dispos 37:847-856. Burlacu CL, Buggy DJ. (2008) Update on local anesthetics: Focus on levobupivacaine. Ther Clin Risk Manag 4:381-392. Campo VL, Bernardes LS, Carvalho I. (2009) Stereoselectivity in drug metabolism: Molecular mechanisms and analytical methods. Curr Drug Metab 10:188-205. Cawley AT, Trout GJ, Kazlauskas R, George AV. (2008) The detection of androstenedione abuse in sport: A mass spectrometry strategy to identify the 4-hydroxyandrostenedione metabolite. Rapid Commun Mass Spectrom 22:4147-4157. Chen G, Blevins-Primeau AS, Dellinger RW, Muscat JE, Lazarus P. (2007a) Glucuronidation of nicotine and cotinine by UGT2B10: Loss of function by the UGT2B10 codon 67 (Asp>Tyr) polymorphism. Cancer Res 67:9024-9029. Chen Y, Kuehl GE, Bigler J, Rimorin CF, Schwarz Y, Shen DD, Lampe JW. (2007b) UGT1A6 polymorphism and salicylic acid glucuronidation following aspirin. Pharmacogenet Genomics 17:571-579. Chiarotto JA, Wainer IW. (1995) Determination of metyrapone and the enantiomers of its chiral metabolite metyrapol in human plasma and urine using coupled achiral-chiral liquid chromatography. J Chromatogr B Biomed Appl 665:147-154. Chouinard S, Yueh MF, Tukey RH, Giton F, Fiet J, Pelletier G, Barbier O, Belanger A. (2008) Inactivation by UDP-glucuronosyltransferase enzymes: The end of androgen signaling. J Steroid Biochem Mol Biol 109:247-253. Chung JY, Cho JY, Yu KS, Kim JR, Lim KS, Sohn DR, Shin SG, Jang IJ. (2008) Pharmacokinetic and pharmacodynamic interaction of lorazepam and valproic acid in relation to UGT2B7 genetic polymorphism in healthy subjects. Clin Pharmacol Ther 83:595-600. Coffman BL, King CD, Rios GR, Tephly TR. (1998) The glucuronidation of opioids, other xenobiotics, and androgens by human UGT2B7Y(268) and UGT2B7H(268). Drug Metab Dispos 26:73-77.

47 Copeland RA. 2005. Evaluation of enzyme inhibitors in drug discovery: A guide for medicinal chemists and pharmacologists. Hoboken, NJ, USA: John Wiley & Sons. Cornish-Bowden A, Dixon HBF, Laidler KJ, Segel IH, Ricard J, Velick SF, Webb EC. (1983) Symbolism and terminology in enzyme kinetics. recommendations 1981. Biochem J 213:561- 571. Cortes A, Cascante M, Cardenas ML, Cornish-Bowden A. (2001) Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: New ways of analysing data. Biochem J 357:263-268. Costantino HR, Leonard AK, Brandt G, Johnson PH, Quay SC. (2008) Intranasal administration of acetylcholinesterase inhibitors. BMC Neurosci 9 Suppl 3:S6. Court MH, Duan SX, Guillemette C, Journault K, Krishnaswamy S, Von Moltke LL, Greenblatt DJ. (2002) Stereoselective conjugation of oxazepam by human UDP-glucuronosyltransferases (UGTs): S-oxazepam is glucuronidated by UGT2B15, while R-oxazepam is glucuronidated by UGT2B7 and UGT1A9. Drug Metab Dispos 30:1257-1265. Court MH, Hao Q, Krishnaswamy S, Bekaii-Saab T, Al-Rohaimi A, von Moltke LL, Greenblatt DJ. (2004) UDP-glucuronosyltransferase (UGT) 2B15 pharmacogenetics: UGT2B15 D85Y genotype and gender are major determinants of oxazepam glucuronidation by human liver. J Pharmacol Exp Ther 310:656-665. Court MH, Hazarika S, Krishnaswamy S, Finel M, Williams JA. (2008) Novel polymorphic human UDP-glucuronosyltransferase (UGT) 2A3: Cloning, functional characterization of enzyme variants, comparative tissue expression, and gene induction. Mol Pharmacol 74:744- 754. Crespi CL, Miller VP. (1999) The use of heterologously expressed drug metabolizing enzymes-- state of the art and prospects for the future. Pharmacol Ther 84:121-131. Cryer B, Feldman M. (1998) Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am J Med 104:413-421. Cubitt HE, Houston JB, Galetin A. (2009) Relative importance of intestinal and hepatic glucuronidation-impact on the prediction of drug clearance. Pharm Res 26:1073-1083. Daly AK, Aithal GP, Leathart JB, Swainsbury RA, Dang TS, Day CP. (2007) Genetic susceptibility to diclofenac-induced hepatotoxicity: Contribution of