0090-9556/06/3407-1102–1108$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 7 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 9621/3119192 DMD 34:1102–1108, 2006 Printed in U.S.A.

HUMAN UDP-, UGT1A8, GLUCURONIDATES DIHYDROTESTOSTERONE TO A MONOGLUCURONIDE AND FURTHER TO A STRUCTURALLY NOVEL DIGLUCURONIDE

Takahiro Murai, Naozumi Samata, Haruo Iwabuchi, and Toshihiko Ikeda

Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan

Received February 3, 2006; accepted March 29, 2006

ABSTRACT: We identified human UDP-glucuronosyltransferase (UGT) isoforms trointestinal UGT, and to a lesser extent with UGT1A1 and UGT1A9. responsible for producing dihydrotestosterone (DHT) diglucu- In contrast, the activity of DHT monoglucuronidation was high and ronide, a novel glucuronide in which the second glucuronosyl moi- was found in UGT2B17, UGT2B15, UGT1A8, and UGT1A4 in de- Downloaded from ety is attached at the C2؅ position of the first glucuronosyl moiety, scending order. Among the 12 UGT isoforms tested, only UGT1A8 leading to diglucuronosyl conjugation of a single hydroxyl group of was capable of producing DHT diglucuronide from DHT. The ki- DHT at the C17 position. Incubation of the DHT monoglucuronide netics of DHT diglucuronidation by microsomes from human liver with 12 cDNA-expressed recombinant human UGT isoforms and and intestine fitted the Michaelis-Menten model, and the Vmax/Km uridine 5؅-diphosphoglucuronic acid resulted in a low but measur- value for the intestinal microsomes was approximately 4 times

able DHT diglucuronidation activity primarily with UGT1A8, a gas- greater than that for the liver microsomes. dmd.aspetjournals.org

Glucuronidation is one of the most important phase II metabolic [a]pyrene (Bevan and Sadler, 1992; Bock et al., 1992), chrysene reactions not only for xenobiotics but also for endogenous substances (Bock et al., 1992), diosmetin (Boutin et al., 1993), phenolphthalein such as steroids and bile acids (Burchell and Coughtrie, 1989; (Griffin et al., 1998), and octylgallate (Soars et al., 2002). Conversely,

Radominska-Pandya et al., 1999). This biotransformation is catalyzed we define “diglucuronide” as a particular glucuronide in which a at ASPET Journals on March 10, 2015 by a superfamily of enzymes, named UDP-glucuronosyltransferase single functional group on the molecule is repeatedly conjugated by (UGT), which is located in the inner membrane of endoplasmic two glucuronosyl groups in tandem (Murai et al., 2005). reticulum. UGTs are classified into either of two families, UGT1 or The first diglucuronide reported was nalmefene diglucuronide, and UGT2, based on the evolutionary divergence (Mackenzie et al., 1997). this metabolite of the narcotic antagonist was detected in dog urine To date, nucleotide sequences encoding 18 human UGT have (Dixon et al., 1989; Murthy et al., 1996). After the discovery of been identified (Miners et al., 2004). nalmefene diglucuronide in vivo, we were the first to report the in UGT catalyzes the transfer of the glucuronosyl moiety from the vitro production of the diglucuronide of 4-hydroxybiphenyl, a non- cofactor uridine 5Ј-diphosphoglucuronic acid (UDPGA) to a suitable planar phenolic compound used commonly as a model substrate for functional group, such as an amino, carboxyl, hydroxyl, or sulfhydryl glucuronidation, using dog liver microsomes (Murai et al., 2002). The group, as well as an acidic carbon on a lipophilic substrate (Miners et diglucuronidation reaction was found to proceed in a stepwise man- al., 2004). As a result of a hydrophilic glucuronosyl moiety being ner, from 4-hydroxybiphenyl to 4-hydroxybiphenyl monoglucuronide introduced into a drug molecule, glucuronides are generally less toxic, and from the monoglucuronide to the diglucuronide. Rat, monkey, and polar compounds and are readily excreted into the bile and/or urine. human liver microsomes showed undetectable 4-hydroxybiphenyl di- However, in some instances, when glucuronides are not hydrophilic glucuronidation activities (Murai et al., 2002). Although the formation enough, they can undergo subsequent glucuronidation to form a of this type of diglucuronide had been found solely for xenobiotics, bisglucuronide or diglucuronide. In this paper, we define “bisglucu- we recently found that physiologically important endogenous sex ronide” as a glucuronide in which two separate functional groups on steroids, namely, androsterone, dihydrotestosterone (DHT), estradiol, the molecule are conjugated at the same time. Bisglucuronides are estriol, estrone, and testosterone, also undergo diglucuronidation in relatively common phase II metabolites of both endogenous and vitro (Murai et al., 2005). For the steroid diglucuronidation, dog liver exogenous substances. Examples include the bisglucuronides of bili- microsomes again had the highest activity, although it was shown for rubin (Gordon et al., 1976), morphine (Yeh et al., 1977), benzo- the first time that monkey and human liver microsomes also have a low but still detectable activity. Of the six steroid substrates tested, Article, publication date, and citation information can be found at only DHT was converted to the diglucuronide at a detectable level by http://dmd.aspetjournals.org. human liver microsomes (Murai et al., 2005). doi:10.1124/dmd.106.009621. To date, human UGT isoforms that are capable of producing the

ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; DHT, dihydrotestosterone; UDPGA, uridine 5Ј-diphosphoglucuronic acid; MS, mass spectrometry; MS/MS, tandem mass spectrometry; HPLC, high-performance liquid chromatography; LC/MS/MS, liquid chromatography-tandem mass spectrometry; ESI, electrospray ionization; COSY, correlated spectroscopy; HSQC, heteronuclear single quantum correlation spectroscopy;

