Abstract
Different pharmacokinetic properties have been observed for the two enantiomers of the entactogen 3,4-methylendioxy-methamphetamine, most probably a result of enantioselective metabolism. The aim of the present work was to study the involvement of human UDP-glucuronyltransferase (UGT) isoforms in the glucuronidation of the enantiomers of its major metabolite 4-hydroxy-3-methoxymethamphetamine (HMMA). First, the reference standards of R- and S-HMMA-O-glucuronide were synthesized semipreparatively using the enzymes of rat liver microsomes, followed by isolation with semipreparative high-performance liquid chromatography and identification using mass spectrometry and NMR. Racemic HMMA was then incubated using heterologously expressed human UGTs and pooled human liver microsomes (HLMs), and the glucuronides were quantified by liquid chromatography-linear ion trap-mass spectrometry. UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were involved in the glucuronidation of HMMA. UGT2B15, UGT2B17, and HLM revealed classic Michaelis-Menten kinetics, whereas UGT1A9 and UGT2B7 showed sigmoidal curves and the respective Eadie-Hofstee plots indicated autoactivation kinetics. UGT2B15 showed the highest affinity and activity. UGT2B15, UGT2B17, and HLMs were not considerably enantioselective but showed slight preferences for S-HMMA. Marked enantioselectivity could only be observed for UGT1A9 with respect to the S-enantiomer and for UGT2B7 with respect to the R-enantiomer. In conclusion, the O-glucuronidation of HMMA in vivo should not be expected to be enantioselective, and the different pharmacokinetic properties may not be caused directly by glucuronidation.
- MDMA, 3,4-methylenedioxymethamphetamine
- DHMA, dihydroxymethamphetamine
- COMT, catechol-O-methyltransferase
- HMMA, 4-hydroxy-3-methoxymethamphetamine
- UGT, UDP-glucuronyltransferase
- SULT, sulfotransferase
- RLM, rat liver microsome
- M6G, morphine-6-β-d-glucuronide
- UDPGA, UDP-glucuronic acid
- HPLC, high-performance liquid chromatography
- ICM, insect cell microsome
- HLM, pooled human liver microsome
- S-HFBPCl, S-heptafluorobutyrylprolyl chloride
- GC/MS, gas chromatography/mass spectrometry
- LC/MS, liquid chromatography/mass spectrometry.
R,S-3,4-Methylenedioxy-methamphetamine [R,S-MDMA, N-methyl-1-(3,4-methylenedioxyphenyl)propane-2-amine], also known as Adam or ecstasy, is a chiral compound and a very popular drug of abuse leading to feelings of euphoria and energy and a desire to socialize (Kalant, 2001). The S-enantiomer of MDMA is known to be more potent than the R-enantiomer in producing the distinctive subjective effects that are characteristic for ecstasy. However, it can induce severe acute toxic symptoms, such as tachycardia, hypertension, hyperthermia, and hepatotoxicity, and severe or even fatal intoxications have been described previously (Fallon et al., 1999; Kalant, 2001).
Concerning chronic toxicity, data from animal experiments strongly suggest that these compounds can cause irreversible damage to serotoninergic nerve terminals in the central nervous system (Kalant, 2001; de la Torre et al., 2004; Monks et al., 2004; Easton and Marsden, 2006). Decreased levels of the serotonin metabolite 5-hydroxyindoleacetic acid (McCann et al., 1994) and serotonin transporters (McCann et al., 1998) were found in recreational MDMA users compared with control subjects with no history of MDMA exposure point in the same direction. However, as the history of drug abuse of the studied MDMA users relied on self-report, it could not be excluded that these findings were (in part) attributable to concomitant abuse of other drugs of abuse (Gouzoulis-Mayfrank and Daumann, 2006). R- and S-MDMA also differ in their dose-response curves for changes in serotonergic function and neurotoxicity, and their in vivo kinetics are known to be different (Fallon et al., 1999; Kalant, 2001; Kraemer and Maurer, 2002; Peters et al., 2003, 2005; Pizarro et al., 2004).