UGT2B7, CYP2C8, and ABCC2 genotypes. Gastroenterology 132:272-281. Deeken JF, Slack R, Marshall JL. (2008) Irinotecan and uridine diphosphate glucuronosyltransferase 1A1 pharmacogenetics: To test or not to test, that is the question. Cancer 113:1502-1510. Dehennin L, Matsumoto AM. (1993) Long-term administration of testosterone enanthate to normal men: Alterations of the urinary profile of androgen metabolites potentially useful for detection of testosterone misuse in sport. J Steroid Biochem Mol Biol 44:179-189. Dixon M. (1953) The determination of enzyme inhibitor constants. Biochem J 55:170-171. Donike M, Bärwald KR, Klostermann K, Schänzer W, Zimmerman J. (1983). Nachweis von exogenem testosterone. Köln: Sport: Leistung und Gesundheit 293-298. Donike M, Ueki M, Kuroda Y, Geyer H, Nolteernsting E, Rauth S, Schänzer W, Schindler U, Volker E, Fujisaki M. (1995) Detection of dihydrotestosterone (DHT) doping: Alterations in the steroid profile and reference ranges for DHT and its 5 alpha-metabolites. J Sports Med Phys Fitness 35:235-250.

48 Dufort I, Labrie F, Luu-The V. (2001) Human types 1 and 3 3 alpha-hydroxysteroid dehydrogenases: Differential lability and tissue distribution. J Clin Endocrinol Metab 86:841- 846. Evans WE, Relling MV. (1999) Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 286:487-491. Finel M, Kurkela M. (2008) The UDP-glucuronosyltransferases as oligomeric enzymes. Curr Drug Metab 9:70-76. Finel M, Li X, Gardner-Stephen D, Bratton S, Mackenzie PI, Radominska-Pandya A. (2005) Human UDP-glucuronosyltransferase 1A5: Identification, expression, and activity. J Pharmacol Exp Ther 315:1143-1149. Fisher MB, Paine MF, Strelevitz TJ, Wrighton SA. (2001) The role of hepatic and extrahepatic UDP-glucuronosyltransferases in human drug metabolism. Drug Metab Rev 33:273-297. Fujiwara R, Nakajima M, Oda S, Yamanaka H, Ikushiro SI, Sakaki T, Yokoi T. (2010) Interactions between human UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A enzymes. J Pharm Sci 99: 442-454. Fujiwara R, Nakajima M, Yamanaka H, Katoh M, Yokoi T. (2007a) Interactions between human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities. Drug Metab Dispos 35:1781-1787. Fujiwara R, Nakajima M, Yamanaka H, Katoh M, Yokoi T. (2008) Product inhibition of UDP- glucuronosyltransferase (UGT) enzymes by UDP obfuscates the inhibitory effects of UGT substrates. Drug Metab Dispos 36:361-367. Fujiwara R, Nakajima M, Yamanaka H, Nakamura A, Katoh M, Ikushiro S, Sakaki T, Yokoi T. (2007b) Effects of coexpression of UGT1A9 on enzymatic activities of human UGT1A isoforms. Drug Metab Dispos 35:747-757. Fujiwara R, Nakajima M, Yamanaka H, Yokoi T. (2009) Key amino acid residues responsible for the differences in substrate specificity of human UDP-glucuronosyltransferase (UGT)1A9 and UGT1A8. Drug Metab Dispos 37:41-46. Girard H, Villeneuve L, Court MH, Fortier LC, Caron P, Hao Q, von Moltke LL, Greenblatt DJ, Guillemette C. (2006) The novel UGT1A9 intronic I399 polymorphism appears as a predictor of 7-ethyl-10-hydroxycamptothecin glucuronidation levels in the liver. Drug Metab Dispos 34:1220-1228. Guillemette C. (2003) Pharmacogenomics of human UDP-glucuronosyltransferase enzymes. Pharmacogenomics J 3:136-158. Haginaka J. (2002) Pharmaceutical and biomedical applications of enantioseparations using liquid chromatographic techniques. J Pharm Biomed Anal 27:357-372. Hanioka N, Hayashi K, Shimizudani T, Nagaoka K, Koeda A, Naito S, Narimatsu S. (2008) Stereoselective glucuronidation of propranolol in human and cynomolgus monkey liver microsomes: Role of human hepatic UDP-glucuronosyltransferase isoforms, UGT1A9, UGT2B4 and UGT2B7. Pharmacology 82:293-303. Hanson LR, Frey WH 2nd. (2008) Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC Neurosci 9 Suppl 3:S5. Hao H, Wang G, Sun J. (2005) Enantioselective pharmacokinetics of ibuprofen and involved mechanisms. Drug Metab Rev 37:215-234.

