DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 DMD ThisFast article Forward. has not been Published copyedited onand formatted.April 23, The 2013 final asversion doi:10.1124/dmd.113.051565 may differ from this version.
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Human UGT2B10 in drug N-glucuronidations: substrate screening and comparison with
UGT1A3 and UGT1A4
Yukiko Kato, Takeshi Izukawa, Shingo Oda, Tatsuki Fukami, Moshe Finel, Tsuyoshi Yokoi, and
Miki Nakajima Downloaded from
dmd.aspetjournals.org
Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, at ASPET Journals on May 31, 2021 Kakuma-machi, Kanazawa 920-1192, Japan (Y. K., T. I., S. O., T. F., T. Y., M. N.), Center for
Drug Research and Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of
Helsinki, Helsinki, Finland (M. F.).
1 Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
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Running title: Significance of UGT2B10 in N-glucuronidation of drugs
To whom correspondence should be addressed:
Miki Nakajima, Ph.D.
Drug Metabolism and Toxicology
Faculty of Pharmaceutical Sciences
Kanazawa University
Kakuma-machi Downloaded from Kanazawa 920-1192, Japan
Tel / Fax +81-76-234-4407
E-mail: [email protected] dmd.aspetjournals.org
Number of text pages: 36
Number of tables: 4 at ASPET Journals on May 31, 2021 Number of figures: 6
Number of references: 35
Number of words in abstracts: 243 words
Number of words in introduction: 423 words
Number of words in discussion: 1053 words
Abbreviations: UGT, UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; HLM, human liver microsomes; HPLC, high performance-liquid chromatography; LC, liquid chromatography; MS/MS, tandem mass spectrometry.
2 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
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Abstract
Recent observations revealed that human UDP-glucuronosyltransferase (UGT) 2B10 catalyzes N-glucuronidation of amine-containing compounds. Knowledge of the substrate specificity and clinical significance of UGT2B10 is still limited. The purpose of this study was to expand the knowledge of UGT2B10 substrates and to evaluate its significance in drug clearance. Downloaded from Using recombinant UGT2B10, we found that it catalyzes the N-glucuronidation of amitriptyline, imipramine, ketotifen, pizotifen, olanzapine, diphenhydramine, tamoxifen, ketoconazole and dmd.aspetjournals.org midazolam. These are drugs that were previously reported to be substrates for UGT1A4 or
UGT1A3 and that contain in their structure either tertiary aliphatic amines, cyclic amines, or an
imidazole group. UGT2B10 was inactive in the glucuronidation of desipramine, nortriptyline, at ASPET Journals on May 31, 2021 carbamazepine and afloqualone. This group of drugs contains secondary or primary amines, and these results suggest that UGT2B10 preferably conjugates tertiary amines. This preference is partial because UGT2B10 did not conjugate the tertiary cyclic amine in trifluoperazine. Kinetic analyses revealed that the affinity and clearance of UGT2B10 for amitriptyline, imipramine, and diphenhydramine are significantly higher than the corresponding values of UGT1A4 and
UGT1A3, although the Vmax values of UGT1A4 toward these drugs are considerably higher.
These findings suggest that UGT2B10 plays a major role in the N-glucuronidation of these drugs at therapeutic concentrations. These results are also supported by inhibition studies with nicotine and hecogenin. In conclusion, this study expands the understanding of the substrate specificity of
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UGT2B10, highlighting its preference for tertiary amines with higher affinities and clearance values than those of UGT1A4 and UGT1A3. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021
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Introduction
UDP-Glucuronosyltransferases (UGT) catalyze glucuronidation of a variety of xenobiotics and endogenous compounds (Turkey and Strassburg, 2000). The 19 functional human UGT enzymes are classified into three subfamilies, UGT1A, UGT2A, and UGT2B. The individual human UGTs show both overlaps and specificity in their substrate preferences. The major Downloaded from reactions that the UGTs catalyze are O- and N-glucuronidations (Chiu and Huskey, 1998). The
N-glucuronidation of primary, secondary, and tertiary amines represents an important pathway in dmd.aspetjournals.org the elimination of many drugs, as indicated by the high amounts of N-glucuronides in human urine (Green and Tephly, 1996; Green et al., 1998; Kaivosaari et al., 2011). It is widely
recognized that the N-glucuronidation of tertiary amines is catalyzed by UGT1A4 and UGT1A3 at ASPET Journals on May 31, 2021
(Green and Tephly, 1998). However, because it was difficult to reconcile the observed biphasic kinetics in the N-glucuronidation of some tertiary amines by human liver microsomes with the low-affinity that UGT1A4 and UGT1A3 exhibited toward the tested compounds, the possibility that another UGT might be responsible for the high-affinity reaction has been raised
(Breyer-Pfaff et al., 2000; Nakajima et al., 2002).
Jin et al (1993) cloned the c-DNA for human UGT2B10, expressed it in COS-7 cells and tested the enzyme activity toward 42 compounds. These compounds mostly contained a hydroxyl group but one of them did contain a primary amine. Since UGT2B10 did not catalyze the glucuronidation of any of the compounds in this study, it was considered an orphan UGT for over
5 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565 ten years. A breakthrough occurred in 2007 with the findings from two different laboratories that
UGT2B10 catalyzes the N-glucuronidation of nicotine and cotinine (Kaivosaari et al., 2007,
Chen et al., 2007). It was later reported that UGT2B10 also catalyzes the N-glucuronidation of nitrosamines (Chen et al., 2008) and R-medetomidine (Kaivosaari et al., 2008). These UGT2B10 substrates are also glucuronidated by UGT1A4, but at much lower affinity. The latter studies
implied a preference of UGT2B10 for tertiary amines and an overlapping substrate specificity Downloaded from with UGT1A4 and UGT1A3 (Kaivosaari et al., 2007, 2008; Chen et al., 2007, 2008). Regardless
of these and later studies (Zhou et al., 2010, Erickson-Ridout et al., 2011), the current dmd.aspetjournals.org understanding of the substrate specificity of UGT2B10 is limited, and its clinical significance remains to be clarified. To this end, we have now screened 14 drugs containing different amine at ASPET Journals on May 31, 2021 structures to find whether or not they are UGT2B10 substrates. Based on these findings, we determined the affinity and clearance for the glucuronidation of representative three substrates by
UGT2B10 and compared them with the corresponding values for reactions catalyzed by
UGT1A4 and UGT1A3.