In vivo studies with racemic MDMA revealed two main metabolic pathways. As shown in Fig. 1, one major pathway includes O-demethylenation to 3,4-dihydroxymethamphetamine [DHMA, N-methyl-1-(3,4-dihydroxyphenyl)propane-2-amine], followed by O-methylation catalyzed by the catechol-O-methyltransferase (COMT) mainly to 4-hydroxy-3-methoxymethamphetamine [HMMA, N-methyl-1-(4-hydroxy-3-methoxyphenyl)propane-2-amine] and conjugation by UDP-glucuronyltransferases (UGTs) to the two diastereomers R- and S-3-methoxymethamphetamine 4-O-glucuronides (R-/S-HMMA-O-glucuronide) or by sulfotransferases (SULTs) to 3-methoxymethamphetamine 4-O-sulfate (Maurer, 1996; Maurer et al., 2000; de la Torre et al., 2004). In urine samples of recreational MDMA users, mainly conjugated HMMA can be detected (Shima et al., 2008). However, it has been shown that after ingestion of racemic MDMA, the S-enantiomer is eliminated at a higher rate than the R-enantiomer (Fallon et al., 1999; Kalant, 2001; Kraemer and Maurer, 2002; Peters et al., 2003, 2005; Pizarro et al., 2004). Enantioselective metabolism is the most likely explanation for the enantioselective pharmacokinetics of MDMA and was observed for the cytochrome P450-mediated phase I metabolism and for the COMT-catalyzed methylation (Meyer et al., 2008; Meyer and Maurer, 2009).
More recently, human UGT enzymes involved in the glucuronidation of racemic HMMA were investigated, and human UGT2B15 was identified as the only isoform with adequate enzyme activity toward the described reaction (Shoda et al., 2009). However, the authors did not take into consideration that two different diastereomeric glucuronides, namely, R- and S-HMMA-O-glucuronides, should be formed.
Concerning all these points, the question arises whether the glucuronidation of HMMA is enantioselective too and contributes also to the pharmacokinetic difference between the respective MDMA enantiomers. Therefore, the two diastereomeric O-glucuronides of HMMA have to be enzymatically synthesized as reference standards using rat liver microsomes (RLMs). The aim of the present study was to elucidate the human UGT isoforms involved in the formation of R- and S-HMMA-O-glucuronides and to obtain enantioselective enzyme kinetic data for the relevant UGT isoforms.
Materials and Methods
Racemic HMMA · HCl was obtained from Lipomed (Bad Saeckingen, Germany), and morphine-6-β-d-glucuronide (M6G) was from Sigma-Aldrich (Steinheim, Germany). Single HMMA glucuronides were synthesized in the authors' laboratory as described below. Sodium bicarbonate was obtained from Fluka (Buchs, Switzerland); UDP-glucuronic acid (UDPGA), d-saccharic acid 1,4-lactone (saccharolactone), alamethicin, and Brij58 were from Sigma-Aldrich; and ZIC-HILIC solid-phase extraction cartridges (1 g, 6 ml) were from Sequant (Marl, Germany). Water and acetonitrile of high-performance liquid chromatography (HPLC) grade were obtained from Thermo Fisher Scientific (Dreieich, Germany). All the other chemicals and reagents used were from Merck (Darmstadt, Germany). The following microsomes were from BD Gentest (Woburn, MA) and delivered by NatuTec (Frankfurt, Germany): baculovirus-infected insect cell microsomes (ICMs; Supersomes) containing 5.0 mg/ml human cDNA-expressed UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, UGT2B17, wild-type baculovirus-infected ICMs (control Supersomes), and pooled human liver microsomes (HLMs; 20 mg of microsomal protein/ml). After delivery, the microsomes were thawed at 37°C, aliquoted, shock-frozen in liquid nitrogen, and stored at −80°C until use. All of the chemicals were of analytical grade or the highest grade available. The derivatization reagent S-heptafluorobutyrylprolyl chloride (S-HFBPCl) was synthesized in the authors' laboratory according to Peters et al. (2002).