HMBC, heteronuclear multiple bond correlation spectroscopy; DMSO, dimethyl sulfoxide; DMSO-d6, deuterated dimethyl sulfoxide. 1102 DIGLUCURONIDATION OF ONE DIHYDROTESTOSTERONE HYDROXYL GROUP 1103 diglucuronide have not been identified. Therefore, the objectives of the same procedure as above. The purity of DHT monoglucuronide, obtained the present study were 1) to identify the human UGT(s) responsible as a white amorphous powder (approximately 8 mg), was more than 95% by for producing the DHT diglucuronide from the DHT monoglucu- UV absorption at 220 and 280 nm. This authentic standard of DHT monoglu- ronide and DHT monoglucuronide from DHT using a panel of 12 curonide was used as the substrate for the UGT assays of glucuronidation of the monoglucuronide, i.e., diglucuronidation, as well as the standard substance different recombinant human isoforms and 2) to determine enzyme for generating the standard curve for the quantification of DHT monoglucu- kinetic parameters for DHT diglucuronidation for microsomes from ronide by LC/MS/MS. human liver and intestine as well as recombinant human UGT. For In Vitro Biosynthesis of DHT Diglucuronide. DHT diglucuronide was these objectives, authentic standards of DHT monoglucuronide and biosynthesized using dog liver microsomes, since dog liver microsomes had diglucuronide were biosynthesized using rat and dog liver micro- the highest activity for producing DHT diglucuronide from DHT among the somes, respectively. rat, dog, monkey, and human liver microsomes (Murai et al., 2005). The incubation mixture for preparing DHT diglucuronide was the same as that for Materials and Methods preparing DHT monoglucuronide, except that dog liver microsomes (1 mg Materials. UDPGA, DHT, estrone 3-O-glucuronide, D-saccharic acid 1,4- /ml) were used instead of the rat liver microsomes and that the total lactone, and alamethicin were purchased from Sigma-Aldrich Co. (St. Louis, incubation volume was 100 ml. After 24 h of incubation, the reaction was MO). D-Saccharic acid 1,4-lactone was used as a ␤-glucuronidase inhibitor. terminated by adding 5 ml of formic acid to the incubation mixture, and the Alamethicin was used as a pore-forming peptide to enhance the access of acidified mixture was chilled on ice (15 min) before centrifugation. The substrates to the UGTs present in the inner membrane of the microsomal supernatant was subjected to a solid phase extraction using two Mega Bond vesicles (Fisher et al., 2000). Deuterated dimethyl sulfoxide (DMSO-d6, 99.9% Elut C18 columns, which had been preconditioned in the same manner as D) was obtained from Isotec Inc. (Miamisburg, OH). Other reagents and described in the preceding section. Each column was eluted with 50 ml of Downloaded from solvents used were either of analytical or of HPLC grade. Microsomes pre- solvent B, and the eluates obtained were combined, lyophilized, and reconsti- pared from baculovirus-infected insect cells expressing human UGT1A1, tuted in a mixture of solvent A and solvent B (80:20) for the purification and UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, isolation of DHT diglucuronide using the same HPLC system used for the UGT2B4, UGT2B7, UGT2B15, and UGT2B17 (Supersomes), as well as the isolation of DHT monoglucuronide. The gradient program for HPLC, starting control microsomal preparation, were obtained from BD Gentest (Woburn, with a mixture of solvent A and solvent B (80:20) and increasing solvent B MA). Pooled human intestinal microsomes prepared from the duodenum and from 20% to 44% linearly over a period of 6 min, was used for isolating DHT dmd.aspetjournals.org jejunum sections of five donors, as well as pooled human liver microsomes diglucuronide (eluted at 3.8 min). After lyophilization of the fraction contain- prepared from 23 donors, were obtained from BD Gentest. Pooled rat liver ing DHT diglucuronide, DHT diglucuronide (approximately 2 mg) was ob- microsomes were also obtained from BD Gentest. Dog liver microsomes used tained as a white amorphous powder. For obtaining authentic standard to were prepared as described previously (Murai et al., 2002). All microsomes generate the standard curve for the quantification of DHT diglucuronide by were stored frozen at approximately Ϫ80°C until use. LC/MS/MS in UGT assays, a portion of the DHT diglucuronide was again In Vitro Biosynthesis of DHT Monoglucuronide. DHT monoglucuronide, reconstituted in the initial mobile phase for HPLC, and the solution was which is not commercially available, was biosynthesized using rat liver mi- subjected to purification and isolation. The authentic standard of DHT diglu- crosomes. For this purpose, we did not use the dog liver microsomes to avoid curonide for quantification was obtained as a white amorphous powder (ap- at ASPET Journals on March 10, 2015 risk of contamination of DHT monoglucuronide with DHT diglucuronide. The proximately 0.6 mg) and its purity was more than 95% by UV absorption at incubation mixture contained Tris-HCl buffer (100 mM, pH 7.5), magnesium 220 and 280 nm. chloride (10 mM), D-saccharic acid 1,4-lactone (5 mM), DHT (2 mM), rat liver Structure Determination of DHT Glucuronides. The DHT monoglucu- microsomes (0.5 mg protein/ml), and UDPGA (5 mM). DHT was added to the ronide and DHT diglucuronide isolated as above were each dissolved in a incubation mixture as a solution in dimethyl sulfoxide (DMSO; final, 1% mixture of acetonitrile and water (50:50) and analyzed by a Waters Q-Tof DMSO). Before starting the incubation, microsomes were treated with ala- Ultima mass spectrometer (Waters, Milford, MA) operated in negative ion methicin (50 ␮g/mg protein) on ice for 15 min. The glucuronidation reaction electrospray ionization (ESI) mode. A capillary voltage of Ϫ2800 V and a cone was started by the addition of UDPGA to make a total incubation volume of voltage of Ϫ80 V were used. For the ESI/MS/MS analysis, argon gas was used 60 ml. After incubation for 24 h at 37°C, the reaction was terminated by adding as the collision gas and collision energy was set at 25 eV. 3 ml of formic acid, and the mixture was placed on ice for 15 min. After DHT monoglucuronide and DHT diglucuronide, each dissolved in DMSO- 1 centrifugation of the acidified mixture, the supernatant was collected and d6, were subjected to NMR analysis. One-dimensional H NMR, correlated applied to a solid phase extraction column (Varian Mega Bond Elut C18, 10 g; spectroscopy (COSY), heteronuclear single quantum correlation spectroscopy Varian, Inc., Palo Alto, CA), which had been washed successively with 50 ml (HSQC), and heteronuclear multiple bond correlation spectroscopy (HMBC) of acetonitrile containing 0.1% formic acid (solvent B) and 10 ml of distilled spectra were obtained using a Unity Inova 500 NMR spectrometer (Varian water containing 0.1% acetic acid (solvent A) for preconditioning. After Inc.), operating at 500 MHz for 1H and 125 MHz for 13C, equipped with a application of the sample, the column was washed with 50 ml of solvent A, Varian nano probe. All measurements were carried out at 25°C. Chemical followed by elution with 50 ml of solvent B. The column eluate was lyophi- shifts were referenced to the signals of the residual DMSO in DMSO-d6 at 2.50 lized, and the residue was reconstituted in a mixture of solvent A and solvent ppm for 1H and 39.5 ppm for 13C. B (70:30) for purification and isolation of DHT monoglucuronide using HPLC. Assay of Glucuronidation of DHT and DHT Monoglucuronide Using

