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Epirubicin Glucuronidation and UGT2B7 Developmental Expression

Pfizer, Kalamazoo, Michigan (M.J.Z.) and St. Louis, Missouri (J.C.S.); and the Department of Pediatrics, Medical College of Wisconsin and Children's Research Institute, Children's Hospital and Health System, Milwaukee, Wisconsin (R.N.H.)

(Received June 7, 2006; accepted September 15, 2006)

	Abstract

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The usefulness of epirubicin in the treatment of adult and childhood malignant diseases is related in part to the potential reduction in cardiac toxicity compared with that of other anthracyclines given at equivalent doses. An important pathway for epirubicin detoxification is UGT2B7-dependent glucuronidation. This study was implemented to provide a preclinical evaluation of the metabolism of epirubicin with respect to age-related changes in epirubicin glucuronidation in pediatric liver microsomes. Rates of epirubicin glucuronidation and levels of UGT2B7 were determined for liver microsomes from four pediatric age categories (n = 32) and one adult age category (n = 8). Both sets of data showed an increase in UGT2B7 activity and content with increasing age. Epirubicin glucuronidation activity in the adult group was statistically higher compared with all pediatric age groups (p 0.01). UGT2B7 expression also was statistically higher in adults compared with children below 11 years of age, with evidence of significant differences in protein levels among the pediatric age categories. A positive correlation (r = 0.68) between UGT2B7 levels and postnatal age was observed, suggesting a progressive increase in UGT2B7 protein expression with increasing age. However, allometric scaling using the power rule suggested no difference in activity between any of the pediatric age categories and the adult, although only a single neonatal sample was included in the analysis. In summary, these in vitro data show differences in epirubicin glucuronidation and UGT2B7 content within pediatric age groups and support the use of epirubicin in pediatric patients at least 6 months of age.

Epirubicin and doxorubicin are structurally highly similar, differing only in the orientation of the 4'-hydroxy group. However, this minor difference leads to significant differences in the pharmacokinetics and metabolism of these drugs. Epirubicin is extensively metabolized by the liver to epirubicin glucuronide (Fig. 1) and epirubicinol, with subsequent biliary excretion of the glucuronide conjugate (approximately 25% of dose) (Weenen et al., 1983, 1984). Using adult human liver microsomes and heterologous enzyme expression systems, this route of epirubicin inactivation was shown by Innocenti et al. (2001) to be catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). Doxorubicin does not share this important detoxification pathway, thus suggesting that epirubicin represents an effective anthracycline possessing a favorable pharmacokinetic profile and fewer toxicological effects (Launchbury and Habboubi, 1993; Robert, 1993). Clinical pharmacokinetic studies have demonstrated an up to 10-fold interpatient variability in epirubicin glucuronidation linked with metabolic clearance, and in vitro metabolism studies have provided evidence that interindividual variability in rates of glucuronidation seems related to variability in UGT2B7 protein levels. For example, the pharmacokinetics of morphine glucuronidation suggest both age-dependent differences in UGT2B7 activity and significant increases in enzyme expression and/or activity at birth. Adjusting for liver size relative to body weight, adult levels of UGT2B7 are reached by 2 to 6 months of age (reviewed in de Wildt et al., 1999). Thus, the developmental pattern for this UGT isoform may provide valuable clinical information in the assessment of epirubicin pediatric pharmacology.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Structural formula of epirubicin and epirubicin glucuronide.