49 Ho KJ. (1995) Urinary beta-glucuronidase in screening and followup of primary urinary tract malignancy. J Urol 154:1335-1338. Hyttel J, Bogeso KP, Perregaard J, Sanchez C. (1992) The pharmacological effect of citalopram residues in the (S)-(+)-enantiomer. J Neural Transm Gen Sect 88:157-160. Itäaho K, Mackenzie P, Ikushiro SI, Miners J, Finel M. (2008) The configuration of the 17- hydroxy group variably influences the glucuronidation of {beta}-estradiol and epiestradiol by human UDP-glucuronosyltransferases. Drug Metab Dispos 36:2307-2315. Izukawa T, Nakajima M, Fujiwara R, Yamanaka H, Fukami T, Takamiya M, Aoki Y, Ikushiro S, Sakaki T, Yokoi T. (2009) Quantitative analysis of UDP-glucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers. Drug Metab Dispos 37:1759-1768. Jakobsson J, Ekstrom L, Inotsume N, Garle M, Lorentzon M, Ohlsson C, Roh HK, Carlstrom K, Rane A. (2006) Large differences in testosterone excretion in korean and swedish men are strongly associated with a UDP-glucuronosyl transferase 2B17 polymorphism. J Clin Endocrinol Metab 91:687-693. Jäntti SE, Kiriazis A, Reinilä RR, Kostiainen RK, Ketola RA. (2007) Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids. Steroids 72:287-296. Jedlitschky G, Cassidy AJ, Sales M, Pratt N, Burchell B. (1999) Cloning and characterization of a novel human olfactory UDP-glucuronosyltransferase. Biochem J 340 (Pt 3):837-843. Jin Y, Duan L, Lee SH, Kloosterboer HJ, Blair IA, Penning TM. (2009) Human cytosolic hydroxysteroid dehydrogenases of the aldo-keto reductase superfamily catalyze reduction of conjugated steroids: Implications for phase I and phase II steroid hormone metabolism. J Biol Chem 284:10013-10022. Juul A, Sorensen K, Aksglaede L, Garn I, Rajpert-De Meyts E, Hullstein I, Hemmersbach P, Ottesen AM. (2009) A common deletion in the uridine diphosphate glucuronyltransferase (UGT) 2B17 gene is a strong determinant of androgen excretion in healthy pubertal boys. J Clin Endocrinol Metab 94:1005-1011. Kakkar T, Boxenbaum H, Mayersohn M. (1999) Estimation of ki in a competitive enzyme- inhibition model: Comparisons among three methods of data analysis. Drug Metab Dispos 27:756-762. Kancheva L, Hill M, Vcelakova H, Vrbikova J, Pelikanova T, Starka L. (2007) The identification and simultaneous quantification of neuroactive androstane steroids and their polar conjugates in the serum of adult men, using gas chromatography-mass spectrometry. Steroids 72:792-801. Karypidis AH, Olsson M, Andersson SO, Rane A, Ekstrom L. (2008) Deletion polymorphism of the UGT2B17 gene is associated with increased risk for prostate cancer and correlated to gene expression in the prostate. Pharmacogenomics J 8:147-151. Kiang TK, Ensom MH, Chang TK. (2005) UDP-glucuronosyltransferases and clinical drug-drug interactions. Pharmacol Ther 106:97-132. Kilford PJ, Stringer R, Sohal B, Houston JB, Galetin A. (2009) Prediction of drug clearance by glucuronidation from in vitro data: Use of combined cytochrome P450 and UDP- glucuronosyltransferase cofactors in alamethicin-activated human liver microsomes. Drug Metab Dispos 37:82-89. King CD, Rios GR, Green MD, Tephly TR. (2000) UDP-glucuronosyltransferases. Curr Drug Metab 1:143-161.