Preparation of RLMs.
Microsomes were prepared from rat liver by differential ultracentrifugation (De Duve, 1971). RLMs were prepared from eight frozen (−80°C) livers of male Wistar rats (Charles River, Sulzfleck, Germany). Protein concentrations of the microsomes were determined according to the method of Lowry (1951).
Enzymatic Synthesis and Extraction of the R- and S-HMMA Glucuronides.
The bioassay consisted of 2.5 mM racemic HMMA · HCl, 2 mg/ml RLMs, 5 mM MgCl2, 5 mM saccharolactone, and 100 μg/mg protein Brij58 in a total volume of 10 ml of 100 mM phosphate buffer, pH 7.4. The reaction was started with the addition of 5 mM UDPGA. After 4.5 h of incubation at 37°C, the glucuronidation was terminated with 10 ml of acetonitrile, and the mixture was centrifuged at 10,000g for 20 min. The supernatant containing the R- and S-HMMA-O-glucuronides was diluted 1:20 with acetonitrile containing 0.1% formic acid and was transferred to solid-phase extraction cartridges (Sequant ZIC-HILIC, 1 g, 6 ml) previously conditioned triply with 5 ml of water and 5 ml of acetonitrile. After pass-through of the supernatant, the cartridges were washed triply with 5 ml of acetonitrile and 5 ml of an acetonitrile/water mixture (90:10, v/v). The cartridges were eluted three times with 2.5 ml of an acetonitrile/water mixture (50:50, v/v), and the acetonitrile part of the eluate was evaporated under reduced pressure at room temperature.
Separation of R- and S-HMMA Glucuronide.
The two diastereomeric compounds R- and S-HMMA-O-glucuronide were separated using a Hewlett Packard (Agilent, Waldbronn, Germany) Series 1050 semipreparative HPLC system consisting of a pump and a variable wavelength detector (λ * 230 nm). The stationary phase consisted of a Phenomenex (Aschaffenburg, Germany) Chirex 3012 column (250 × 4.6 mm, 5 μm) and the mobile phase of a mixture of 20% 10 mM ammonium formate buffer acidified with 0.1% formic acid and 80% acetonitrile acidified with 0.1% formic acid at a flow rate of 1 ml/min. The eluates were separated in aliquots of 50 μl, and the fractions with the two glucuronides were collected. The HPLC fractions containing the single diastereomers were diluted 1:1 with acetonitrile and submitted to solid-phase extraction using ZIC-HILIC cartridges as described above. The cartridges were eluted three times with 2.5 ml of water, and the combined eluates of each glucuronide were lyophilized.
Confirmation of Configuration.
The elution order of the diastereomeric glucuronides on the Chirex 3012 column was determined using 100 μl of a solution of the isolated first-eluting HMMA glucuronide. After dilution with 900 μl of water, the pH was adjusted to pH 5.2 with acetic acid, and the mixture was incubated at 50°C for 1.5 h with 100 μl of a mixture (100,000 Fishman units/ml) of glucuronidase (EC 3.2.1.31) and arylsulfatase (EC 3.1.6.1) from Helix pomatia L. Afterward, 10 μl of this solution and 10 μl of a 10 μM solution of racemic HMMA · HCl were worked up as described previously by Meyer and Maurer (2009). In brief, the analytes were derivatized with S-HFBPCl and analyzed by gas chromatography/mass spectrometry (GC/MS) using negative-ion chemical ionization. Selected-ion monitoring was performed with the ions m/z 487 and 781 for derivatized HMMA. Retention time of the isolated glucuronide after conjugate cleavage and derivatization was compared with the retention times of derivatized R- and S-HMMA, which were previously described by Meyer and Maurer (2009).