An LC-10AVP system (Shimadzu Corporation, Kyoto, Japan), consisting of Recombinant Human UGT Isoforms. Activities of DHT monoglucuronida- two LC-10ADVP pumps, a DGU-12A on-line degasser, a CTO-10AVP column tion (glucuronidation of DHT to DHT monoglucuronide) and DHT diglucu- oven, an SPD-10AVP UV/Vis detector, and an SCL-10AVP system controller, ronidation (glucuronidation of DHT monoglucuronide to DHT diglucuronide) was used for the isolation. The reconstituted solution as aliquots (200–250 ␮l were measured using 12 cDNA-expressed recombinant human UGT isoforms each) was repeatedly injected into the HPLC system. Separations were carried (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, ϫ out on an Alltech (Alltech Associates, Deerfield, IL) Alltima C18 column (7 UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17). Microsomes 33 mm, 3 ␮m in particle size) maintained at 30°C in the column oven. The from insect cells without cDNA were used as the negative control. The initial mobile phase used was a mixture of solvent A and solvent B (70:30) and incubation mixture, prepared in triplicate for each UGT isoform, contained was delivered to the HPLC system at a flow rate of 2.5 ml/min. After sample Tris-HCl buffer (100 mM, pH 7.5), magnesium chloride (10 mM), D-saccharic injection, the proportion of solvent B in the mobile phase was increased acid 1,4-lactone (5 mM), substrate (20 ␮M DHT for monoglucuronidation linearly from 30% to 35% over a period of 5 min. The fraction containing DHT assay; 500 ␮M DHT monoglucuronide for diglucuronidation assay), recom- monoglucuronide, eluted at 3.1 min in the UV chromatogram (280 nm), was binant UGTs (0.1 mg protein/ml for monoglucuronidation assay; 1 mg pro- manually collected into a chilled glass container. After lyophilization of the tein/ml for diglucuronidation assay), and UDPGA (5 mM), in a final volume fraction, crude DHT monoglucuronide obtained was again reconstituted in the of 100 ␮l. The substrate, DHT or DHT monoglucuronide, was added as a initial mobile phase, and the solution was subjected to purification following solution in DMSO (final: 1% DMSO). Before starting the incubation, the 1104 MURAI ET AL.

microsomes were treated with alamethicin (50 ␮g/mg protein) on ice for 15 TABLE 1 min. According to preliminary experiments, monoglucuronide and diglucu- 1H and 13C NMR chemical shifts of dihydrotestosterone monoglucuronide ronide formation was linear up to 30 min and 360 min, respectively. The The experiments were conducted at 25°C using DMSO-d as a solvent. reaction was started by the addition of UDPGA, allowed to proceed for 30 min 6 (monoglucuronidation assay) or 240 min (diglucuronidation assay) at 37°C on Carbon No. 1H 13C a shaking incubator, and terminated by the addition of 100 ␮l of acetonitrile. 1 1.25, 1.92 37.7 After addition of 10 ␮l of the internal standard solution (5000 ng/ml estrone 2 2.08, 2.41 37.4 glucuronide in water), the samples were placed on ice. After centrifugation, the 3 210.4 supernatants were transferred to other tubes and concentrated using a centri- 4 1.88, 2.29 43.9 fugal evaporator. The residue was reconstituted in 1000 ␮l (monoglucuronida- 5 1.44 45.8 ␮ 6 1.26 28.0 tion assay) or 60 l (diglucuronidation assay) of 10 mM ammonium formate 7 0.84, 1.61 30.6 in a mixture of water and methanol (50:50), and the supernatant (20 ␮l) 8 1.37 34.5 obtained by centrifugation was subjected to LC/MS/MS quantification of the 9 0.70 52.9 DHT monoglucuronide or DHT diglucuronide. 10 35.2 Furthermore, to investigate whether each single UGT isoform could pro- 11 1.31, 1.51 20.3 12 1.10, 1.89 36.6 duce DHT diglucuronide from DHT, we incubated DHT (100 ␮M) with 13 42.6 recombinant human UGT isoform (1 mg protein/ml) in the presence of 14 0.92 49.9 UDPGA for 24 h, and the DHT diglucuronide produced was determined 15 1.16, 1.47 22.6 by LC/MS/MS. 16 1.47, 1.85 28.3