	Materials and Methods

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Chemical and Reagents. Primary antisera containing an anti-peptide antibody against UGT2B7 and horseradish peroxidase-conjugated goat anti-rabbit antibody were obtained from BD Biosciences (Bedford, MA). Ten percent polyacrylamide Tris-HCl (1.0 mm Criterion) resolving gels and polyvinylidene difluoride transfer membranes (Immun-Blot) were obtained from Bio-Rad Laboratories (Hercules, CA). Premixed 10x Tris/glycine/SDS running buffer, 10x Tris/glycine transfer buffer, and Tween 20 also were obtained from Bio-Rad Laboratories. Sample buffers and reducing agents (NuPAGE) were obtained from Invitrogen (Carlsbad, CA). Enhanced chemiluminescence (ECL) blotting reagents and detection film (Hyperfilm ECL) were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Tris-buffered saline buffer (BupH Tris-buffered saline) was purchased from Pierce Chemical Company (Rockford, IL). Epirubicin HCl and alamethicin were obtained from Pharmacia (Nerviano, Italy and Kalamazoo, MI, respectively). Daunorubicin, ß-glucuronidase, and UDP-glucuronic acid (UDPGA) were obtained from Sigma-Aldrich (St. Louis, MO). All other reagents and materials were obtained from common commercial sources at the highest grade available.

Microsomes. Pediatric liver samples were obtained and microsome fractions prepared as described previously (Koukouritaki et al., 2002). The age categories for the donor liver microsome samples are given in Table 1. Pooled human liver microsomes were obtained from Xenotech LLC (Kansas City, KS) and individual adult liver microsomes were obtained from the Pfizer/Pharmacia liver microsome bank. Microsomes from insect cells infected with baculovirus expressing recombinant UGT2B7 were obtained from BD Biosciences Discovery Labware (Bedford, MA).

Epirubicin Glucuronidation Assay. Kinetic reactions with expressed UGT2B7 and pooled human liver microsomes were performed using a 100-µl reaction mixture containing 0.5 mg/ml microsomal protein, 5.0 mM UDPGA, and 25 to 1200 µM epirubicin in 50 mM Tris-HCl/8 mM MgCl₂ (pH 7.5) buffer containing alamethicin (50 µg/mg protein) and incubated in a shaking water bath at 37°C for 15 min. Reactions with pediatric and adult liver microsomes were performed using 500 µM epirubicin, 0.2 mg/ml protein, and 30-min incubation times under the same conditions described above. Chilled samples were preincubated at 37°C for 3 min and all reactions were initiated with the addition of 5 mM UDPGA to the reaction mixture. The reaction was terminated by the addition of 25 µl of ice-cold acetonitrile/0.03% phosphoric acid containing 100 µM daunorubicin as the internal standard and centrifuged at 10,000g for 3 min. A 50-µl aliquot of the supernatant was used for high-performance liquid chromatography analysis. Preliminary experiments showed the glucuronidation assay was linear under conditions of a 30-min incubation and 0.5 mg of microsomal protein.

Epirubicin, epirubicin glucuronide, and daunorubicin were detected using a PerkinElmer Series 200 high-performance liquid chromatography system (PerkinElmer, Wellesley, MA) with fluorescence detection at 480 (_ex) and 560 (_em) nm using a Waters 474 fluorescence scanning detector (Waters Corp., Milford, MA). Separation was accomplished at room temperature using a reversed phase Supelcosil LC-CN column (4.6 x 250 mm, 5 µm; Supelco Inc., Bellefonte, PA) preceded by a SecurityGuard phenyl guard cartridge (Phenomenex, Torrance, CA) and a mobile phase consisting of 30% acetonitrile and 70% 50 mM sodium dihydrogen phosphate (pH 4.0) at a flow rate of 0.8 ml/min. The quantitation of the epirubicin glucuronide metabolite was performed by comparing chromatographic peak heights to a standard curve of 0.5 to 50.0 nmol epirubicin/ml constructed, because of the unavailability of pure epirubicin glucuronide. The assumption that the fluorescence of epirubicin glucuronide was equal to that of epirubicin is based on the comparative fluorescence spectra and has been used in related studies (Barker et al., 1996). Identification of the epirubicin glucuronide chromatographic peak by enzymatic hydrolysis with ß-glucuronidase was performed by treatment of a human liver microsome incubation sample with 1000 IU of ß-glucuronidase (type VII, from Escherichia coli) in 0.1 M sodium phosphate buffer, pH 6.8 at 37°C overnight and analyzed as described above.