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Materials and Methods
Materials. UDPGA, alamethicin, hecogenin acetate, nortriptyline hydrochloride, and
β-glucuronidase from Helix pomatia (Type H-2) were purchased from Sigma-Aldrich (St. Louis,
MO). Amitriptyline, imipramine hydrochloride, ketotifen fumarate, trifluoperazine dihydrochloride, desipramine hydrochloride, carbamazepine, diphenhydramine hydrochloride, Downloaded from and midazolam were from Wako Pure Chemicals (Osaka, Japan). Olanzapine and ketoconazole were purchased from Toronto Research Chemicals (Toronto, Canada). Afloqualone was kindly dmd.aspetjournals.org provided by Mitsubishi Tanabe Pharma (Osaka, Japan). Microsomes from 15 individual human livers (HG03, HG32, HG43, HG64, HG70, HG95, HH1, HH6, HH13, HH18, HH31, HH35,
HH40, HH47, and HH89) and pooled human liver microsomes (HLM) (n = 50) were obtained at ASPET Journals on May 31, 2021 from BD Gentest (Woburn, MA). Cellfectin Reagent and Sf9 cells were purchased from
Invitrogen (Carlsbad, CA). Mouse anti-tetra-His monoclonal antibody was obtained from
QIAGEN (Valencia, CA). All other reagents were of the highest grade commercially available.
Construction of Expression Vectors for His-Tagged UGT1A3, UGT1A4, and UGT2B10.
Expression vectors for human UGT1A3 or UGT1A4 containing a His tag (6 histidines) at the C terminus were constructed using the Bac-to-Bac Baculovirus Expression System (Invitrogen,
Carlsbad, CA). Briefly, The UGT1A3 and UGT1A4 cDNAs were digested from the previously constructed pTARGET/UGT1A3 or pTARGET/UGT1A4 vectors (Koga et al., 2011; Fujiwara et al., 2007) and used to replace the UGT1A1 cDNA in the pFastBac1/His-UGT1A1 vector
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(Kurkela et al., 2007); this resulted in the pFastBac/UGT1A3 and pFastBac/UGT1A4 vectors.
The nucleotide sequences of the constructed plasmids were confirmed by DNA sequencing analyses (Long-Read Tower DNA sequencer; GE Healthcare). The expression vector for human
UGT2B10 containing a His tag (6 histidines) (pFastBac/UGT2B10 vector) was previously constructed (Kurkela et al., 2007). These pFastBac1 vectors were transformed into DH10Bac
competent cells, followed by transposition of the inserts into bacmid DNA. Downloaded from
Expression of His-UGT1A3, UGT1A4, and UGT2B10 in Sf9 Cells. Spodoptera frugiperda dmd.aspetjournals.org Sf9 cells (Invitrogen) were grown in Sf-900 II SFM containing 10% fetal bovine serum at 27°C.
The recombinant bacmid DNAs were transfected into the Sf9 cells using the Cellfectin Reagent
(Invitrogen), and the recombinant baculoviruses were harvested from the cell culture medium 72 at ASPET Journals on May 31, 2021 hr after transfection. Cells were routinely harvested 5 days after the infection of the recombinant baculoviruses, washed twice with PBS, and stored at -80°C until analysis. Cell homogenates were prepared by suspension in Tris-buffered saline [25 mM Tris-HCl buffer (pH 7.4), 138 mM
NaCl, and 2.7 mM KCl] followed by three cycles of freezing and thawing to disrupt the cells.
The suspensions were then homogenized with ten strokes in a Teflon-glass homogenizer. The protein concentrations of the homogenates were determined according to Bradford (1976).
Glucuronidation Assays. The typical incubation mixture (200 µl total volume), containing 50
mM Tris-HCl (pH 7.4), 10 mM MgCl2, 25 µg/ml alamethicin, 0.5 mg/ml HLM or 1 mg/ml cell homogenates containing recombinant human UGT, and test compound was kept on ice for 10
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min. The compounds and their concentrations, which were set at approximately their Km value in
HLM, are shown in Table 1. The mixture was pre-incubated at 37°C for 3 min and reaction was initiated by the addition of UDPGA (final concentration of 5 mM). After incubation at 37°C for
120 min, the reaction was terminated by the addition of 100 µl of ice-cold acetonitrile. Following removal of the protein by centrifugation at 10,000 g for 5 min, a 20-µl portion of the sample was
subjected to LC-MS/MS analysis. Downloaded from
N-Glucuronides Detection by LC-MS/MS. The LC equipment was comprised of an HP1100 dmd.aspetjournals.org system including a binary pump, an automatic sampler, and a column oven (Agilent
Technologies, Santa Clara CA), which was equipped with a ZORBAX SB-C18 column (2.1 × 50
mm, 3.5 µm, Agilent Technologies). The column temperature was set at 25°C and the flow rate at ASPET Journals on May 31, 2021 was 0.2 ml/min. The mobile phase was 0.1% formic acid (A) and methanol including 0.1% formic acid (B). The conditions for elution were as follows: 20% B (0 -2.0 min), 20 - 90% B
(2.01 -3 min), 90% B (3.01-10 min), and 20% B (10.01-18 min). The LC was connected to a PE
Sciex API2000 tandem mass spectrometer (AB Sciex, Framingham, MA) operated in the positive electrospray ionization mode. The turbo gas was maintained at 550°C. Nitrogen was used as the nebulizing, turbo, and curtain gas at 70, 40, and 50 psi, respectively. Parent and/or fragment ions were filtered in the first quadrupole and dissociated in the collision cell using nitrogen as the collision gas. The different transitions selected for each of the compounds are detailed in Table 1.
The analytical data were processed using Analyst software (version 1.5; Applied Biosystems,
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Foster City, CA). Because authentic standards for the glucuronides were not available, we used the peak area values as the index of activity when comparing the 3 enzymes.
Quantification of Amitriptyline, Imipramine, and Diphenhydramine N-Glucuronides.
HPLC analyses were performed to quantify the amitriptyline, imipramine, and diphenhydramine
N-glucuronides. A 50-µl portion of the sample described above was subjected to HPLC analysis. Downloaded from The equipment consisted of an L-2130 pump (Hitachi, Tokyo, Japan), an L-2200 autosampler
(Hitachi), an L-2400 UV detector (Hitachi), a D-2000 HPLC System Manager program (Hitachi), dmd.aspetjournals.org and an L-2300 column oven, which was equipped with a Mightysil RP-18GP column (4.6 × 150 mm; 5 µm; Kanto Chemical, Tokyo, Japan). The flow rate was 1 ml/min and the column
temperature was 35°C. The mobile phases and the UV wavelength are shown in Table 2. The at ASPET Journals on May 31, 2021 eluate containing each glucuronide was collected with reference to the retention time. A part of the eluate (200 µl) was incubated with 1000 U/ml β-glucuronidase at 37°C for 24 h. The reaction was terminated by the addition of 100 µl of ice-cold acetonitrile. After removal of the protein by centrifugation at 10,000g for 5 min, a 50-µl aliquot of the sample was subjected to HPLC analysis. Since the conversion of the glucuronide to the parent compound was partial (< 50%), the peak area of the remaining glucuronide in the treated sample was compared with that in a control sample that was not treated with β-glucuronidase. The content of the re-generated parent compound in the β-glucuronidase-treated sample could be determined using a standard curve made by the authentic standard. Since the content of the re-generated parent compound should
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DMD #51565 correspond to that of the hydrolyzed glucuronide, we could calculate the content of the glucuronide in the collected eluate. The collected eluate containing the glucuronide, at a known concentration, was used to prepare a standard curve for LC-MS/MS analyses.
Kinetics Analyses. Kinetic analyses of amitriptyline, imipramine, and diphenhydramine
N-glucuronidation by recombinant UGTs or HLM were performed. The substrate concentration Downloaded from ranged in each case was 10-1000 µM. The kinetic parameters were estimated from the fitted curve to a Michaelis-Menten, two-enzyme Michaelis-Menten, substrate inhibition or Hill dmd.aspetjournals.org equation (Bowalgaha et al., 2005) using a computer program (KaleidaGraph; Synergy Software,
Reading, PA) designed for nonlinear regression analysis. The kinetic models that best fitted to
2 appropriate curves with the highest R values were chosen based on the Levenberg-Marquardt at ASPET Journals on May 31, 2021 algorithm in the KaleidaGraph program. Eadie-Hofstee plots were drawn to visually reveal the suitability of biphasic kinetics in each analysis. For reactions exhibiting Michaelis-Menten
kinetics and substrate inhibition, the intrinsic clearance (CLint) was calculated as Vmax/Km. For
reactions exhibiting sigmoidal kinetics, the maximum clearance (CLmax) was calculated as Vmax •
(n - 1)/(S50 • n(n - 1)1/n) to estimate the highest clearance value (Houston and Kenworthy, 2000).
Data are expressed as the means ± SD of three independent determinations.
Inhibition Analyses of N-Glucuroidation in HLM. The concentrations of substrates were
determined according to the Km1 and Km2 values in HLM and were as follows: amitriptyline, 20 and 400 µM; imipramine, 50 and 400 µM; diphenhydramine, 20 and 500 µM. For selective
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DMD #51565 inhibition of UGT1A4 and UGT2B10, we used hecogenin (Uchaipichat et al., 2006; Zhou et al.,
2010) and nicotine (Zhou et al., 2010), respectively. Hecogenin (dissolved in dimethyl sulfoxide), and nicotine (dissolved in methanol), were added to the incubation mixtures and their final concentrations were 10 µM and 500 µM, respectively. The final solvent concentration was 2%
(v/v) for both inhibitors; the control incubation mixture contained the same solvent
concentration. Downloaded from
Immunoblot Analyses of Recombinant His-tagged UGTs. The Sf9 cell homogenates dmd.aspetjournals.org expressing His-tagged UGTs (2 to 45 µg) were separated on 10% SDS-polyacrylamide gels and electrotransferred onto polyvinylidene difluoride membranes, Immobilon-P (Millipore, Billerica,
MA). The membranes were probed with the monoclonal tetra-His antibody (Qiagen, Valencia, at ASPET Journals on May 31, 2021
CA) and then with an IRDye 680LT goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE).
The band intensity was quantified using the Odyssey infrared imaging system (LI-COR
Biosciences, Lincoln, NE).
Statistical Analysis. Correlation analysis was determined by the Spearman rank correlation method and p values lower than 0.05 were considered statistically significant.
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Results
UGT2B10 substrates. In order to expand the knowledge of the importance of UGT2B10 in drug glucuronidation we have tested its activity toward drugs that contain different amine structures.
Analysis of previous studies implied a preference of UGT2B10 for tertiary amines and substrate specificity overlaps with UGT1A4 and UGT1A3 (Kaivosaari et al., 2007, 2008; Chen et al., Downloaded from 2007, 2008). Therefore, we first chose to examine amitriptyline and imipramine, tricyclic antidepressants that were previously reported to be substrates of UGT1A4 and UGT1A3 dmd.aspetjournals.org (Breyer-Pfaff et al., 1997; Nakajima et al., 2002). We also selected other tricyclic compounds for the current analyses, namely ketotifen, pizotifen, olanzapine, trifluoperazine, desipramine,
nortriptyline, and carbamazepine, of which the former four compounds contain tertiary amines. at ASPET Journals on May 31, 2021
Ketotifen, olanzapine, and trifluoperazine were reported to be substrates of UGT1A4
(Breyer-Pfaff et al., 2000; Linnet, 2002), but there is no information about which UGT enzyme(s) could catalyze pizotifen glucuronidation. Desipramine and nortriptyline carry secondary amines and were previously reported not to be glucuronidated by UGT1A4 (Green and Tephly, 1996). Carbamazepine, a substrate of UGT2B7, contains a primary amine (Stains et al., 2004). Afloqualone, diphenhydramine, tamoxifen, ketoconazole, and midazolam were also examined in this study for glucuronidation by UGT2B10 because these drugs have amine structures and were reported to be substrates for UGT1A4 (Kaji and Kume, 2005; Green and
Tephly, 1996; Kaku et al., 2004; Bourcier et al., 2010; Klieber et al., 2008).
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The results revealed that UGT2B10 catalyzes the N-glucuronidations of amitriptyline, imipramine, ketotifen, pizotifen, olanzapine, diphenhydramine, tamoxifen, ketoconazole, and midazolam. The structures of these compounds are shown in Fig. 1, along with their conjugation site (indicated by solid black arrows). The tested compounds that were not glucuronidated by
UGT2B10 are also shown in Fig. 1. The conjugation sites in the non-UGT2B10 substrates, and
the human UGT(s) that conjugates then, are indicated by open white arrows . These results Downloaded from suggest that UGT2B10 has a clear preference for tertiary amines over secondary or primary
amines. The negative results with trifluoperazine, however, indicate that not every drug with a dmd.aspetjournals.org tertiary amine is a good substrate for UGT2B10 (Fig. 1).
The nine compounds that were found to be substrates for UGT2B10 were subsequently at ASPET Journals on May 31, 2021 subjected to activity assays with UGT1A3 and UGT1A4. The substrate concentrations in these experiments are listed in Table 1 and the obtained peak areas are shown in Fig. 2; peak areas are shown because authentic glucuronide standards were not available. In all these examinations,
UGT1A4 exhibited the highest activity, whereas the relative activity of UGT1A3 and UGT2B10 varied between the compounds. It should be noted that the comparison of the rate values of the different enzymes without accounting for their relative expression levels might be misleading.
Therefore, for the detailed enzyme kinetic studies of the glucuronidation of amitriptyline, imipramine, and diphenhydramine, the relative expression levels of UGT1A3, UGT1A4, and
UGT2B10 were determined and used in the calculations.
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Enzyme kinetics of N-Glucuronidation of Amitriptyline, Imipramine, and Diphenhydramine by recombinant UGT1A3, UGT1A4, and UGT2B10. We compared the affinities and clearance values of UGT1A3, UGT1A4, and UGT2B10 for the glucuronidation of amitriptyline, imipramine, and diphenhydramine. To normalize the activity with UGT protein expression levels, we performed immunoblot analyses of the recombinant UGTs using a monoclonal antibody toward
the C-terminal histidine tag that these recombinant enzymes carry (Fig. 3A). The expression level Downloaded from of UGT2B10, per mg protein, was taken as 1.0 and the relative expression levels of UGT1A4 and
UGT1A3 were calculated to be 9.4 and 34.3, respectively. Using these relative expression values dmd.aspetjournals.org to normalize activity, the enzyme kinetics of the three UGT enzymes for the glucuronidation of amitriptyline, imipramine and diphenhydramine were examined (Figs. 3B-D). For the three tested at ASPET Journals on May 31, 2021 substrates, UGT1A4 exhibited the highest Vmax values, although relatively high substrate concentrations were required to reach high turnover rates by this enzyme. Amitriptyline and diphenhydramine N-glucuronidations by UGT1A4 followed Michaelis-Menten kinetics, while imipramine N-glucuronidation by UGT1A4 fitted best to the Hill equation. In the case of
UGT1A3, amitriptyline and imipramine N-glucuronidations fitted best to the Hill equation, whereas diphenhydramine N-glucuronidation by UGT1A3 was barely detectable and the activity was too low to calculate any kinetic parameters.
In contrast to both UGT1A3 and UGT1A4, UGT2B10 exhibited substrate inhibition kinetics for all the three substrate, while the diphenhydramine N-glucuronidation was difficult to
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DMD #51565 fit. For this reason, the apparent kinetic parameters for diphenhydramine were estimated only from the results with up to 100 µM, a part of the V vs. S curve that fitted best to the
Michaelis-Menten equation. A clear outcome of the kinetic assays was that at substrate concentrations below 100 µM, the glucuronidation rates of UGT2B10 were the highest of the three tested enzymes for all the tested substrates. This result is mainly due to the prominently
lower Km values and the higher clearance values of UGT2B10, in comparison to both UGT1A4 Downloaded from and UGT1A3, in the glucuronidation of these three substrates (Table 3). dmd.aspetjournals.org Kinetics of Amitriptyline, Imipramine, and Diphenhydramine Glucuronidation by HLM.
The results of the kinetic analyses of amitriptyline, imipramine, and diphenhydramine
glucuronidation by HLM are shown in Fig. 4. For all three of the tested substrates, the at ASPET Journals on May 31, 2021 glucuronidation kinetics fitted best to the two-enzyme Michaelis-Menten equation. The
Eadie-Hofstee plots were biphasic, supporting previous reports for imipramine (Nakajima et al.,
2002) and diphenhydramine (Breyer-Pfaff et al., 1997). The Km values for the high affinity
component of these multiple enzyme reactions were close to the Km values that we have
determined for UGT2B10, whereas the Km values for the low affinity component were close to
the Km values of UGT1A4.
Inhibition Studies. To confirm the contribution of UGT2B10 and UGT1A4 to the glucuronidation reactions in HLM at low and high substrate concentrations, respectively, we performed inhibition studies (Fig. 5). Using recombinant UGT1A3, UGT1A4, and UGT2B10,
16 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565 we first confirmed that 10 µM hecogenin selectively inhibited UGT1A4, whereas 500 µM nicotine selectively inhibited UGT2B10 (Fig. 5). Under these assay conditions, we investigated the effects of the two inhibitors on amitriptyline, imipramine, and diphenhydramine glucuronidation by HLM at low and high substrate concentrations. The results demonstrated that hecogenin inhibited the activities by HLM only at high substrate concentrations, whereas
nicotine inhibited the activities by HLM only at low substrate concentrations (Fig. 5). These Downloaded from results strongly support the conclusion from the kinetic analyses that UGT2B10 and UGT1A4
are responsible for the high and low affinity components in the HLM-catalyzed reactions, dmd.aspetjournals.org respectively.
Correlation Analysis of N-Glucuronidation using a panel of 15 HLM. To verify the at ASPET Journals on May 31, 2021 significance of UGT2B10 and UGT1A4 in amitriptyline, imipramine, and diphenhydramine
N-glucuronidations by HLM at low and high substrate concentrations, we performed a correlation analysis (Fig. 6). The glucuronidation of the three drugs by 15 individual HLM were determined at substrate concentrations of 20 and 1000 µM; these concentrations were selected
based on the Km or S50 values for the recombinant UGT2B10 and UGT1A4, respectively (Figs.
3B-D and Table 3). The amitriptyline, imipramine, and diphenhydramine glucuronidation rates at the 20 µM substrate concentration varied from 14 to 51 pmol/min/mg, 36 to 150 pmol/min/mg, and 58 to 210 pmol/min/mg, showing 3.6, 4.2, and 3.6-fold variability, respectively. These three glucuronidation rates were significantly (p < 0.001) correlated with each other (r = 0.775, 0.964,
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DMD #51565 and 0.779). The activity rates at the 1000 µM substrate concentration ranged from 110 to 340 pmol/min/mg, 220 to 800 pmol/min/mg, and 580 to 2400 pmol/min/mg, showing 3.1, 3.6, and
4.1-fold variability, respectively. These activities were also significantly (p < 0.001) correlated with each other (r = 0.983, 0.946, and 0.911). Interestingly, however, for a given substrate, the correlation between the activities at 20 µM and 1000 µM substrate concentration was weak,
suggesting that the enzymes responsible for the activities were different. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021
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Discussion
In this study, we sought to expand the knowledge of the substrate specificity and clinical significance of UGT2B10. Starting with aromatic amines, we found that UGT2B10 has the ability to catalyze the N-glucuronidations of amitriptyline, imipramine, ketotifen, pizotifen, olanzapine, diphenhydramine, tamoxifen, ketoconazole, and midazolam. These drugs, except Downloaded from pizotifen for which there is no report, were previously reported as substrates of other human
UGTs, UGT1A4 and/or UGT1A3. Amitriptyline and imipramine were shown by Zhou et al., in dmd.aspetjournals.org 2010, also to be substrates of UGT2B10, but their work was unpublished and unknown to us at the start of the current study. A common property among these UGT2B10 substrates is the
presence of a tertiary amine. UGT2B10 did not catalyze the N-glucuronidations of at ASPET Journals on May 31, 2021 carbamazepine, afloqualone, desipramine and nortriptyline, all of which have a primary or secondary amine. Thus, it appears that, while UGT1A4 can metabolize primary and secondary amines (Green and Tephly, 1996; Kaji and Kume, 2005) in addition to tertiary amines, UGT2B10 exhibits a clear preference for tertiary amines. However, the substrate preference of UGT2B10 should presently be regarded as a hypothesis that could be accepted or rejected following studies with yet-untested compounds.
Ketoconazole and midazolam are N-glucuronidated at a tertiary amine within an imidazole ring. The ability of UGT2B10 to conjugate a tertiary amine within an imidazole ring was previously shown for medetomidine (Kaivosaari et al., 2008). It is interesting that UGT2B10 can
19 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565 efficiently form a glucuronide at the tertiary amine within the imidazole ring of R-medetomidine, while this enzyme can form two glucuronides in the imidazole ring of S-medetomidine. These reactions include both glucuronidation on a tertiary amine and glucuronidation on a secondary amine. However, these reactions occur at relatively low rates (Kaivosaari et al., 2008). In addition, it was unexpected that UGT2B10 does not glucuronidate trifluoperazine, that has a tertiary cyclic
amine and is a typical substrate of UGT1A4. Taken together, our findings may suggest that the Downloaded from length of side chains, and the configuration and spatial orientation of the compound in the active
site of the enzyme are also important determinants of UGT2B10 glucuronidation in addition to dmd.aspetjournals.org the chemistry of the amine.
As for carbamazepine and afloqualone toward which recombinant UGT2B10 showed no at ASPET Journals on May 31, 2021 activity, we could detect their glucuronides when HLM was used as an enzyme source, revealing the validity of our assays. However, in the cases of desipramine and nortriptyline, we could not detect N-glucuronides even with HLM as the enzyme source. For these compounds, there is currently no report of detected glucuronide in either human plasma or urine. Therefore, it is feasible that desipramine and nortriptyline do not undergo glucuronidation in the human body, or the formed glucuronides are highly unstable.
For the amitriptyline, imipramine, and diphenhydramine N-glucuronidations, the Km values
of UGT2B10 were close to those of the high affinity component in HLM, whereas the Km values of UGT1A4 were close to those of low affinity component (Tables 2 and 3). Zhou et al (2010)
20 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565 reported similar results for amitriptyline and imipramine N-glucuronidations by UGT2B10 and
UGT1A4. However, the Vmax and clearance values of UGT2B10 and UGT1A4 cannot be compared because the values in Zhou et al., 2010 were calculated as per mg protein, without normalization according to relative expression levels (that could not be done in the study by
Zhou et al., 2010). The ability to perform such normalization is an advantage of our study system
that employs His-tagged recombinant UGTs. Using glucuronidation rate normalization, we found Downloaded from that the clearances of amitriptyline, imipramine, and diphenhydramine by N-glucuronidations of
UGT2B10 were higher than those by UGT1A4 by 12-fold, 57-fold, and 2.9-fold, respectively. dmd.aspetjournals.org
The reported steady-state plasma concentrations of amitriptyline, imipramine and
diphenhydramine in patients receiving therapeutic doses were approximately 0.2, 0.6, and 0.2 at ASPET Journals on May 31, 2021
µM, respectively (Bailey and Jatlow, 1976a; Bailey and Jatlow, 1976b; Blyden et al., 1986).
Based on these plasma concentrations and the determined Km values, it appears that UGT2B10 is the major enzyme responsible for their glucuronidation in vivo. We have also tested whether or not UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7,
UGT2B15 and UGT2B17 have the ability to catalyze the glucuronidation of these drugs, but found no evidence for such an activity (data not shown).
UGT2B10 exhibited a prominently higher affinity, for the amitriptyline, imipramine, and diphenhydramine N-glucuronidations, than either UGT1A4 or UGT1A3. This observation is in line with previous studies demonstrating that UGT2B10 has a higher affinity than UGT1A4 for
21 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565 all the substrates that both enzymes glucuronidate, namely nicotine (Kaivosaari et al., 2007), clomipramine (Zhou et al., 2010), trimipramine (Zhou et al., 2010), medetomidine (Kaivosaari et al., 2008), and tobacco-specific nitrosamines (Chen et al., 2008). It is expected that this phenomenon will be observed in the other UGT2B10 substrates that were identified in this study, such as ketotifen, pizotifen, olanzapine, tamoxifen, ketoconazole, midazolam, and in UGT2B10
substrates that will be identified in the future. Downloaded from
Previously, we determined the expression levels of all the UGT genes that are expressed in dmd.aspetjournals.org the human liver (Izukawa et al., 2009) and found that the mRNA of UGT2B10 (19.6 ± 13.4% of all UGTs) was more abundant than the mRNA of UGT1A4 (5.5 ± 3.2%) and UGT1A3 (1.4 ±
1.4%). Although we have to consider the possibility of a poor correlation between mRNA levels at ASPET Journals on May 31, 2021 and protein expression in UGTs (Izukawa et al., 2009; Oda et al., 2012), the quantitative mRNA data appear to support the significance of UGT2B10 in N-glucuronidations in human livers. In a panel of 25 human liver samples, a huge interindividual variability (506-fold) was observed in the levels of UGT2B10 mRNA (Izukawa et al., 2009). In contrast, the present study showed that the interindividual variability in the amitriptyline, imipramine, and diphenhydramine
N-glucuronidations was, at most, 4-fold. This discrepancy might partly be due to the amplifying characteristic of the real-time PCR method. In addition, the possibility of post-transcriptional regulation cannot be ruled out at this stage.
In conclusion, we found that UGT2B10 has the ability to metabolize compounds having
22 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565 tertiary aliphatic amines, cyclic amines and tertiary amine within an imidazole ring. For amitriptyline, imipramine, and diphenhydramine N-glucuronidations, UGT2B10 exhibited a higher affinity and a higher clearance than UGT1A4. The current results significantly expand the knowledge of UGT2B10 and its substrate specificity. In addition, they demonstrate that
UGT2B10 should be ranked highly as an enzyme catalyzing the N-glucuronidation of clinically
important drugs. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021
23 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565
Authorship Contributions
Participated in research design: Kato, Izukawa, Fukami, Yokoi, and Nakajima
Conducted experiments: Kato, Izukawa, and Oda
Contributed new reagents or analytic tools: None
Performed data analysis: Kato, Izukawa, and Nakajima
Wrote or contributed to the writing of the manuscript: Kato, Izukawa, Finel, Yokoi, and Nakajima Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021
24 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565
References
Bailey DN and Jatlow PI (1976a) Gas-chromatographic analysis for therapeutic concentrations
of amitriptyline and nortriptyline in plasma, with use of a nitrogen detector. Clin Chem
22:777-781.
Bailey DN and Jatlow PI (1976b) Gas-chromatographic analysis for therapeutic concentration of
imipramine and desipramine in plasma, with use of a nitrogen detector. Clin Chem Downloaded from
22:1697-1701.
Blyden GT, Greenblatt DJ, Scavone JM, and Shader RI (1986) Pharmacokinetics of dmd.aspetjournals.org
diphenhydramine and a demethylated metabolite following intravenous and oral
administration. J Clin Pharmacol 26:529-533. at ASPET Journals on May 31, 2021
Bourcier K, Hyland R, Kempshall S, Jones R, Maximilien J, Irvine N, and Jones B (2010)
Investigation into UDP-glucuronosyltransferase (UGT) enzyme kinetics of imidazole-
and triazole-containing antifungal drugs in human liver microsomes and recombinant
UGT enzymes. Drug Metab Dispos 38:923-929.
Bowalgaha K, Elliot DJ, Mackenzie PI, Knights KM, Swedmark S, and Miners JO (2005)
S-Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and
recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the
elimination of naproxen. Br J Clin Pharmacol 60:423-433.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities
25 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565
of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
Breyer-Pfaff U, Fischer D, and Winne D (1997) Biphasic kinetics of quaternary ammonium
glucuronide formation from amitriptyline and diphenhydramine in human liver
microsomes. Drug Metab Dispos 25:340-345.
Breyer-Pfaff U, Mey U, Green MD, and Tephly TR (2000) Comparative N-glucuronidation
kinetics of ketotifen and amitriptyline by expressed human Downloaded from
UDP-glucuronosyltransferases and liver microsomes. Drug Metab Dispos 28:869-872.
Chen G, Blevins-Primeau AS, Dellinger RW, Muscat JE, and Lazarus P (2007) Glucuronidation dmd.aspetjournals.org
of nicotine and cotinine by UGT2B10: loss of function by the UGT2B10 codon 67
(Asp>Tyr) polymorphism. Cancer Res 67:9024-9029. at ASPET Journals on May 31, 2021
Chen G, Dellinger RW, Sun, Spratt TE, and Lazarus P (2008) Glucuronidation of
tobacco-specific nitrosamines by UGT2B10. Drug Metab Dispos 36:824-830.
Chiu SH and Huskey SW (1998) Species differences in N-glucuronidation. Drug Metab Dispos
26:838-847.
Erickson-Ridout KK, Zhu J, and Lazarus P (2011) Olanzapine metabolism and the significance
of UGT1A448v and UGT2B1067Y variants. Pharmacogenet Genomics 21:539-551.
Fujiwara R, Nakajima M, Yamanaka H, Katoh M, and Yokoi T (2007) Interactions between
human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities. Drug Metab
Dispos 35:1781-1787.
26 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565
Green MD, King CD, Mojarrabi B, Mackenzie PI, and Tephly TR (1998) Glucuronidation of
amines and other xenobiotics catalyzed by expressed human
UDP-glucuronosyltransferase 1A3. Drug Metab Dispos 26:507-512.
Green MD and Tephly TR (1996) Glucuronidation of amines and hydroxylated xenobiotics and
endobiotics catalyzed by expressed human UGT1.4 protein. Drug Metab Dispos
24:356-363. Downloaded from
Green MD and Tephly TR (1998) Glucuronidation of amine substrates by purified and expressed
UDP-glucuronosyltransferase proteins. Drug Metab Dispos 26:860-867. dmd.aspetjournals.org
Hawes EM (1998) N+-glucuronidation, a common pathway in human metabolism of drugs with a
tertiary amine group. Drug Metab Dispos 26:830-837. at ASPET Journals on May 31, 2021
Houston JB and Kenworthy KE (2000) In vitro-in vivo scaling of CYP kinetic data not consistent
with the classical Michaelis-Menten model. Drug Metab Dispos 28:246-254.
Izukawa T, Nakajima M, Fujiwara R, Yamanaka H, Fukami T, Takamiya M, Aoki Y, Ikushiro S,
Sakaki T, and Yokoi T (2009) Quantitative analysis of UDP-glucuronosyltransferase
(UGT) 1A and UGT2B expression levels in human livers. Drug Metab Dispos
37:1759-1768.
Jin CJ, Miners JO, Lillywhite KJ, and Mackenzie PI (1993) cDNA cloning and expression of two
new members of the human liver UDP-glucuronosyltransferase 2B subfamily. Biochem
Biophys Res Commun 194:496-503.
27 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565
Kaivosaari S, Finel M, and Koskinen M (2011) N-glucuronidation of drugs and other xenobiotics
by human and animal UDP-glucuronosyltransferases. Xenobiotica 41:652-669.
Kaivosaari S, Toivonen P, Aitio O, Sipilä J, Koskinen M, Salonen JS, and Finel M (2008) Regio-
and stereospecific N-glucuronidation of medetomidine: the differences between UDP
glucuronosyltransferase (UGT) 1A4 and UGT2B10 account for the complex kinetics of
human liver microsomes. Drug Metab Dispos 36:1529-1537. Downloaded from
Kaivosaari S, Toivonen P, Hesse LM, Koskinen M, Court MH, and Finel M (2007) Nicotine
glucuronidation and the human UDP-glucuronosyltransferase UGT2B10. Mol Pharmacol dmd.aspetjournals.org
72:761-768.
Kaji H and Kume T (2005) Characterization of afloqualone N-glucuronidation: species differences at ASPET Journals on May 31, 2021
and identification of human UDP-glucuronosyltransferase isoform(s). Drug Metab Dispos
33:60-67.
Kaku T, Ogura K, Nishiyama T, Ohnuma T, Muro K, and Hiratsuka A (2004) Quaternary
ammonium-linked glucuronidation of tamoxifen by human liver microsomes and
UDP-glucuronosyltransferase 1A4. Biochem Pharmacol 67:2093-2102.
Klieber S, Hugla S, Ngo R, Arabeyre-Fabre C, Meunier V, Sadoun F, Fedeli O, Rival M, Bourrie
M, Guillou F, Maurel P, and Fabre G (2008) Contribution of the N-glucuronidation
pathway to the overall in vitro metabolic clearance of midazolam in humans. Drug Metab
Dispos 36:851-862.
28 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565
Koga T, Fujiwara R, Nakajima M, and Yokoi T (2011) Toxicological evaluation of acyl
glucuronides of nonsteroidal anti-inflammatory drugs using human embryonic kidney
293 cells stably expressing human UDP-glucuronosyltransferase and human hepatocytes.
Drug Metab Dispos 39:54-60.
Kurkela M, Patana AS, Mackenzie PI, Court MH, Tate CG, Hirvonen J, Goldman A, and Finel M
(2007) Interactions with other human UDP-glucuronosyltransferases attenuate the Downloaded from
consequences of the Y485D mutation on the activity and substrate affinity of UGT1A6.
Pharmacogenet Genomics 17:115-126. dmd.aspetjournals.org
Linnet K (2002) Glucuronidation of olanzapine by cDNA-expressed human
UDP-glucuronosyltransferases and human liver microsomes. Hum Psychopharmacol at ASPET Journals on May 31, 2021
17:233-238.
Nakajima M, Tanaka E, Kobayashi T, Ohashi N, Kume T, and Yokoi T (2002) Imipramine
N-glucuronidation in human liver microsomes: biphasic kinetics and characterization of
UDP-glucuronosyltransferase isoforms. Drug Metab Dispos 30:636-642.
Oda S, Nakajima M, Hatakeyama M, Fukami T, and Yokoi T (2012) Preparation of a specific
monoclonal antibody against human UDP-glucuronosyltransferase (UGT) 1A9 and
evaluation of UGT1A9 protein levels in human tissues. Drug Metab Dispos
40:1620-1627.
Staines AG, Coughtrie MW, and Burchell B (2004) N-glucuronidation of carbamazepine in
29 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565
human tissues is mediated by UGT2B7. J Pharmacol Exp Ther 311:1131-1137.
Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism,
expression, and disease. Annu Rev Pharmacol Toxicol 40:581-616.
Uchaipichat V, Mackenzie PI, Elliot DJ, and Miners JO (2006) Selectivity of substrate
(trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin,
phenylbutazone, quinidine, quinine, and sulfinpyrazone) “probes” for human Downloaded from
UDP-glucuronosyltransferases. Drug Metab Dispos 34:449-456.
Zhou D, Guo J, Linnenbach AJ, Booth-Genthe CL, and Grimm SW (2010) Role of human dmd.aspetjournals.org
UGT2B10 in N-glucuronidation of tricyclic antidepressants, amitriptyline, imipramine,
clomipramine, and trimipramine. Drug Metab Dispos 38: 863-870. at ASPET Journals on May 31, 2021
30 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
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Figure legends
Fig. 1. Chemical structures of drugs containing an amine used for the screening of UGT2B10 substrates. Arrows indicate the position of N-glucuronidation. The UGT enzymes, which were previously reported to have this activity, are shown. The compounds with solid arrows were glucuronidated by UGT2B10, whereas the compounds with open arrows were not glucuronidated Downloaded from by UGT2B10.
Fig. 2. Comparison of the N-glucuronidation of drugs by UGT2B10 with that by UGT1A3 and dmd.aspetjournals.org
UGT1A4. Recombinant UGT1A3, UGT1A4, and UGT2B10 (0.5 mg/ml) were incubated with various compounds and UDPGA for 120 min. The substrate concentrations are shown in Table 1. at ASPET Journals on May 31, 2021 Peak area values of the formed N-glucuronide from the LC-MS/MS analysis were used as the indices of activity. Each column represents the mean of duplicate determinations.