Quantification of DHT Glucuronides by LC/MS/MS. For the quantifi- 17 3.56 87.5 Downloaded from cation of DHT monoglucuronide and DHT diglucuronide by LC/MS/MS, a 18 0.75 11.2 19 0.96 10.8 Waters Alliance 2795 separations module was used as the HPLC system. 1Ј 4.23 103.3 Chromatographic separation was carried out using an Inertsil ODS-3 column 2Ј 2.95 73.2 (2.1 ϫ 75 mm, 5 ␮m in particle size; GL Sciences, Inc., Tokyo Japan), which 3Ј 3.14 76.0 was maintained at 35°C. Two mobile phases, 10 mM ammonium formate in a 4Ј 3.28 71.3 mixture of water and methanol (50:50; solvent 1) and 10 mM ammonium 5Ј 3.53 75.4

Ј dmd.aspetjournals.org formate in a mixture of water and methanol (10:90; solvent 2), were used at a 6 170.4 flow rate of 200 ␮l/min. A linear gradient elution program, starting with solvent 1 as the initial mobile phase, increasing solvent 2 linearly from 0% to 100% over a period of 3 min, and holding 100% solvent 2 for 9 min, was used. Chemical Structure of DHT Diglucuronide. The negative ion The detection was performed by a Micromass Quattro II triple quadrupole ESI/MS of DHT diglucuronide, biosynthesized using DHT and dog Ϫ mass spectrometer (Micromass, Manchester, UK) with an ESI interface oper- liver microsomes, exhibited deprotonated molecule [M Ϫ H] at m/z ated in the negative ion mode. The quantification was accomplished in the 641, demonstrating that the molecular weight is greater than that of multiple reaction monitoring mode by monitoring a transition pair of m/z DHT by a mass of two glucuronosyl moieties. In the ESI/MS/MS 3 3 at ASPET Journals on March 10, 2015 465 85 for DHT monoglucuronide, m/z 641 351 for DHT diglucuronide, spectrum of DHT diglucuronide, a characteristic fragment ion was 3 and m/z 445 175 for estrone glucuronide (internal standard). Argon was used detected at m/z 351, demonstrating that the two glucuronosyl moieties as the collision gas. The MS operating conditions were optimized as follows: 1 capillary voltage, Ϫ2500 V; source temperature, 100°C; and desolvation gas introduced were connected in tandem (Fig. 1). In the H NMR temperature, 400°C. Cone voltage and collision energy were set at Ϫ55 V and spectrum, two anomeric protons were observed at 4.37 and 4.43 ppm. 40 eV for DHT monoglucuronide, Ϫ55 V and 25 eV for DHT diglucuronide, Detailed analysis of two-dimensional COSY, HSQC, and HMBC and Ϫ40 V and 16 eV for estrone glucuronide, respectively. spectral data enabled unambiguous assignments of all protons and Enzyme Kinetics and Data Analysis. Pooled human liver microsomes (1 carbons in DHT diglucuronide (Table 2). The regiochemistry of the mg protein/ml), pooled human intestinal microsomes (1 mg protein/ml), and two glucuronosyl moieties was elucidated by HMBC correlations human UGT1A8 Supersomes (1 mg protein/ml) were incubated with various (Fig. 2). The first glucuronosyl moiety was attached to the C17 concentrations of DHT monoglucuronide (50–2000 ␮M) in the presence of position of DHT, as confirmed by the key HMBC correlation peak UDPGA. According to preliminary experiments, DHT diglucuronide forma- Ј tion was linear up to 360 min. The incubation was performed in triplicate for from the H1 proton (4.37 ppm) to the C17 carbon (88.9 ppm). The second glucuronidation occurred at the C2Ј position of the first 240 min at 37°C. The apparent enzyme kinetic parameters, Km (Michaelis

constant), Vmax (maximum velocity), and Ksi (substrate inhibition constant), glucuronosyl moiety, which was determined by two key HMBC were estimated by nonlinear regression analysis using WinNonlin Professional correlation peaks from the H1Љ proton (4.43 ppm) to the C2Ј carbon (Ver. 3.1; Pharsight Corporation, Mountain View, CA). The plots of the (82.3 ppm) and from the H2Ј proton (3.25 ppm) to the C1Љ carbon velocities (V) versus substrate concentrations ([S]) were fitted either to the (104.5 ppm). Michaelis-Menten equation, V ϭ V ϫ [S]/(K ϩ [S]), or to the substrate max m DHT Monoglucuronidation Activities in Recombinant Human inhibition equation, V ϭ V /(1 ϩ (K /[S]) ϩ ([S]/K )). max m si UGT Isoforms. Activities of DHT monoglucuronidation (glucu- Results ronidation of DHT to DHT monoglucuronide) in 12 recombinant ␮ Chemical Structure of DHT Monoglucuronide. The negative ion human UGT isoforms were examined using DHT (20 M) as a ESI/MS of DHT monoglucuronide, biosynthesized using DHT and rat substrate. Under the condition used, decrease of DHT monoglucu- liver microsomes, exhibited deprotonated molecule [M Ϫ H]Ϫ at m/z ronide by further glucuronidation was small enough to be negligible. 465, demonstrating that the molecular weight is greater than that of As shown in Fig. 3A, UGT2B17 exhibited the highest DHT mono- DHT by a mass of one glucuronosyl moiety. In the 1H NMR spec- glucuronidation activity (1626 Ϯ 51 pmol/min/mg protein). trum, one anomeric proton was observed at 4.23 ppm. Complete UGT2B15 (388 Ϯ 10 pmol/min/mg protein), UGT1A8 (127 Ϯ 2 proton and carbon chemical shift assignments of DHT monoglucu- pmol/min/mg protein), and UGT1A4 (21 Ϯ 4 pmol/min/mg protein) ronide (Table 1) were accomplished by the analysis of the two- also exhibited the monoglucuronidation activities, in descending or- dimensional NMR data (COSY, HSQC, and HMBC). All spectral data der. All other isoforms (UGT1A1, UGT1A3, UGT1A6, UGT1A7, obtained were consistent with the chemical structure of DHT mono- UGT1A9, UGT1A10, UGT2B4, and UGT2B7) had very low (Ͻ10 glucuronide. pmol/min/mg protein) monoglucuronidation activities. DIGLUCURONIDATION OF ONE DIHYDROTESTOSTERONE HYDROXYL GROUP 1105