50 Kohle C, Bock KW. (2009) Coordinate regulation of human drug-metabolizing enzymes, and conjugate transporters by the Ah receptor, pregnane X receptor and constitutive androstane receptor. Biochem Pharmacol 77:689-699. Kong SY, Ki CS, Yoo BC, Kim JW. (2008) UGT1A7 haplotype is associated with an increased risk of hepatocellular carcinoma in hepatitis B carriers. Cancer Sci 99:340-344. Kuhnle A, Linderoth TR, Hammer B, Besenbacher F. (2002) Chiral recognition in dimerization of adsorbed cysteine observed by scanning tunnelling microscopy. Nature 415:891-893. Kurkela M, Garcia-Horsman JA, Luukkanen L, Mörsky S, Taskinen J, Baumann M, Kostiainen R, Hirvonen J, Finel M. (2003) Expression and characterization of recombinant human UDP- glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme. J Biol Chem 278:3536-3544. Kurkela M, Hirvonen J, Kostiainen R, Finel M. (2004) The interactions between the N-terminal and C-terminal domains of the human UDP-glucuronosyltransferases are partly isoform- specific, and may involve both monomers. Biochem Pharmacol 68:2443-2450. Kurkela M, Patana AS, Mackenzie PI, Court MH, Tate CG, Hirvonen J, Goldman A, Finel M. (2007) Interactions with other human UDP-glucuronosyltransferases attenuate the consequences of the Y485D mutation on the activity and substrate affinity of UGT1A6. Pharmacogenet Genomics 17:115-126. Kuuranne T, Kurkela M, Thevis M, Schänzer W, Finel M, Kostiainen R. (2003) Glucuronidation of anabolic androgenic steroids by recombinant human UDP-glucuronosyltransferases. Drug Metab Dispos 31:1117-1124. Labrie F, Luu-The V, Labrie C, Belanger A, Simard J, Lin SX, Pelletier G. (2003) Endocrine and intracrine sources of androgens in women: Inhibition of breast cancer and other roles of androgens and their precursor dehydroepiandrosterone. Endocr Rev 24:152-182. Lacko M, Roelofs HM, Te Morsche RH, Voogd AC, Ophuis MB, Peters WH, Manni JJ. (2009) Genetic polymorphisms in the tobacco smoke carcinogens detoxifying enzyme UGT1A7 and the risk of head and neck cancer. Head Neck 31:1274-1281. Laethem ME, Belpaire FM, Bogaert MG. (1996) Direct high-performance liquid chromatography determination of diastereomeric oxprenolol glucuronides. J Chromatogr B Biomed Appl 675:251-255. Lampe JW, Bigler J, Bush AC, Potter JD. (2000) Prevalence of polymorphisms in the human UDP-glucuronosyltransferase 2B family: UGT2B4(D458E), UGT2B7(H268Y), and UGT2B15(D85Y). Cancer Epidemiol Biomarkers Prev 9:329-333. Lampe JW, Bigler J, Horner NK, Potter JD. (1999) UDP-glucuronosyltransferase (UGT1A1*28 and UGT1A6*2) polymorphisms in Caucasians and Asians: Relationships to serum bilirubin concentrations. Pharmacogenetics 9:341-349. Lampe JW, Li SS, Potter JD, King IB. (2002) Serum beta-glucuronidase activity is inversely associated with plant-food intakes in humans. J Nutr 132:1341-1344. Levesque E, Beaulieu M, Green MD, Tephly TR, Belanger A, Hum DW. (1997) Isolation and characterization of UGT2B15(Y85): A UDP-glucuronosyltransferase encoded by a polymorphic gene. Pharmacogenetics 7:317-325. Lewis BC, Mackenzie PI, Elliot DJ, Burchell B, Bhasker CR, Miners JO. (2007) Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation. Biochem Pharmacol 73:1463-1473.