Electrophoresis and Immunoblotting. Proteins were separated by SDS-polyacrylamide gel electrophoresis using 10% acrylamide resolving gels and a Tris-glycine running buffer (Laemmli 1970). A 2- to 3-µg aliquot of microsomal protein from each sample was analyzed. Separation was carried out using the Criterion Precast Gel Electrophoresis System (Bio-Rad Laboratories) and proteins were transferred to polyvinylidene difluoride membranes using the Criterion blotter according to the manufacturer's instructions. Membranes were blocked with 5% nonfat dry milk (NFDM) in Tris-buffered saline (TBS; 25 mM Tris, 150 mM sodium chloride, pH 7.5). The transfer membranes were incubated for 1 h at room temperature with UGT2B7 primary antibody diluted 1:500 in TBS containing 1% NFDM and 0.05% Tween 20. The membranes were then washed several times with TBS containing 0.1% Tween 20 and incubated for 1 h at room temperature with the horseradish peroxidase-labeled secondary antibody diluted 1:500 in TBS containing 1% NFDM and 0.05% Tween 20. Membranes were then washed several times with TBS containing 0.1% Tween 20 and processed for UGT2B7 detection by enhanced chemiluminescence according to the manufacturer's instructions. The luminescence produced was detected by exposure to film and scanned using a Bio-Rad GS-710 calibrated imaging densitometer. Using a standard curve representing 0.01 to 0.25 µg of expressed human UGT2B7 distributed on each membrane, immunoreactive protein bands were quantified using linear regression (Quantity One software; Bio-Rad Laboratories).

Statistical Analysis. Data from rates of epirubicin glucuronidation and UGT2B7 protein levels were analyzed with the statistical analysis package Unistat V5.0 (Unistat, London UK). These data were analyzed by the Kruskal-Wallis nonparametric analysis of variance (Hollander and Wolfe, 1999). Multiple comparisons were made using the t distribution on the mean ranks. Correlation analysis was performed using GraphPad Prism V3.0 (GraphPad Software Inc., San Diego CA). Epirubicin glucuronidation activities that were normalized by scaling to a 70-kg individual using the power rule (Holford, 1996) were compared using one-way analysis of variance (SigmaStat V 3.11; SysStat Software, Point Richmond, CA). An value of p < 0.05 was accepted as significant.

	Results

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Kinetic Analysis of Epirubicin Glucuronidation. A description of incubation conditions and kinetic parameters for epirubicin glucuronidation has been reported (Innocenti et al., 2001); however, the limiting quantities of each sample for the current study necessitated incubations using substantially reduced concentrations of microsomal protein. In brief, under conditions of saturating substrate, less than 4% of the parent compound was consumed using lower protein concentrations (incubations of human liver microsome samples at 0.2 mg protein/ml or 20 µg of total protein and expressed UGT2B7 at 0.5 mg/ml or 50 µg of total protein) for the respective incubation durations. Kinetic evaluations of epirubicin glucuronidation were performed using the above conditions with increasing concentrations of substrate. The resulting activities were fit to a single enzyme model (r² = 0.9936) (data not shown). For a pooled adult human liver microsome sample, apparent K_m (K_m,app) and maximal reaction rate (V_max) were 213 µM and 3.56 nmol epirubicin glucuronide formed/min/mg protein. For expressed UGT2B7, the calculated K_m was 46 µM with a V_max of 0.86 nmol/min/mg protein.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Box and whisker plot analysis of in vitro epirubicin glucuronidation activity as a function of age. Activity levels (pmol epirubicin glucuronide formed/min/mg microsomal protein) were quantitated as described under Materials and Methods. The bottom and top of the boxes represent the 25th and 75th percentiles, respectively. The solid horizontal line within each box represents the median value. The horizontal dash within each box represents the mean value. The lower whisker is equal to the maximum of 1) lower quartile minus 1.5 times the interquartile range (IQR) and 2) the minimum observation within the IQR. The upper whisker is equal to the minimum of 1) the 75th percentile plus 1.5 times the IQR and 2) the maximum observation within the IQR.