TABLE 2 1H and 13C NMR chemical shifts of dihydrotestosterone diglucuronide

The experiments were conducted at 25°C using DMSO-d6 as a solvent. Carbon No. 1H 13C 1 1.25, 1.94 37.6 2 2.10, 2.41 37.3 3 210.2 4 1.88, 2.28 43.9 5 1.44 45.8 6 1.26 28.0 7 0.83, 1.60 30.6 8 1.34 34.4 9 0.67 53.0 10 35.0 11 1.29, 1.46 20.1 12 0.95, 1.89 35.9 13 42.3 14 0.87 49.7 15 1.15, 1.46 22.7 16 1.48, 1.84 28.5

17 3.45 88.9 Downloaded from 18 0.68 10.9 19 0.97 10.7 1Ј 4.37 102.0 2Ј 3.25 82.3 3Ј 3.40 75.2 4Ј 3.35 71.0 5Ј 3.58 74.8

6Ј 169.7 dmd.aspetjournals.org 1Љ 4.43 104.5 2Љ 3.04 74.8 3Љ 3.20 75.1 FIG. 1. ESI/MS/MS spectrum and fragmentation scheme of DHT diglucuronide. 4Љ 3.29 71.4 The regiochemistry of the two glucuronosyl moieties was elucidated by NMR 5Љ 3.57 76.0 analysis. 6Љ 169.9

DHT Diglucuronidation Activities in Recombinant Human UGT Isoforms. Activities of DHT diglucuronidation (glucuronida- at ASPET Journals on March 10, 2015 tion of DHT monoglucuronide to DHT diglucuronide) in 12 recom- binant human UGT isoforms were examined using the biosynthesized DHT monoglucuronide (500 ␮M) as a substrate. Among the UGT isoforms tested, primarily UGT1A8 (0.047 Ϯ 0.004 pmol/min/mg protein), and secondarily UGT1A1 (0.014 Ϯ 0.001 pmol/min/mg protein) and UGT1A9 (0.012 Ϯ 0.003 pmol/min/mg protein) showed low but measurable activities for DHT diglucuronidation (Fig. 3B). All other isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17) showed undetectable diglucuronidation activities under the conditions used. UGT Isoforms Capable of Producing DHT Diglucuronide from DHT. The capability of each UGT isoform to produce DHT diglucu- ronide from DHT (glucuronidation of DHT to DHT monoglucuronide, and further to DHT diglucuronide) was investigated using 12 recom- FIG. 2. Chemical structure of DHT diglucuronide. Key correlations in the HMBC binant human UGT isoforms. The incubation of DHT (100 ␮M) with spectrum are shown by arrows. 12 recombinant human UGTs in the presence of UDPGA for 24 h resulted in the production of DHT diglucuronide (5.56 Ϯ 0.34 ng/ml) binant human UGT1A8. Over the substrate concentration range exclusively in the sample after incubation with UGT1A8 (Fig. 3C), tested, the kinetics of DHT diglucuronidation by human liver micro- consistent with the data showing that only UGT1A8 had the activities somes and human intestinal microsomes fit Michaelis-Menten kinet- of both DHT monoglucuronidation and diglucuronidation (Fig. 3, A ics (Fig. 4, A and B). However, the kinetic data of UGT1A8, which and B). The y-axis unit in Fig. 3C represents DHT diglucuronide showed decreasing velocity with increasing substrate concentration, concentration instead of rate as in Fig. 3, A and B because 1) the result were best described by a substrate inhibition kinetic model (Fig. 4C). demonstrates the capacity of each UGT isoform to form DHT diglu- Enzyme kinetic parameters, obtained by fitting data to the appropriate curonide after long incubation where linearity is not verified, and 2) kinetic model, are summarized in Table 3. The apparent Km values for there are two enzymatic steps involved. human liver and intestinal microsomes were 527.9 Ϯ 66.5 ␮M and Kinetics of DHT Diglucuronidation in Pooled Human Liver 653.4 Ϯ 69.0 ␮M, respectively, being comparable to each other. In

Microsomes, Pooled Human Intestinal Microsomes, and Recom- contrast, the apparent Vmax value for human intestinal microsomes binant Human UGT1A8. Enzyme kinetic studies were performed by (1.303 Ϯ 0.056 pmol/min/mg protein) was approximately 5 times incubating DHT monoglucuronide (50–2000 ␮M) with pooled human larger than that for human liver microsomes (0.256 Ϯ 0.012 pmol/ liver microsomes, pooled human intestinal microsomes, and recom- min/mg protein). The Vmax/Km value for human intestinal microsomes 1106 MURAI ET AL. Downloaded from dmd.aspetjournals.org at ASPET Journals on March 10, 2015

FIG. 3. Glucuronidation of DHT to DHT monoglucuronide (A), DHT monoglucu- ronide to DHT diglucuronide (B), and DHT to DHT diglucuronide (C) by recom- binant human UGT enzymes. Each column represents the mean Ϯ standard devi- FIG. 4. Enzyme kinetics for the formation of DHT diglucuronide from DHT mono- ␮ ation of triplicate incubations. A, DHT (20 M) was incubated with each human glucuronide catalyzed by human liver microsomes (A), human intestinal micro- UGT (0.1 mg protein/ml) and UDPGA (5 mM) for 30 min at 37°C. B, DHT somes (B), and recombinant human UGT1A8 (C). Open circles denote experimental ␮ monoglucuronide (500 M) was incubated with each human UGT (1 mg protein/ observations. The solid line in A and B, and the dotted line in C represent predicted ␮ ml) and UDPGA (5 mM) for 240 min at 37°C. C, DHT (100 M) was incubated values calculated from Michaelis-Menten kinetics. The solid line in C represents with each human UGT (1 mg protein/ml) and UDPGA (5 mM). predicted values calculated from substrate inhibition kinetics.

Ϫ (1.994 ϫ 10 3 ␮l/min/mg protein) was approximately 4 times larger somes, respectively, and their chemical structures were analyzed by Ϫ than that for human liver microsomes (0.486 ϫ 10 3 ␮l/min/mg mass spectrometry and nuclear magnetic resonance spectroscopy. The

protein). In contrast, the Km, Vmax, Vmax/Km, and Ksi values for negative ion MS/MS spectrum of DHT diglucuronide clearly indi- recombinant UGT1A8 were 382.3 Ϯ 79.7 ␮M, 0.107 Ϯ 0.015 pmol/ cated that the two glucuronosyl moieties are linked in tandem because Ϫ min/mg protein, 0.279 ϫ 10 3 ␮l/min/mg protein, and 964.4 Ϯ 231.7 the characteristic fragment ion at m/z 351, which corresponds to a ␮M, respectively. dehydrated diglucuronosyl anion, was observed in the spectrum (Fig. 1). The regiochemistry of the two glucuronosyl moieties in DHT Discussion diglucuronide, which was unable to be elucidated by analyzing the First, in this study, authentic DHT monoglucuronide and DHT MS/MS spectrum, was determined using two-dimensional NMR anal- diglucuronide were biosynthesized using rat and dog liver micro- ysis (Fig. 2). It was determined that the first glucuronosyl moiety in DIGLUCURONIDATION OF ONE DIHYDROTESTOSTERONE HYDROXYL GROUP 1107

TABLE 3 Kinetic parameters for DHT diglucuronidation in human liver microsomes (HLM), human intestinal microsomes (HIM), and recombinant human UGT1A8 DHT monoglucuronide (50–2000 ␮M) was incubated with 1 mg/ml microsomes in the presence of UDPGA at 37°C for 240 min. Kinetic parameters were calculated by fitting triplicate data to the appropriate kinetic model. Each value represents the estimate Ϯ the standard error of the estimate. Numbers in parentheses are kinetic values for reference.

a Enzyme Km Vmax Vmax/Km Ksi ␮M pmol/min/mg protein ␮l/min/mg protein ␮M HLMb 527.9 Ϯ 66.5 0.256 Ϯ 0.012 0.486 ϫ 10Ϫ3 HIMb 653.4 Ϯ 69.0 1.303 Ϯ 0.056 1.994 ϫ 10Ϫ3 UGT1A8c 382.3 Ϯ 79.7 (0.107 Ϯ 0.015) (0.279 ϫ 10Ϫ3) (964.4 Ϯ 231.7)

a Substrate inhibition constant. b Michaelis-Menten model. c Substrate inhibition model.

DHT diglucuronide was attached to the C17 position of DHT and that liver and intestinal microsomes, the kinetics of UGT1A8 was atypical, the second glucuronosyl moiety was attached at the C2Ј position of and was well described by the substrate inhibition model (Fig. 4C). By the first glucuronosyl moiety. Interestingly, the regiochemistry of the comparing the apparent kinetic parameters of UGT1A8 to those of ␤ two glucuronosyl moieties in DHT diglucuronide [ -D-glucuronopy- intestinal microsomes, the Km value was within a 2-fold range, but Љ3 Ј ␤ ranosyl-(1 2 )- -D-glucuronopyranosyl] is common to all the pre- there was a 7-fold difference in the intrinsic clearance, calculated as Downloaded from viously characterized diglucuronides of nalmefene (Dixon et al., Vmax/Km (Table 3). The higher diglucuronidation activity in the hu- 1989), 4-hydroxybiphenyl (Murai et al., 2002), and estrone and tes- man intestinal microsomes compared with the human liver micro- tosterone (Murai et al., 2005), as well as some plant saponins includ- somes might be due to the higher expression level of intestinal DHT ing glycyrrhizic acid (Kitagawa et al., 1993; Yoshida et al., 1993). diglucuronidation enzyme, namely, either UGT1A8 or UGT1A1, or These accumulating observations suggest that the 2Ј-hydroxyl group both, in intestinal microsomes. The kinetic data for UGT1A8, in terms of specific monoglucuronides could particularly be recognized as a of the substrate inhibition, differed from those for the intestinal dmd.aspetjournals.org functional group for further glucuronidation by some UGT enzymes microsomes. It is reported that UGT enzymes exist as UGT oligomers in animals and the plant enzymes involved in the biosynthesis of some in the endoplasmic reticulum membrane and that these oligomers may saponins. act as cooperative multisubunit enzymes and thus affect the kinetics of In the present study, we incubated the biosynthesized DHT mono- UGT-catalyzed reactions (Miners et al., 2004). The UGT oligomers glucuronide with 12 recombinant human UGT isoforms in the pres- reported are not only homo-oligomers, consisting of single UGT ence of UDPGA, and demonstrated for the first time that primarily enzymes (Meech and Mackenzie, 1997; Ghosh et al., 2001; Kurkela et UGT1A8, and secondarily UGT1A1 and UGT1A9 have low but al., 2003), but also hetero-oligomers, consisting of different UGT measurable activities of forming structurally novel DHT diglucu- enzymes (Ikushiro et al., 1997; Ishii et al., 2001). It is even reported at ASPET Journals on March 10, 2015 ronide (Fig. 3B). Regarding the monoglucuronidation of DHT by that hetero-oligomer formation of UGT with a cytochrome P450 human UGT isoforms (Fig. 3A), UGT2B17, UGT2B15, UGT1A8, altered kinetic parameters of the UGT reactions (Takeda et al., 2005). and UGT1A4 were found to be active, consistent with the findings of Based on this accumulating evidence, it is presumed that the produc- previous reports (Beaulieu et al., 1996; Levesque et al., 1997; Cheng tion of DHT diglucuronide in human liver and intestinal microsomes et al., 1998; Ehmer et al., 2004). Among the UGTs tested, it was only is mostly catalyzed by UGT hetero-oligomers. The findings of these UGT1A8 that exhibited both DHT monoglucuronidation and DHT previous reports would explain the atypical kinetic behavior of diglucuronidation activities (Fig. 3, A and B). This was corroborated UGT1A8 in DHT diglucuronidation. by the fact that only UGT1A8 produced detectable DHT diglucu- In conclusion, we identified a structurally novel DHT diglucuronide ronide after incubation with DHT for 24 h (Fig. 3C). The UGT and demonstrated that the formation of DHT diglucuronide from DHT isoforms tested except for UGT1A8 produced no DHT diglucuronide monoglucuronide is catalyzed primarily by UGT1A8, a gastrointesti- from DHT, because they lacked either DHT monoglucuronidation or nal UGT, and secondarily by UGT1A1 and UGT1A9. The DHT DHT diglucuronidation activity, or both activities. diglucuronidation was more efficiently catalyzed by human intestinal According to the data obtained using 12 human UGT isoforms, microsomes than by human liver microsomes, suggesting a large DHT diglucuronidation activity was found for 3 UGTs, namely, contribution of UGT1A8 to this reaction. Very low activities for DHT UGT1A1, UGT1A8, and UGT1A9. Because UGT1A1 and UGT1A9 diglucuronidation in UGT isoforms compared with those for DHT are hepatic UGTs, diglucuronidation activity in human liver micro- monoglucuronidation (Fig. 3, A and B) indicate that further investi- somes would be due to one or both of these isoforms. Conversely, gation should be conducted to determine whether or not this novel UGT1A8 has been reported to be an extrahepatic UGT whose mRNA diglucuronidation reaction takes place under in vivo conditions. expression is found in gastrointestinal tissues such as the jejunum, Acknowledgments. We are grateful to Caroline Bertorelli for ileum, and colon, but not in the liver (Cheng et al., 1998; Tukey and editing the manuscript. We appreciate Shin-ichi Inaba for helpful Strassburg, 2001). Therefore, we compared the enzyme kinetics of discussion on kinetic data analysis. DHT diglucuronidation (glucuronidation of DHT monoglucuronide to DHT diglucuronide) using human liver and intestinal microsomes, References together with recombinant UGT1A8. As shown in Fig. 4, A and B, the Beaulieu M, Levesque E, Hum DW, and Belanger A (1996) Isolation and characterization of a kinetics of human liver and intestinal microsomes fitted the Michae- novel cDNA encoding a human UDP-glucuronosyltransferase active on C19 steroids. J Biol lis-Menten model. The affinities of both human liver and intestinal Chem 271:22855–22862. Bevan DR and Sadler VM (1992) Quinol diglucuronides are predominant conjugated metabolites microsomes for DHT monoglucuronide, as assessed by Km, were found in bile of rats following intratracheal instillation of benzo[a]pyrene. Carcinogenesis similar (Table 3). The maximal rate of diglucuronidation, as assessed 13:403–407. Bock KW, Gschaidmeier H, Seidel A, Baird S, and Burchell B (1992) Mono- and diglucuronide by Vmax, was approximately 5 times greater in human intestinal formation from chrysene and benzo[a]pyrene phenols by 3-methylcholanthrene-inducible microsomes than in human liver microsomes. However, unlike human phenol UDP-glucuronosyltransferase (UGT1A1). Mol Pharmacol 42:613–618. 1108 MURAI ET AL.