Identity and Purity Confirmation of the Isolated Glucuronides.
For identity check, a small amount of each diastereomer was dissolved in water and analyzed by liquid chromatography/mass spectrometry (LC/MS) as described below.
For structure confirmation by 1H NMR, solutions of S-HMMA glucuronide (4 mg/0.5 ml) and R-HMMA glucuronide (2 mg/0.5 ml) were prepared in D2O. 1H NMR spectra (500 MHz) were recorded on a Bruker (Bremen, Germany) DRX 500 at 300 K. The chemical shifts were given in δ values (ppm) relative to acetone at δ2.22.
For purity check of the respective diastereomers, a small amount of each glucuronide was dissolved in water and analyzed by HPLC/UV using an Hewlett Packard 1050 series HPLC system with UV detection at 230 and 280 nm using a Merck LiChroCART Superspher 60 RP Select B column (2.0 × 125 mm, 5 μm) and a Phenomenex Chirex 3012 (250 × 4.6 mm, 5 μm) column. The mobile phase consisted of 10 mM ammonium formate buffer acidified with 0.1% formic acid and acetonitrile acidified with 0.1% formic acid. The isocratic composition was 98:2 (v/v) with a flow rate of 0.5 ml/min and 20:80 (v/v) with a flow rate of 1 ml/min, respectively. The injection volume was 15 μl for analysis at 230 nm and 25 μl for analysis at 280 nm.
Microsomal Incubations.
Incubation mixtures (final volume, 50 μl) consisted of 100 mM phosphate buffer, pH 7.4, 5 mM MgCl2, alamethicin (50 μg/mg protein), and 0.25 to 1 mg/ml UGT-containing microsomes and substrate at 37°C. The substrate was added after dilution of a 50 mM aqueous stock solution in the above-mentioned phosphate buffer. Reactions were started by addition of 5 mM UDPGA and terminated with 50 μl of acetonitrile.
Initial Screening Studies.
Incubations were performed with 500 μM R,S-HMMA and 1 mg/ml UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 for 30 min. Further composition of the incubation mixtures is described under Microsomal Incubations.
Kinetic Studies.
The kinetic constants of HMMA O-glucuronidation were derived from incubations with an incubation time of 25 min (recombinant UGTs) and 15 min (HLMs). The protein concentrations were 0.25 mg/ml for UGT2B15; 0.5 for UGT1A9, UGT2B7, and UGT2B15; and 1 mg/ml for HLMs. These concentrations were within the linear range of the glucuronide formation.
Enzyme kinetic constants were estimated by nonlinear curve-fitting using GraphPad Software Inc. (San Diego, CA) Prism 5.00 software. The normalized initial velocity data from the enzyme kinetic studies were fitted either to the Michaelis-Menten equation (eq. 1), where v is the initial velocity of the reaction, S the substrate concentration, Vmax the maximal velocity, and Km the substrate concentration at half Vmax; or to the Hill equation (eq. 2), where Km is the substrate concentration at 0.5 Vmax and n is the Hill coefficient. The best kinetic model was selected, considering the randomness of the residuals, the S.E. of the estimates, and the correlation coefficients. In addition, Eadie-Hofstee plots were used to check for allosteric kinetics.
Sample Preparation for Microsomal Incubations.
After termination of the incubation, 10 μl of the internal standard (M6G, 5 μM) was added to each incubation mixture; the samples were vortexed and centrifuged (10,000g, 10 min); 100 μl of the supernatants was transferred to autosampler vials; and 10-μl aliquots were injected into the LC/MS system.
LC/MS Conditions.
The analytes were separated using an Accela LC system (Thermo Fisher Scientific) consisting of a degasser, a quaternary pump, and an autosampler coupled to a Thermo Fisher Scientific LXQ Linear Ion Trap MS system equipped with a heated electrospray ionization source. The collected data were processed by Xcalibur 2.0.7 (Thermo Fisher Scientific).