Immunoquantitation of UGT2B7 Protein. Since previous studies have identified UGT2B7 as the primary catalyst of epirubicin glucuronidation, the ontogeny of UGT2B7 expression was investigated. Figure 3 shows that a 25-fold dynamic range of UGT2B7 immunoquantitation could be obtained using the expressed enzyme, since detection of the 0.005-µg sample was not reliable (Fig. 3, lanes 1–6). An immunoreactive protein of molecular weight similar to that of expressed UGT2B7 was observed for six of eight samples from the <1-year pediatric category (Fig. 3, lanes 8–15) and from all other human liver microsome samples analyzed (data not shown). Figure 4 shows the box and whisker plots for human liver microsome UGT2B7 protein levels by age category. The results follow a pattern similar to that observed for rates of epirubicin glucuronidation by age category in that a statistical difference was observed between the adult group compared with each of the pediatric groups except 12 to 17 years. The latter approached significance (p = 0.057). Average levels of UGT2B7 protein showed an age-dependent increase, and the differences in the pediatric age groups were significant for the 12- to 17-year age group compared with the <1- and 1- to 5-year age groups (p < 0.001). The 6- to 11-year age group also showed a statistical difference compared with the <1-year age (p < 0.001) and the 1- to 5-year group (p = 0.0086).

View larger version (9K): [in this window] [in a new window]   FIG. 3. Immunoblot analysis of UGT2B7 protein in human liver microsomes. Lanes 1 to 6 contain 0.005, 0.01, 0.025, 0.050, 0.10, and 0.25 µg, respectively, of expressed UGT2B7. Lane 7 contains UGT insect control microsomal protein, and lanes 8 to 15 include samples from the <1-year postnatal age group (3 µg of total protein per analysis). Lane 16 contains pooled human liver microsomes (3 µg). Electrophoresis and immunodetection using an antibody to UGT2B7 were conducted as described under Materials and Methods.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Box and whisker plot analysis of immunodetectable UGT2B7 determined for pediatric and adult liver microsomal samples. Protein levels (µg expressed UGT2B7/mg microsomal protein) were quantitated as described under Materials and Methods. The bottom and top of the boxes represent the 25th and 75th percentiles, respectively. The solid horizontal line within each box represents the median value. The horizontal dash within each box represents the mean value. The lower whisker is equal to the maximum of 1) lower quartile minus 1.5 times the IQR and 2) the minimum observation within the IQR. The upper whisker is equal to the minimum of 1) the 75th percentile plus 1.5 times the IQR and 2) the maximum observation within the IQR.

View larger version (13K): [in this window] [in a new window]   FIG. 5. Correlation between epirubicin glucuronidation and UGT2B7 protein content in adult and pediatric liver microsomes. The correlation coefficient (r = 0.86) was statistically significant (p < 0.0001; r2 = 0.74, n = 40).

View larger version (13K): [in this window] [in a new window]   FIG. 6. Correlation between epirubicin glucuronidation activity and UGT2B7 content and postnatal age. A, correlation between epirubicin glucuronidation activity and postnatal age (PNA) in pediatric liver microsomes (n = 32). Coefficient of determination (r2) = 0.13. The correlation coefficient (r) = 0.36, p = 0.047. B, correlation between UGT2B7 content and PNA in pediatric liver microsomes (n = 32). Coefficient of determination (r2) = 0.47. The correlation coefficient (r) = 0.68, p < 0.0001.

	Discussion

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The use of the anthracycline, doxorubicin, as a chemotherapeutic agent in children has generated considerable concern regarding long-term safety. The development of the less toxic anthracycline derivative, epirubicin, has resulted in a more widespread use of this class of agents for childhood cancer. However, a careful comparison between doxorubicin and epirubicin pharmacokinetics and pharmacodynamics suggests the decreased toxicity of the latter is due to its metabolism and clearance by UGT2B7. Thus, an assessment of the postnatal developmental expression pattern of human hepatic UGT2B7 and, more specifically, the possible changes in activity toward epirubicin, was considered important in the assessment of age-dependent pharmacokinetic changes and variability of this drug in the pediatric population.