Boutin JA, Meunier F, Lambert PH, Hennig P, Bertin D, Serkiz B, and Volland JP (1993) In vivo Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Belanger A, Fournel-Gigleux S, and in vitro glucuronidation of the flavonoid diosmetin in rats. Drug Metab Dispos 21:1157– Green M, Hum DW, Iyanagi T, et al. (1997) The UDP superfamily: 1166. recommended nomenclature update based on evolutionary divergence. Pharmacogenetics Burchell B and Coughtrie MWH (1989) UDP-. Pharmacol Ther 43: 7:255–269. 261–289. Meech R and Mackenzie PI (1997) UDP-Glucuronosyltransferase, the role of the amino terminus Cheng Z, Radominska-Pandya A, and Tephly TR (1998) Cloning and expression of human in dimerization. J Biol Chem 272:26913–26917. UDP-glucuronosyltransferase (UGT) 1A8. Arch Biochem Biophys 356:301–305. Miners JO, Smith PA, Sorich MJ, McKinnon RA, and Mackenzie PI (2004) Predicting human Dixon R, Hsiao J, Hsu HB, Smulkowski M, Tze-Ming-Chan, Pramanik B, and Morton J (1989) drug glucuronidation parameters: application of in vitro and in silico modeling approaches. Isolation of a novel morphinan 3-O-diglucuronide metabolite from dog urine. Pharm Res (NY) Annu Rev Pharmacol Toxicol 44:1–25. 6:28–32. Murai T, Iwabuchi H, and Ikeda T (2005) Repeated glucuronidation at one hydroxyl group leads Ehmer U, Vogel A, Schutte JK, Krone B, Manns MP, and Strassburg CP (2004) Variation of to structurally novel diglucuronides of steroid sex hormones. Drug Metab Pharmacokinet hepatic glucuronidation: novel functional polymorphisms of the UDP-glucuronosyltransferase 20:282–293. UGT1A4. Hepatology 39:970–977. Murai T, Tsuruta F, Terao T, Ikeda T, and Iwabuchi H (2002) Formation of a structurally novel, Fisher MB, Campanale K, Ackermann BL, VandenBranden M, and Wrighton SA (2000) In vitro serial diglucuronide of 4-hydroxybiphenyl by further glucuronidation of a monoglucuronide in glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. dog liver microsomes. Drug Metab Pharmacokinet 17:457–466. Drug Metab Dispos 28:560–566. Murthy SS, Mathur C, Kvalo LT, Lessor RA, and Wilhelm JA (1996) Disposition of the opioid Ghosh SS, Sappal BS, Kalpana GV, Lee SW, Chowdhury JR, and Chowdhury NR (2001) antagonist, nalmefene, in rat and dog. Xenobiotica 26:779–792. Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltrans- Radominska-Pandya A, Czernik PJ, Little JM, Battaglia E, and Mackenzie PI (1999) Structural ferase-1 (UGT1A1) and its functional implications. J Biol Chem 276:42108–42115. and functional studies of UDP-glucuronosyltransferases. Drug Metab Rev 31:817–899. Gordon ER, Goresky CA, Chang TH, and Perlin AS (1976) The isolation and characterization of Soars MG, Mattiuz EL, Jackson DA, Kulanthaivel P, Ehlhardt WJ, and Wrighton SA (2002) bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile. Biochem J Biosynthesis of drug glucuronides for use as authentic standards. J Pharmacol Toxicol 155:477–486. Methods 47:161–168. Griffin RJ, Godfrey VB, and Burka LT (1998) Metabolism and disposition of phenolphthalein in Takeda S, Ishii Y, Iwanaga M, Mackenzie PI, Nagata K, Yamazoe Y, Oguri K, and Yamada H male and female F344 rats and B6C3F1 mice. Toxicol Sci 42:73–81. (2005) Modulation of UDP-glucuronosyltransferase function by cytochrome P450: evidence Ikushiro S, Emi Y, and Iyanagi T (1997) Protein-protein interactions between UDP- for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4. Mol

glucuronosyltransferase isozymes in rat hepatic microsomes. Biochemistry 36:7154–7161. Pharmacol 67:665–672. Downloaded from Ishii Y, Miyoshi A, Watanabe R, Tsuruda K, Tsuda M, Yamaguchi-Nagamatsu Y, Yoshisue K, Tukey RH and Strassburg CP (2001) Genetic multiplicity of the human UDP-glucuronosyltrans- Tanaka M, Maji D, Ohgiya S, et al. (2001) Simultaneous expression of guinea pig UDP- ferases and regulation in the gastrointestinal tract. Mol Pharmacol 59:405–414. glucuronosyltransferase 2B21 and 2B22 in COS-7 cells enhances UDP-glucuronosyltrans- Yeh SY, Gorodetzky CW, and Krebs HA (1977) Isolation and identification of morphine 3- and ferase 2B21-catalyzed morphine-6-glucuronide formation. Mol Pharmacol 60:1040–1048. 6-glucuronides, morphine 3,6-diglucuronide, morphine 3-ethereal sulfate, normorphine and Kitagawa I, Hori K, Taniyama T, Zhou JL, and Yoshikawa M (1993) Saponin and sapogenol. normorphine 6-glucuronide as morphine metabolites in humans. J Pharm Sci 66:1288–1293. XLVII. On the constituents of the roots of Glycyrrhiza uralensis Fischer from northeastern Yoshida K, Kameda K, and Kondo T (1993) Diglucuronoflavones from purple leaves of Perilla China. (1). Licorice-saponins A3, B2 and C2. Chem Pharm Bull (Tokyo) 41:43–49. ocimoides. Phytochemistry 33:917–919. Kurkela M, Garcia-Horsman JA, Luukkanen L, Morsky S, Taskinen J, Baumann M, Kostiainen

R, Hirvonen J, and Finel M (2003) Expression and characterization of recombinant human dmd.aspetjournals.org UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than Address correspondence to: Takahiro Murai, Drug Metabolism and Pharma- other UGTs and was purified as an active dimeric enzyme. J Biol Chem 278:3536–3544. Levesque E, Beaulieu M, Green MD, Tephly TR, Belanger A, and Hum DW (1997) Isolation and cokinetics Research Laboratories, Sankyo Co., Ltd., 1-2-58, Hiromachi, Shina- characterization of UGT2B15(Y85): a UDP-glucuronosyltransferase encoded by a polymor- gawa-ku, Tokyo 140-8710, Japan. E-mail: [email protected] phic gene. Pharmacogenetics 7:317–325. at ASPET Journals on March 10, 2015