The LC conditions were as follows: Phenomenex Chirex 3012 column (250 × 4.6 mm, 5 μm); column temperature, 35°C; isocratic mobile phase consisted of 30% 10 mM aqueous ammonium formate buffer containing 0.1% (v/v) formic acid and 70% acetonitrile containing 0.1% (v/v) formic acid; and flow rate, 0.5 ml/min for identity confirmation and 1.0 ml/min for quantification. The total run times were 25 or 12 min, respectively.
The MS conditions were as follows: electrospray ionization, positive mode; sheath gas, nitrogen at flow rate of 40 arbitrary units; auxiliary gas, nitrogen at flow rate of 25 arbitrary units; vaporizer temperature, 300°C; source voltage, 3.00 kV; ion transfer capillary temperature, 380°C; capillary voltage, 9.0 V; and tube lens voltage, 75 V. For identity confirmation, collision-induced dissociation/MS/MS experiments were performed on precursor ions selected from MS1 using information-dependent acquisition: MS1 was performed in the full-scan mode (m/z 100–400). MS2 was performed in the information-dependent acquisition mode: MS2 on the most intense signal from MS1 with normalized collision energies of 35%. Other settings were as follows: minimum signal threshold: MS1, 50 counts; isolation width, 2.00 u; activation Q, 0.25; activation time, 30 ms; dynamic exclusion mode, repeat counts 2; repeat duration, 15 s; exclusion list, 50; and exclusion duration, 15 s.
For quantification, the MS1 was performed in the full-scan mode (m/z 100–500). Daughter ion scans (m/z 50–400 for HMMA, m/z 125–400 for M6G, m/z 100–400 for R-/S-HMMA-O-glucuronide) were performed using collision-induced dissociation with normalized collision energies of 35% on respective precursor ions m/z 196, m/z 462, and m/z 372. The following ions in MS2 were used for quantification: m/z 165 for HMMA, m/z 286 for M6G, and m/z 196 for R-/S-HMMA-O-glucuronide. Calibration curves were constructed plotting peak area ratios (S-HMMA glucuronide and R-HMMA glucuronide versus internal standard) of spiked calibrators versus their concentrations (0.01, 0.1, 1.0, 5.0, 10, 25, and 50 μM). For quantification, a weighted (1/X) linear regression model was used.
Results
Enzymatic Synthesis of R- and S-HMMA Glucuronides.
Initial experiments showed that RLMs were the most efficient microsomal source for enzymatic synthesis of R- and S-HMMA-O-glucuronides (data not shown). Optimal incubation conditions were evaluated (data not shown) and shown to be best for 5 mM MgCl2, 5 mM saccharolactone, 100 μg/mg protein Brij58, 2 mg/ml microsomal protein, 5 mM UDPGA, 2.5 mM racemic HMMA, and an incubation time of 4.5 h. Under these conditions, HMMA was completely converted to the respective diastereomeric glucuronides. The two diastereomers were separated on a Phenomenex Chirex 3012 chiral column (Fig. 2A), and the elution order of R- and S-HMMA-O-glucuronide was determined after enzymatic cleavage of the fraction I, derivatization with S-HFBPCl, and GC/MS analysis. The retention time was compared with derivatized racemic HMMA (Fig. 2B) as described by Meyer and Maurer (2009), where the S-enantiomer elutes before the R-enantiomer. Figure 2C shows the worked-up fraction I, indicating that it corresponded to S-HMMA-O-glucuronide. The final yield was 4 mg for S-HMMA-O-glucuronide (87%) and 2 mg for R-HMMA-O-glucuronide (44%). The purity of the products determined by HPLC/UV (230 and 280 nm) was >90%.