Fig. 3. Amitriptyline, imipramine, and diphenhydramine N-glucuronidations by recombinant
UGT1A3, UGT1A4, and UGT2B10. (A) Immunoblot analyses of the recombinant UGT1A3,
UGT1A4, and UGT2B10 using anti-tetra-His antibody. The relative expression levels of
UGT2B10, UGT1A4, and UGT1A3 were calculated as 1, 9.4, and 34.3, respectively, using the calibration curves. Each point represents the mean of duplicate determinations. Kinetics of amitriptyline (B), imipramine (C), and diphenhydramine (D) N-glucuronidation by UGT1A3,
UGT1A4, and UGT2B10 using the normalized activities. Data are mean ± SD of three
31 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #51565 independent determinations.
Fig. 4. Kinetics of amitriptyline (A), imipramine (B), and diphenhydramine (C)
N-glucuronidation in HLM. An Eadie-Hofstee plot is shown in the inset. Pooled HLM (0.5 mg/ml) were incubated with 10-1000 μM substrate and 5 mM UDPGA at 37°C for 120 min.
Data are mean ± SD of three independent determinations. Downloaded from
Fig. 5. Effects of hecogenin and nicotine on the amitriptyline (A), imipramine (B), and diphenhydramine (C) N-glucuronidations by recombinant UGTs and HLM. (A) The amitriptyline dmd.aspetjournals.org concentrations for UGT1A3, UGT1A4, UGT2B10, high and low affinity components in HLM were 600 µM, 600 µM, 10 µM, 20 µM, and 400 µM, respectively. (B) The imipramine at ASPET Journals on May 31, 2021 concentrations for UGT1A3, UGT1A4, UGT2B10, high and low affinity components in HLM were 600 µM, 400 µM, 100 µM, 50 µM, and 400 µM, respectively. (C) The diphenhydramine concentrations for UGT1A4 and UGT2B10, high and low affinity components in HLM were 600
µM, 60 µM, 20 µM, and 500 µM, respectively. Data are mean ± SD of three independent determinations.
Fig. 6. Correlation coefficients between the amitriptyline, imipramine, and diphenhydramine
N-glucuronidation in a panel of 15 HLM. The correlation analyses were performed using the
Spearman rank correlation method. The Spearman correlation coefficients (r) are shown. *p <
0.05; **p < 0.01; and ***p < 0.005.
32 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
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Table 1. Compound concentrations optimized for each compound and the mass/charge (m/z) values to detect the glucuronide in LC-MS/MS analyses. Incubation conditions Transitions Compounds Compound concn. (µM) Q1-Q3 (m/z) Tricyclic amines Amitriptyline 100 454-278 Imipramine 100 457-281 Ketotifen 100 485-309 Pizotifen 100 473-296 Olanzapine 500 489-313 Trifluoperazine 50 585-409
Desipramine 100 443 -266 Downloaded from Nortriptyline 100 440-264 Carbamazepine 100 413-237 Other amines
Afloqualone 1000 461-284 dmd.aspetjournals.org Diphenhydramine 100 432-256 Tamoxifen 100 548-372 Ketoconazole 10 709-532 Midazolam 10 502-326 at ASPET Journals on May 31, 2021
33 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version.
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Table 2. The mobile phases and the UV wavelengths in this study.
Compounds Mobile phase UV wavelength Amitriptyline 30% acetonitrile/20 mM ammonium dihydrogen phosphate 214 nm Imipramine 25% acetonitrile/50 mM ammonium dihydrogen phosphate 214 nm
Diphenhydramine 19% acetonitrile/10 mM ammonium acetate 220 nm
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021
34 DMD #51565 Table 3. Kinetic parameters for amitriptyline, imipramine, and diphenhydramine N-glucuronidation by recombinant His-tagged UGT1A3, UGT1A4, and UGT2B10. N.A., not applicable.
Km or S50 Vmax Normalized Vma CLint or CLmax Ki n Kinetic model This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. (µM) (pmol/min/mg) (pmol/min/mg) (nL/min/mg) (µM)
Amitriptyline DMD FastForward.PublishedonApril23,2013asDOI:10.1124/dmd.113.051565 UGT1A3 490 ± 42 740 ± 71 22 ± 2.1 23 ± 3.8 2.3 ± 0.4 Hill UGT1A4 450 ± 72 530 ± 120 56 ± 13 130 ± 370 Michaelis-Menten UGT2B10 7.5 ± 5.1 7.5 ± 2.0 7.5 ± 2.0 1600 ± 1500 130 ± 28 Substrate inhibition Imipramine UGT1A3 760 ± 110 24 ± 5.6 0.7 ± 0.2 0.6 ± 0.1 1.2 ± 0.0 Hill UGT1A4 500 ± 55 55 ± 2.1 5.8 ± 0.2 8.2 ± 0.7 1.1 ± 0.0 Hill UGT2B10 7.2 ± 5.5 2.5 ± 0.3 2.5 ± 0.3 470 ± 260 380 ± 250 Substrate inhibition Diphenhydramine UGT1A3 N.A. N.A. N.A. N.A. N.A UGT1A4 470 ± 26 150 ± 8.2 16 ± 0.9 34 ± 1.4 Michaelis-Menten UGT2B10 37 ± 13 3.6 ± 1.0 3.6 ± 1.0 99 ± 8.7 Michaelis-Menten
35
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Table 4. Kinetic parameters for amitriptyline, imipramine, and diphenhydramine N-glucuronidations in HLM.
High affinity Low affinity
Km1 Vmax1 Km2 Vmax2 (µM) (pmol/min/mg) (µM) (pmol/min/mg) Amitriptyline 3.6 ± 1.1 270 ± 76 390 ± 2.0 2000 ± 430 Imipramine 9.3 ± 2.7 270 ± 14 370 ± 120 1200 ± 12 Diphenhydramine 9.2 ± 3.1 210 ± 110 310 ± 120 1100 ± 190
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021
36 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021 DMD Fast Forward. Published on April 23, 2013 as DOI: 10.1124/dmd.113.051565 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 31, 2021