51 Li X, Yu YN, Zhu GJ, Qian YL. (2001) Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells. World J Gastroenterol 7:841-845. Luan LJ, Shao Q, Ma JY, Zeng S. (2005) Stereoselective urinary excretion of S-(-)- and R-(+)- propranolol glucuronide following oral administration of RS-propranolol in chinese han subjects. World J Gastroenterol 11:1822-1824. Lucas R, Alcantara D, Morales JC. (2009) A concise synthesis of glucuronide metabolites of urolithin-B, resveratrol, and hydroxytyrosol. Carbohydr Res 344:1340-1346. Luukkanen L, Kilpelainen I, Kangas H, Ottoila P, Elovaara E, Taskinen J. (1999) Enzyme-assisted synthesis and structural characterization of nitrocatechol glucuronides. Bioconjug Chem 10:150-154. Luukkanen L, Taskinen J, Kurkela M, Kostiainen R, Hirvonen J, Finel M. (2005) Kinetic characterization of the 1A subfamily of recombinant human UDP-glucuronosyltransferases. Drug Metab Dispos 33:1017-1026. Mackenzie PI, Gregory PA, Gardner-Stephen DA, Lewinsky RH, Jorgensen BR, Nishiyama T, Xie W, Radominska-Pandya A. (2003) Regulation of UDP glucuronosyltransferase genes. Curr Drug Metab 4:249-257. Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Belanger A, Fournel-Gigleux S, Green M, Hum DW, Iyanagi T, Lancet D, Louisot P, Magdalou J, Chowdhury JR, Ritter JK, Schachter H, Tephly TR, Tipton KF, Nebert DW. (1997) The UDP glycosyltransferase gene superfamily: Recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 7:255-269. Mackenzie PI, Walter Bock K, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners JO, Owens IS, Nebert DW. (2005) Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet Genomics 15:677-685. McCarroll SA, Hadnott TN, Perry GH, Sabeti PC, Zody MC, Barrett JC, Dallaire S, Gabriel SB, Lee C, Daly MJ, Altshuler DM, International HapMap Consortium. (2006) Common deletion polymorphisms in the human genome. Nat Genet 38:86-92. Meech R, Mackenzie PI. (2009) UGT3A: Novel UDP-glycosyltransferases of the UGT superfamily. Drug Metab Rev. In Press. Menard V, Eap O, Harvey M, Guillemette C, Levesque E. (2009) Copy-number variations (CNVs) of the human sex steroid metabolizing genes UGT2B17 and UGT2B28 and their associations with a UGT2B15 functional polymorphism. Hum Mutat 30:1310-1319. Midha KK, Roscoe RM, Wilson TW, Cooper JK, Loo JC, Ho-Ngoc A, McGilveray IJ. (1983) Pharmacokinetics of glucuronidation of propranolol following oral administration in humans. Biopharm Drug Dispos 4:331-338. Miley MJ, Zielinska AK, Keenan JE, Bratton SM, Radominska-Pandya A, Redinbo MR. (2007) Crystal structure of the cofactor-binding domain of the human phase II drug-metabolism enzyme UDP-glucuronosyltransferase 2B7. J Mol Biol 369:498-511. Miners JO, Knights KM, Houston JB, Mackenzie PI. (2006) In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: Pitfalls and promises. Biochem Pharmacol 71:1531-1539. Miners JO, Smith PA, Sorich MJ, McKinnon RA, Mackenzie PI. (2004) Predicting human drug glucuronidation parameters: Application of in vitro and in silico modeling approaches. Annu Rev Pharmacol Toxicol 44:1-25.

52 Murai T, Iwabuchi H, Ikeda T. (2005) Repeated glucuronidation at one hydroxyl group leads to structurally novel diglucuronides of steroid sex hormones. Drug Metab Pharmacokinet 20:282-293. Nakajima M, Yamanaka H, Fujiwara R, Katoh M, Yokoi T. (2007) Stereoselective glucuronidation of 5-(4'-hydroxyphenyl)-5-phenylhydantoin by human UDP- glucuronosyltransferase (UGT) 1A1, UGT1A9, and UGT2B15: Effects of UGT-UGT interactions. Drug Metab Dispos 35:1679-1686. Nakamura A, Nakajima M, Yamanaka H, Fujiwara R, Yokoi T. (2008) Expression of UGT1A and UGT2B mRNA in human normal tissues and various cell lines. Drug Metab Dispos 36:1461- 1464. Nishimura M, Naito S. (2006) Tissue-specific mRNA expression profiles of human phase I metabolizing enzymes except for cytochrome P450 and phase II metabolizing enzymes. Drug Metab Pharmacokinet 21:357-374. Oatis JE,Jr, Baker JP, McCarthy JR, Knapp DR. (1983) Synthesis and chromatographic separation of the glucuronides of (R)- and (S)-propranolol. J Med Chem 26:1687-1691. Ohno S, Nakajin S. (2009) Determination of mRNA expression of human UDP- glucuronosyltransferases and application for localization in various human tissues by real-time reverse transcriptase-polymerase chain reaction. Drug Metab Dispos 37:32-40. Oleson L, Court MH. (2008) Effect of the beta-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases. J Pharm Pharmacol 60:1175-1182. Olson KC, Dellinger RW, Zhong Q, Sun D, Amin S, Spratt TE, Lazarus P. (2009) Functional characterization of low-prevalence missense polymorphisms in the UDP- glucuronosyltransferase1A9 gene. Drug Metab Dispos 37:1999-2007. Operana TN, Tukey RH. (2007) Oligomerization of the UDP-glucuronosyltransferase 1A proteins: Homo- and heterodimerization analysis by fluorescence resonance energy transfer and co- immunoprecipitation. J Biol Chem 282:4821-4829. Osborne R, Joel S, Trew D, Slevin M. (1990) Morphine and metabolite behavior after different routes of morphine administration: Demonstration of the importance of the active metabolite morphine-6-glucuronide. Clin Pharmacol Ther 47:12-19. Ouzzine M, Barre L, Netter P, Magdalou J, Fournel-Gigleux S. (2003) The human UDP- glucuronosyltransferases: Structural aspects and drug glucuronidation. Drug Metab Rev 35:287-303. Owens IS, Ritter JK. (1995) Gene structure at the human UGT1 locus creates diversity in isozyme structure, substrate specificity, and regulation. Prog Nucleic Acid Res Mol Biol 51:305-338. Patana AS. (2009) Academic dissertation: The human UDP-glucuronosyltransferases: Studies on substrate binding and catalytic mechanism. University of Helsinki. Patana AS, Kurkela M, Finel M, Goldman A. (2008) Mutation analysis in UGT1A9 suggests a relationship between substrate and catalytic residues in UDP-glucuronosyltransferases. Protein Eng Des Sel 21:537-543. Peng SH, Segura J, Farre M, de la Torre X. (2000) Oral testosterone administration detected by testosterone glucuronidation measured in blood spots dried on filter paper. Clin Chem 46:515- 522.