The purified glucuronides were identified using LC/MS as shown in Fig. 3. The chromatograms of ion m/z 372 (M+H+; Fig. 3, A and B) showed S- and R-HMMA-O-glucuronide at a retention time of 14.6 and 15.6 min, respectively. The isolated R-HMMA-O-glucuronide contained less than 4% of the S-diastereomer. For further structure confirmation, 1H NMR spectra of both diastereomers were recorded with the following results: R-HMMA-O-glucuronide: 1H NMR (500 MHz, D2O) δ7.150 (1H, d, J * 8.3, H-5), 7.006 (1H, d, J * 1.9, H-2), 6.882 (1H, dd, J * 8.3, 1.9, H-6), 5.117 (1H, d, J * 7.4, H-1"), 3.880 (3H, s, OCH3), 3.841 (1H, m, H-3"), 3.680 to 3.560 (3H, m, H-2″, H-4″, H-5″), 3.527 (1H, m, H-2′), 3.011 (1H, dd, J * 14.0, 6.6, H-1′a), 2.889 (1H, dd, J * 14.0, 7.6, H-1′b), 2.689 (3H, s, NCH3), 1.282 (3H, d, J * 6.7, H-3′); S-HMMA-O-glucuronide: 1H NMR (500 MHz, D2O) δ7.153 (1H, d, J * 8.3, H-5), 7.001 (1H, d, J * 1.9, H-2), 6.885 (1H, dd, J * 8.3, 1.9, H-6), 5.117 (1H, d, J * 7.4, H-1″), 3.880 (3H, s, OCH3), 3.842 (1H, m, H-3″), 3.680 to 3.560 (3H, m, H-2″, H-4″, H-5″), 3.523 (1H, m, H-2′), 3.017 (1H, dd, J * 14.0, 6.5, H-1′a), 2.879 (1H, dd, J * 14.0, 7.8, H-1′b), 2.691 (3H, s, NCH3), and 1.275 (3H, d, J * 6.7, H-3′). Significant differences in the chemical shifts for the R- and S-diastereomers could not be observed under the used conditions. The 1H NMR spectra were in agreement with those published by Shima et al. (2007) after chemical synthesis of the mixture of R- and S-HMMA-O-glucuronide.
LC/MS for Diastereoselective Quantification.
The applied LC/MS conditions for quantification provided separation of HMMA, M6G, and R- and S-HMMA-O-glucuronide. The chosen target ions of the respective daughter spectra were selective for the analytes under these conditions as proven with blank samples (control microsomes without substrate and standard) and zero samples (control microsomes without substrate but with internal standard). The method showed good linearity in a range of 0.01 to 50 μM R- and S-HMMA-O-glucuronide (R2 > 0.998). Matrix effect studies, comparing the peak areas of the glucuronides in neat standard solutions with those in spiked incubation mixtures containing the same concentrations of the glucuronides, gave no indication for ion suppression or enhancement (n * 5).
Initial Activity Screening.
As shown in Fig. 4, O-glucuronidation of racemic HMMA was catalyzed by UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17.
Kinetic Studies.
Twelve HMMA concentrations ranging from 0.01 to 20 mM were used in the kinetic analysis of UGTs and HLMs. The Km, Vmax, Vmax/Km values, and the Hill coefficients n are listed in Table 1. The data for UGT2B15, UGT2B17, and HLM followed the expected classic hyperbolic Michaelis-Menten kinetics as shown in Fig. 5. In contrast, UGT1A9 and UGT2B7 were best described by the Hill equation indicating sigmoidal kinetics. Marked enantioselectivity [Vmax/Km(S-HMMA)/Vmax/Km(R-HMMA) >1.5 or <0.67] was observed for UGT1A9 toward S-HMMA and UGT2B7 toward R-HMMA.
As shown in Fig. 7, HMMA glucuronidation in the range of 1 to 10 μM by UGT2B7, UGT2B17, and HLMs was not considerably enantioselective, but there was a slight preference for the S-diastereomer. No preference could be observed for UGT2B15, and only UGT1A9 revealed a relevant selectivity for glucuronidation of S-HMMA.