The kinetic constants determined in the current study (K_m,app = 213 µM and V_max = 3.56 nmol/min/mg for pooled microsomes; K_m = 46 µM and V_max = 0.86 nmol/min/mg for expressed recombinant protein) are different from those previously reported (K_m values of 568 µM and 149 µM for human liver microsomes and expressed UGT2B7, respectively). These comparatively higher affinity constants may be attributable to the higher microsomal protein concentration used by these investigators (3 mg/ml microsomal protein). Although not measured experimentally in the current study, nonspecific microsomal protein binding is well documented for producing increased relative affinity parameters for in vitro studies (Obach, 1999; Kalvass et al., 2001). The 4-fold difference in apparent K_m values between human liver microsomes and the expressed microsomal UGT2B7 enzyme has been noted previously and may be attributable to 1) the involvement of an unidentified higher-affinity human liver UGT form in the glucuronidation of epirubicin, 2) the influence of incubation conditions or the lipid environment of the microsomal preparations (Remmel and Burchell, 1993; Fisher et al., 2000; Soars et al., 2003), or 3) alteration of UGT kinetics and activity due to oligomerization (Ikushiro et al., 1997; Meech and Mackenzie, 1997; Ishii et al., 2001).

An examination of epirubicin glucuronidation activity in pediatric liver microsomes demonstrated a median activity in tissue from individuals less than 1 year of age that was approximately 10% of that observed in tissue from adults. Between 1 year of age and puberty (11 years of age), median enzyme activity was 25% of that seen in adults, whereas between 12 and 17 years of age, the observed median activity was approximately 50% of the observed adult value. These relative values are corroborated by the immunological determination of UGT2B7 content, which correlated well with enzyme activity (r = 0.86). However, if one normalizes the pediatric data to a 70-kg individual using the power rule, no significant differences are observed among the different developmental age groups or between any of the pediatric age groups and adults (Table 3) (p = 0.138). These data are consistent with morphine clearance in pediatric patients by glucuronidation, a well documented marker of UGT2B7 activity (Coffman et al., 1997). Morphine clearance was 5-fold higher in children 1 to 16 years of age versus neonates, but clearance values matching adults were attained between 6 and 30 months when calculated using a per-kg size model (Choonara et al., 1989). When scaled using the power model, adult levels of morphine clearance were attained between 2 and 6 months of age (Anderson et al., 1997). In the current data set, only one of the tissue samples analyzed was from a neonate, only two were from individuals less than 2 months of age, and the median age of all samples less than 1 year of age was 4.8 months.

In summary, epirubicin glucuronidation activity and content clearly vary directly as a function of increasing age. However, when pediatric data were normalized using power allometric scaling, epirubicin glucuronidation activity in all pediatric age categories was not found to be statistically different from activities determined for the adult liver samples. These data are consistent with data on the ontogeny of morphine glucuronidation wherein adult activity values are attained by 2 to 6 months of age. Thus, the data presented in the current study would support the use of epirubicin as a cancer chemotherapeutic agent in pediatric patients older than 6 months of age.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.011387.

ABBREVIATIONS: IQR, interquartile range; NFDM, nonfat dry milk; PNA, postnatal age; TBS, Tris-buffered saline; UDPGA, uridine diphosphate glucuronic acid; UGT, UDP-glucuronosyl transferase.

Address correspondence to: Matthew J. Zaya, Pfizer, Inc., 7000 Portage Rd., Kalamazoo, MI 49001. E-mail: matthew.j.zaya{at}pfizer.com

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.011387v1 34/12/2097    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zaya, M. J. Articles by Stevens, J. C. Search for Related Content PubMed PubMed Citation Articles by Zaya, M. J. Articles by Stevens, J. C.