53 Perera MA, Innocenti F, Ratain MJ. (2008) Pharmacogenetic testing for uridine diphosphate glucuronosyltransferase 1A1 polymorphisms: Are we there yet? Pharmacotherapy 28:755- 768. Petit FM, Hebert M, Gajdos V, Capel L, M'Rad R, Labrune P. (2008) Large deletion in UGT1A1 gene encompassing the promoter and the exon 1 responsible for Crigler-Najjar type I syndrome. Haematologica 93:1590-1591. Pozo OJ, Van Eenoo P, Van Thuyne W, Deventer K, Delbeke FT. (2008) Direct quantification of steroid glucuronides in human urine by liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr A 1183:108-118. Radominska-Pandya A, Little JM, Pandya JT, Tephly TR, King CD, Barone GW, Raufman JP. (1998) UDP-glucuronosyltransferases in human intestinal mucosa. Biochim Biophys Acta 1394:199-208. Radominska-Pandya A, Ouzzine M, Fournel-Gigleux S, Magdalou J. (2005) Structure of UDP- glucuronosyltransferases in membranes. Methods Enzymol 400:116-147. Rao PN, Burdett JE,Jr, Moore PH,Jr, Horton R. (1987) Isolation and identification of androstanediol glucuronide from human plasma. J Steroid Biochem 28:565-569. Reddy BS, Weisburger JH, Wynder EL. (1974) Fecal bacterial beta-glucuronidase: Control by diet. Science 183:416-417. Rowland A, Gaganis P, Elliot DJ, Mackenzie PI, Knights KM, Miners JO. (2007) Binding of inhibitory fatty acids is responsible for the enhancement of UDP-glucuronosyltransferase 2B7 activity by albumin: Implications for in vitro-in vivo extrapolation. J Pharmacol Exp Ther 321:137-147. Rowland A, Knights KM, Mackenzie PI, Miners JO. (2008) The "albumin effect" and drug glucuronidation: Bovine serum albumin and fatty acid-free human serum albumin enhance the glucuronidation of UDP-glucuronosyltransferase (UGT) 1A9 substrates but not UGT1A1 and UGT1A6 activities. Drug Metab Dispos 36:1056-1062. Schänzer W. (1996) Metabolism of anabolic androgenic steroids. Clin Chem 42:1001-1020. Schänzer W, Donike M. (1993) Metabolism of anabolic steroids in man: Synthesis and use of reference substances for identification of anabolic steroid metabolites. Anal Chim Acta 275:23- 48. Schulze JJ, Lorentzon M, Ohlsson C, Lundmark J, Roh HK, Rane A, Ekström L. (2008a) Genetic aspects of epitestosterone formation and androgen disposition: Influence of polymorphisms in CYP17 and UGT2B enzymes. Pharmacogenet Genomics 18:477-485. Schulze JJ, Lundmark J, Garle M, Ekström L, Sottas PE, Rane A. (2008b) Substantial advantage of a combined bayesian and genotyping approach in testosterone doping tests. Steroids 74:365- 368. Schulze JJ, Lundmark J, Garle M, Skilving I, Ekström L, Rane A. (2008c) Doping test results dependent on genotype of UGT2B17, the major enzyme for testosterone glucuronidation. J Clin Endocrinol Metab 93:2500-2506. Shackleton C. (2009) Steroid analysis and doping control 1960-1980: Scientific developments and personal anecdotes. Steroids 74:288-295. Shipkova M, Armstrong VW, Oellerich M, Wieland E. (2003) Acyl glucuronide drug metabolites: Toxicological and analytical implications. Ther Drug Monit 25:1-16. Silber B, Holford NH, Riegelman S. (1982) Stereoselective disposition and glucuronidation of propranolol in humans. J Pharm Sci 71:699-704. 54 Skar V, Skar AG, Stromme JH. (1988) Beta-glucuronidase activity related to bacterial growth in common bile duct bile in gallstone patients. Scand J Gastroenterol 23:83-90. Skopp G, Potsch L. (2004) An investigation of the stability of free and glucuronidated 11-nor- delta9-tetrahydrocannabinol-9-carboxylic acid in authentic urine samples. J Anal Toxicol 28:35-40. Sneitz N, Court MH, Zhang X, Laajanen K, Yee KK, Dalton P, Ding X, Finel M. (2009) Human UDP-glucuronosyltransferase UGT2A2: CDNA construction, expression, and functional characterization in comparison with UGT2A1 and UGT2A3. Pharmacogenet Genomics 19:923-934. Soars MG, Mattiuz EL, Jackson DA, Kulanthaivel P, Ehlhardt WJ, Wrighton SA. (2002) Biosynthesis of drug glucuronides for use as authentic standards. J Pharmacol Toxicol Methods 47:161-168. Soars MG, Ring BJ, Wrighton SA. (2003) The effect of incubation conditions on the enzyme kinetics of udp-glucuronosyltransferases. Drug Metab Dispos 31:762-767. Somers GI, Lindsay N, Lowdon BM, Jones AE, Freathy C, Ho S, Woodrooffe AJ, Bayliss MK, Manchee GR. (2007) A comparison of the expression and metabolizing activities of phase I and II enzymes in freshly isolated human lung parenchymal cells and cryopreserved human hepatocytes. Drug Metab Dispos 35:1797-1805. Sottas PE, Saudan C, Schweizer C, Baume N, Mangin P, Saugy M. (2008) From population- to subject-based limits of T/E ratio to detect testosterone abuse in elite sports. Forensic Sci Int 174:166-172. Span PN, Voller MC, Smals AG, Sweep FG, Schalken JA, Feneley MR, Kirby RS. (1999) Selectivity of finasteride as an in vivo inhibitor of 5alpha-reductase isozyme enzymatic activity in the human prostate. J Urol 161:332-337. Starlard-Davenport A, Lyn-Cook B, Radominska-Pandya A. (2008) Identification of UDP- glucuronosyltransferase 1A10 in non-malignant and malignant human breast tissues. Steroids 73:611-620. Stevenson DE, Hubl U. (1999) Optimization of beta-D-glucuronide synthesis using UDP- glucuronyl transferase. Enzyme Microb Technol 24:388-396. Strahm E, Sottas PE, Schweizer C, Saugy M, Dvorak J, Saudan C. (2009) Steroid profiles of professional soccer players: An international comparative study. Br J Sports Med 43:1126- 1130. Strassburg CP. (2008) Pharmacogenetics of Gilbert's syndrome. Pharmacogenomics 9:703-715. Swanson C, Lorentzon M, Vandenput L, Labrie F, Rane A, Jakobsson J, Chouinard S, Belanger A, Ohlsson C. (2007a) Sex steroid levels and cortical bone size in young men are associated with a uridine diphosphate glucuronosyltransferase 2B7 polymorphism (H268Y). J Clin Endocrinol Metab 92:3697-3704. Swanson C, Mellstrom D, Lorentzon M, Vandenput L, Jakobsson J, Rane A, Karlsson M, Ljunggren O, Smith U, Eriksson AL, Belanger A, Labrie F, Ohlsson C. (2007b) The uridine diphosphate glucuronosyltransferase 2B15 D85Y and 2B17 deletion polymorphisms predict the glucuronidation pattern of androgens and fat mass in men. J Clin Endocrinol Metab 92:4878-4882. Tang KS, Lee CM, Teng HC, Huang MJ, Huang CS. (2008) UDP-glucuronosyltransferase 1A7 polymorphisms are associated with liver cirrhosis. Biochem Biophys Res Commun 366:643- 648.

55 Tephly TR, Burchell B. (1990) UDP-glucuronosyltransferases: A family of detoxifying enzymes. Trends Pharmacol Sci 11:276-279. Thomas SS, Li SS, Lampe JW, Potter JD, Bigler J. (2006) Genetic variability, haplotypes, and htSNPs for exons 1 at the human UGT1A locus. Hum Mutat 27:717. Thompson JA, Hull JE, Norris KJ. (1981) Glucuronidation of propranolol and 4'- hydroxypropranolol. substrate specificity and stereoselectivity of rat liver microsomal glucuronyltransferases. Drug Metab Dispos 9:466-471. Tukey RH, Strassburg CP. (2000) Human UDP-glucuronosyltransferases: Metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581-616. Tukey RH, Strassburg CP. (2001) Genetic multiplicity of the human UDP- glucuronosyltransferases and regulation in the gastrointestinal tract. Mol Pharmacol 59:405- 414. Turgeon D, Carrier JS, Levesque E, Hum DW, Belanger A. (2001) Relative enzymatic activity, protein stability, and tissue distribution of human steroid-metabolizing UGT2B subfamily members. Endocrinology 142:778-787. Uchaipichat V, Mackenzie PI, Guo XH, Gardner-Stephen D, Galetin A, Houston JB, Miners JO. (2004) Human udp-glucuronosyltransferases: Isoform selectivity and kinetics of 4- methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid. Drug Metab Dispos 32:413-423. Vietri M, De Santi C, Pietrabissa A, Mosca F, Pacifici GM. (2000) Inhibition of human liver phenol sulfotransferase by nonsteroidal anti-inflammatory drugs. Eur J Clin Pharmacol 56:81- 87. Villeneuve L, Girard H, Fortier LC, Gagne JF, Guillemette C. (2003) Novel functional polymorphisms in the UGT1A7 and UGT1A9 glucuronidating enzymes in caucasian and african-american subjects and their impact on the metabolism of 7-ethyl-10- hydroxycamptothecin and flavopiridol anticancer drugs. J Pharmacol Exp Ther 307:117-128. Walle T, Walle UK, Olanoff LS. (1985) Quantitative account of propranolol metabolism in urine of normal man. Drug Metab Dispos 13:204-209. Wells PG, Mackenzie PI, Chowdhury JR, Guillemette C, Gregory PA, Ishii Y, Hansen AJ, Kessler FK, Kim PM, Chowdhury NR, Ritter JK. (2004) Glucuronidation and the UDP- glucuronosyltransferases in health and disease. Drug Metab Dispos 32:281-290. Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR, Ball SE. (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates: A pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos 32:1201-1208. Wilson W,3rd, Pardo-Manuel de Villena F, Lyn-Cook BD, Chatterjee PK, Bell TA, Detwiler DA, Gilmore RC, Valladeras IC, Wright CC, Threadgill DW, Grant DJ. (2004) Characterization of a common deletion polymorphism of the UGT2B17 gene linked to UGT2B15. Genomics 84:707-714. Wsol V, Skalova L, Szotakova B. (2004) Chiral inversion of drugs: Coincidence or principle? Curr Drug Metab 5:517-533. Xue Y, Sun D, Daly A, Yang F, Zhou X, Zhao M, Huang N, Zerjal T, Lee C, Carter NP, Hurles ME, Tyler-Smith C. (2008) Adaptive evolution of UGT2B17 copy-number variation. Am J Hum Genet 83:337-346.

56 Yang TL, Chen XD, Guo Y, Lei SF, Wang JT, Zhou Q, Pan F, Chen Y, Zhang ZX, Dong SS, Xu XH, Yan H, Liu X, Qiu C, Zhu XZ, Chen T, Li M, Zhang H, Zhang L, Drees BM, Hamilton JJ, Papasian CJ, Recker RR, Song XP, Cheng J, Deng HW. (2008) Genome-wide copy- number-variation study identified a susceptibility gene, UGT2B17, for osteoporosis. Am J Hum Genet 83:663-674. Yin H, Bennett G, Jones JP. (1994) Mechanistic studies of uridine diphosphate glucuronosyltransferase (abstract). Chem Biol Interact 90:47-58. Yost GS, Finley BL. (1985) Stereoselective glucuronidation as a probe of induced forms of UDP- glucuronyltransferase in rabbits. Drug Metab Dispos 13:5-8. Yu L, Qian M, Liu Y, Yao T, Zeng S. (2009) Stereoselective metabolism of propranolol glucuronidation by human UDP-glucuronosyltransferases 2B7 and 1A9. Chirality. In Press. Zipper J, Wheeler RG, Potts DM, Rivera M. (1983) Propranolol as a novel, effective spermicide: Preliminary findings. Br Med J (Clin Res Ed) 287:1245-1246.

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