Discussion
Reference standards of the HMMA glucuronides needed for enzyme kinetic studies were not commercially available. Therefore, the two diastereomers of HMMA-O-glucuronide were synthesized enzymatically by RLMs. Such bioassays for glucuronide synthesis are a versatile alternative to classic chemical synthesis, especially for milligram quantities and have been used previously for a wide range of substrates (Soars et al., 2002; Uutela et al., 2009). HMMA-O-glucuronide was previously synthesized by chemical synthesis by Shima et al. (2007) and by enzymatic synthesis as well (Shoda et al., 2009), but the two diastereomers were not isolated separately. The final yield using chemical synthesis was 6%, which is somewhat low compared with the enzymatic synthesis presented by Shoda et al. (2009) with 71% and to the yields in this study with 87% for the S-diastereomer and 44% for the R-diastereomer.
The incubation conditions chosen for the initial screening of 12 human UGT isoforms were applicable for checking the general involvement of particular isoforms. The microsomes were not allowed to sit on ice for preincubation with alamethicin because preliminary experiments showed no difference for this treatment as already described by Oleson and Court (2008). In contrast to Shoda et al. (2009), UGT1A10 was not found to be involved in the HMMA glucuronidation. One reason for this observation might be the uncommonly high substrate concentration used by Shoda et al. (2009). In the study presented here, enzyme kinetic data not only for UGT2B15 but also for UGT1A9, UGT2B7, and UGT2B17 were recorded. Because of their very low activity, kinetics of UGT1A1, UGT1A3, UGT1A8, and UGT2B4 could not be studied. Different concentrations of UDPGA (1.0–7.5 mM) were tested, and 5 mM was a saturating concentration for all the isoenzymes. It is unfortunate that common protein concentration could not be chosen for all of the isoforms because the glucuronide formation by UGT1A9, UGT2B7, and UGT2B17 was too low to obtain glucuronide concentrations suitable for reliable quantification. Duration and protein content of all the incubations were within the linear range of glucuronide formation (data not shown), and less than 20% of substrate was metabolized in all the incubations.
The kinetic data for UGT2B15, UGT2B17, and HLMs followed classic Michaelis-Menten plots (Fig. 5). In contrast, UGT1A9 and UGT2B7 revealed sigmoidal curves, and the respective Eadie-Hofstee plots (Fig. 6) indicated autoactivation kinetics (Hill coefficients are given in Table 1). Such autoactivation kinetics have been previously described for glucuronidation in HLMs (Hutzler and Tracy, 2002) but also for recombinant UGTs, e.g., UGT2B7 toward 4-methylumbelliferone and 1-naphthol (Uchaipichat et al., 2008) and UGT1A10 toward dopamine glucuronidation (Itäaho et al., 2009).
The Km values for the formation of R- and S-HMMA-O-glucuronide by UGT2B15 were in a similar range as previously described for the mixture of both diastereomers (Shoda et al., 2009). In contrast, the respective Vmax values were approximately 5-fold higher in our study, which could be explained by different incubation times and different activities of the used enzymes. According to our studies, the metabolite formation was linear only up to 30 min, whereas Shoda et al. (2009) used a considerably longer incubation time (120 min). Measuring outside the linear range may lead to false estimation of the metabolite formation. UGT2B15 turned out to have the highest affinity and the highest capacity of all the isoforms tested (Table 1). Vmax values for UGT2B15 are approximately 10-fold higher than for the other isoforms. The respective Km values for all the isoforms seem to be very high compared with other glucuronidation reactions (Kiang et al., 2005; Mazur et al., 2009) and are not expected to be reached after intake of recreational user doses of MDMA, leading to plasma concentrations of MDMA in the range of 1 μM (194 μg/l) (Fallon et al., 1999; de la Torre et al., 2000, 2004; Logan and Couper, 2001; Peters et al., 2003, 2005; Pizarro et al., 2004) but can reach 10 μM and higher in severe intoxications (Peters et al., 2003; Schifano, 2004). Therefore, the glucuronidation activities of all the relevant recombinant UGTs and HLMs were interpolated in a range of 1 to 10 μM substrate concentration (Fig. 7) to consider the activities within a relevant concentration range showing metabolite formation activities comparable with previously published data for other substrates.
Comparing the Vmax/Km values (Table 1) with values described for other glucuronidations, these were in similar ranges (Court et al., 2002; Ohno et al., 2008; Itäaho et al., 2009; Mazur et al., 2009). The higher this value, the better the overall effectiveness of the respective reaction should be. This fact sounds reasonable, as the catalytic efficiency value is higher with increasing affinity (low Km) and increasing velocity (high Vmax).
Enantioselective metabolism is the most likely explanation for the enantioselective pharmacokinetics of MDMA. Enantioselectivity was observed for N-demethylation and demethylenation by CYP2C19, demethylenation by CYP2D6 (Meyer et al., 2008), and for the COMT-catalyzed methylation of DHMA to HMMA, with a preference for the S-enantiomers (Meyer and Maurer, 2009). The study presented here is the first providing enantioselective enzyme kinetic data for MDMA phase II metabolism, the O-glucuronidation of HMMA.
In the case of glucuronidation marked enantioselectivity [Vmax/Km(S-HMMA)/Vmax/Km(R-HMMA) >1.5 or <0.67] was only observed for UGT1A9 toward S-HMMA and UGT2B7 toward R-HMMA. When looking at the interpolated values in the range of 1 to 10 μM, no considerable enantioselectivity by UGT2B7, UGT2B17, and HLM was detected, but there was a slight preference for S-HMMA. No preference could be observed for UGT2B15, and only UGT1A9 revealed a relevant selectivity for glucuronidation of S-HMMA. It is interesting to note that the marked enantioselectivity for UGT2B7 toward R-HMMA did not seem to be relevant in the low concentration range, in which S-HMMA was glucuronidated even with a slightly higher activity than R-HMMA. With respect to the presented data, the O-glucuronidation of HMMA in vivo should not be expected to be enantioselective. Nevertheless, the S-enantiomer of MDMA was eliminated at a higher rate than its R-enantiomer (Fallon et al., 1999; Kalant, 2001; Kraemer and Maurer, 2002; Peters et al., 2003, 2005; Pizarro et al., 2004). Considering the results of the presented study, these pharmacokinetic differences can mainly be explained by enantioselective demethylenation to DHMA and methylation to HMMA (Meyer et al., 2008; Meyer and Maurer, 2009) or additionally by possible enantioselective sulfation.
UGT2B15 was the most abundant isoform involved in the glucuronidation of HMMA and is known to be polymorphically expressed. Two distinct allelic variants caused by a single nucleotide polymorphism are known, UGT2B15*1 and UGT2B15*2, whereas UGT2B15*1 is less frequent in whites compared with other populations, such as Asian, Hispanic, or African (0.45–0.64, respectively) (Guillemette, 2003). For S-oxazepam, a specific substrate for UGT2B15, a 5-fold higher conjugating activity was described for the *1 allele (Court et al., 2002), whereas for the glucuronidation of androgens, an increased activity of the *2 allele was described previously (Lévesque et al., 2001). Because UGT2B15 was the main isoform involved in the O-glucuronidation, possible interindividual differences in HMMA glucuronidation in vivo can be expected and should be further investigated.
Acknowledgments.
We thank Dr. Stefan Böttcher, Gabriele Ulrich, Armin A. Weber, Carsten Schroeder, Dirk K. Wissenbach, Annekathrin Otto, and Daniela Remane for assistance and helpful discussions.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.029215
- Received June 22, 2009.
- Accepted August 7, 2009.
- Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics