Characterization of the Major Bropirimine Oxidative Metabolites Formed In Vitro
Abstract
Bropirimine (2-amino-5-bromo-6-phenyl-4-pyrimidinone) is a member of a class of antineoplastic agents known as aryl pyrimidinones. In human liver microsomal incubations, bropirimine oxidative metabolism is characterized by the formation of three metabolites. Mass spectrometric analysis of the incubation mixture revealed three bropirimine oxidative metabolites, identified as the bropirimine dihydrodiol,p-hydroxybropirimine, and m-hydroxybropirimine.In vitro studies using human liver microsomes and recombinant cytochrome P450 isoforms were performed to identify the P450 enzyme(s) responsible for bropirimine oxidation. Coincubation with the selective CYP1A2 inhibitor alpha-naphthoflavone abolished bropirimine metabolism in human liver microsomes. Furthermore, when screened against a panel of cDNA expressed cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), bropirimine was metabolized to both p- and m-hydroxybropirimine exclusively in incubations with cDNA-expressed CYP1A2 microsomes. Mechanistic studies using cDNA-expressed CYP1A2 microsomes fortified with microsomal epoxide hydrolase revealed that all three bropirimine oxidative metabolites appear to be the result of a common arene oxide, which serves as a substrate for microsomal epoxide hydrolase to generate the dihydrodiol or rearranges to yield p- andm-hydroxybropirimine.
Bropirimine (2-amino-5-bromo-6-phenyl-4-pyrimidinone) is a member of a class of antineoplastic agents known as aryl pyrimidinones and is currently under clinical development for the treatment of bladder cancer (Sarosdy et al., 1992). In humans, clearance of bropirimine primarily occurs via phase-II conjugation with uridine diphosphate-glucuronic acid. In addition to phase-II metabolism, there appears to be a fraction of the drug that undergoes oxidative biotransformation, presumably by member(s) of the cytochrome P450 superfamily (Hauer et al., 1997). The purpose of the current study was to characterize the primary P4501-derived metabolites of bropirimine in human liver microsomal preparations and to identify the P450 enzyme(s) responsible for their formation. To accomplish this, three types of in vitro experiments were conducted to identify which human P450 enzyme(s) are responsible for metabolizing bropirimine: (1) determine the sample-to-sample variation of bropirimine oxidation by a bank of human liver microsomes, followed by an analysis of correlations with the sample-to-sample variation in the activity of the major P450 enzymes expressed in human liver microsomes; (2) an investigation of bropirimine metabolism by human liver microsomes in the presence of chemicals that inhibit specific P450 enzymes; and (3) an analysis of bropirimine metabolism by cDNA-expressed human P450 and microsomal epoxide hydrolase enzymes.
Methods
Chemicals.
Bropirimine specifically labeled with carbon-14 (fig.1), (specific activity 102.2 μCi/mg) and authentic phenolic metabolite standards forp- and m-hydroxybropirimine were obtained from Pharmacia & Upjohn (Kalamazoo, MI) (Skulnick et al., 1985). The radiochemical purity of [14C]bropirimine was found to be greater than 99% as determined by HPLC with radiochemical detection. Quinidine, p-nitrophenol, cysteine,N-acetyl-cysteine, cyclohexene oxide, ketoconazole, coumarin, sulfaphenazole, alpha-naphthoflavone, and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO). (S)-mephenytoin was a gift from Dr. W.F. Trager (Department of Medicinal Chemistry, University of Washington). All other reagents and solvents were of analytical grade.
Human Liver Microsomes and cDNA-Expressed Enzymes.
Human livers were acquired from the Arizona Organ Bank (Phoenix, AZ) and the International Institute for the Advancement of Medicine (IIAM, Exton, PA). Liver microsomal protein isolation and the specific catalytic activity of individual isoforms of P450 were determined as previously described (Wienkers et al., 1996). Microsomes from a human B-lymphoblastoid cell line expressing CYP1A2, 2C9, 2C19, 2E1, 2D6, and 3A4, microsomal epoxide hydrolase (mEH), and control (minus vector) microsomes were purchased from Gentest (Woburn, MA).
Identification of Bropirimine Metabolites.
[14C]bropirimine was incubated with microsomal protein (1 mg/ml incubation) in 100 mM potassium phosphate buffer, pH 7.4, to give a final incubation volume of 300 μl. After a 3-min preincubation period, reactions were initiated by the addition of NADPH and the reactions continued at 37°C for 30 min in a shaking water bath. The reaction was terminated by adding 100 μl of a 20% perchloric acid solution to the incubation. Incubation tubes were vortex-mixed for 20 sec, centrifuged at 2100g for 25 min at 27°C, and the supernatant was analyzed directly by HPLC with radiochemical or electrospray ionization-mass spectrometry detection (Hauer et al., 1997).
HPLC Analysis of Bropirimine Metabolites.
Analytical separation of bropirimine and metabolites were achieved using a HPLC system equipped with a PE410 pump and a PE ISS-200 autosampler (Perkin Elmer, Norwalk, CT) equipped with a Zorbax SB-C8 (250 × 4.6 mm, 5 μm particle size) reversed phase analytical column (Mac-Mod Analytical, Chadds Ford, PA). The mobile phase consisted of solvent A (90:10 water:methanol containing 0.5% acetic acid) and solvent B (10:90 water:methanol containing 0.5% acetic acid). Initial mobile phase conditions (10% B) at a rate of 1.0 ml/min was held for 1.0 min, followed by a linear gradient to 75% B in 20.0 min; the final conditions were held for 5.0 min. Quantitation of bropirimine and its metabolites were detected with a FLO-ONE beta Series A500 flow-through radioactivity detector (Packard/Radiomatic, Meriden, CT). The fractional contribution of each metabolite to total radioactivity was used to calculate rates of metabolite formation.
Variation of Bropirimine Metabolite Formation.
The relative rates of metabolism of bropirimine (550 μM) to form bropirimine dihydrodiol and p- andm-hydroxybropirimine were determined in nine different human liver microsomal preparations, previously characterized for specific P450 substrate activities (Wienkers et al., 1996). Incubations and a sample workup were carried out as described above. Correlation coefficients (r2) for enzyme activities were determined by linear regression analysis using the graphing/statistical program Prism (GraphPad Software Inc., San Diego, CA).
Chemical Inhibition Experiments.
Incubations containing bropirimine were performed using pooled human liver microsomes, [14C]bropirimine (50 μM) and one of the following P450 enzyme substrates/inhibitors: α-naphthoflavone (100 μM), coumarin (200 μM),p-nitrophenol (100 μM), sulfaphenazole (9 μM), (S)-mephenytoin (200 μM), quinidine (5 μM), ketoconazole (5 μM), cyclohexene oxide (1000 μM), l-cysteine (1000 μM), andN-acetyl-l-cysteine (1000 μM) were examined for their effects on bropirimine metabolism. All inhibitors were dissolved in methanol and were added to the incubations to achieve a final concentration of methanol of 1%. Control incubations (minus inhibitor) also contained 1% methanol. Incubation conditions and a sample workup were carried out as described above.
Bropirimine/P450 Isoform Screen.
[14C]bropirimine (550 μM) was incubated for 60 min in 0.3 mg of microsomal protein microsomes from a human lymphoblastoid cell line expressing one of the following enzymes: CYP1A2, 2C9, 2C19, 2E1, 2D6, and 3A4. A sample workup and chromatographic analysis were carried out as described above.
Mechanism of Dihydrodiol Formation.
Incubations were performed using a fixed concentration of [14C]bropirimine (50 μM) and CYP1A2 (0.25 mg protein) in the presence of varying amounts of microsomal epoxide hydrolase (0, 0.25, 0.50, 0.75, and 1.0 mg protein). Total incubation protein remained constant for all incubations by compensating low mEH protein samples with protein derived from control (minus vector) microsomes. Incubation conditions and a sample workup were carried out as described above.
Results and Discussion
The intent of the current in vitro study was to understand the molecular basis of bropirimine oxidative metabolism. In the presence of human liver microsomes, bropirimine is metabolized to form three major products (fig. 2). Formation of all three bropirimine metabolites were dependent upon addition of NADPH, and formation of metabolites were proportional with time and protein concentration (results not shown). Based upon cochromatography with reference compounds and liquid chromatography-electrospray ionization/mass spectrometry analysis, these metabolites were identified as the previously described (Haueret al., 1997) bropirimine in vivometabolites: p- and m-hydroxybropirimine and bropirimine dihydrodiol. Sample-to-sample variation in bropiriminein vitro oxidation was determined in microsomal preparations from nine different human livers.
Comparison of the rates of formation for p- andm-hydroxybropirimine were highly correlated (r2 = 0.95). However, the rates of formation for the two phenolic metabolites did not correlate with bropirimine dihydrodiol formation. Moreover, none of the bropirimine metabolites correlated with the activities for any of cytochrome P450 enzymes determined in the population of livers (table1). Interestingly, if the rates of formation for all three bropirimine oxidative products were combined to represent a single oxidase activity, the resulting value correlated with CYP1A2 activity (r2 = 0.85, data not shown). The effect of coincubations with various P450 inhibitors on thein vitro oxidation of bropirimine (50 μM) is described in table 2. In human liver microsomal incubations, formation of bropirimine dihydrodiol and phenolic metabolites were strongly inhibited (>90%) in the presence of the potent CYP1A2 inhibitor alpha-naphthoflavone (Chang et al., 1994), while co-incubation of bropirimine with other P450 inhibitors resulted in little or no inhibition of bropirimine oxidation (table 2). The results of the inhibition study coupled with the correlation data suggest that CYP1A2 is the principal cytochrome P450 enzyme responsible for the formation of all three bropirimine metabolites.
To further characterize the enzymatic origin of bropirimine oxidationin vitro, experiments were conducted with microsomes prepared from a human lymphoblastoid cell line selectively expressing one of the following cytochrome P450 isoforms: CYP1A2, 2C9, 2C19, 2E1, 2D6, and 3A4. Incubations with β-lymphoblast microsomal preparations containing cDNA-expressed CYP1A2 were found to oxidize bropirimine top- and m-hydroxybropirimine, while bropirimine oxidase activity was not detected in control (minus cDNA insert) microsomes or with microsomes containing cDNA-expressed CYP2C9, 2C19, 2E1, 2D6, and 3A4 (data not shown). Moreover, the CYP1A2-generated bropirimine phenolic metabolites were formed in a ratio consistent with those obtained in human liver microsomes, while the incubations were devoid of any detectable dihydrodiol formation (fig. 2). These results confirm the original observation, that CYP1A2 is the principle bropirimine oxidase.
Enzymatic aromatic hydroxylation may proceed via three mechanisms: (1) radical cation pathway, (2) the addition-rearrangement pathway, or (3) the epoxide pathway (Darbyshire et al., 1996). In the case of bropirimine oxidation, it appears that each metabolite stems from a common arene oxide intermediate, which rearranges to yield the phenols or serves as a substrate for epoxide hydrolase to generate the dihydrodiol. The role of mEH in bropirimine dihydrodiol formation was investigated by incubating fixed amounts of [14C]bropirimine and cDNA-expressed CYP1A2 with varying concentrations of cDNA-expressed mEH. Although the mixing of microsomal preparations is not an optimal means for generating a membrane preparation containing CYP1A2 and epoxide hydrolase, in this experiment bropirimine dihydrodiol formation appeared to be proportional to mEH concentration (fig.3). The implication of a common arene oxide intermediate is observed in the bropirimine inhibitor study (table 2), in which cyclohexene oxide, an inhibitor of epoxide hydrolase (Griffin et al., 1995), inhibited bropirimine dihydrodiol formation by about 38%, while causing an increase in both phenolic metabolites (∼15%).
Reactions involving epoxides generally occur via direct interactions between the epoxide moiety and nucleophilic macromolecules (Tingle et al., 1993). However, coincubation of two nucleophilic compounds, cysteine and N-acetyl-cysteine, with bropirimine in the presence of human liver microsomes, did not result in a quantitative difference in the amount of bropirimine metabolites formed (table 2). Therefore, it appears that detoxification of the bropirimine arene oxide intermediate is rapid and occurs by chemical rearrangement of the epoxide to form the phenolic metabolites and by formation of the dihydrodiol by epoxide hydrolase. Thus, based upon the results of the current in vitro studies using human liver microsomes and cDNA-expressed enzymes, formation of all three bropirimine oxidative metabolites proceed primarily through a CYP1A2-dependent transient arene oxide intermediate.
Footnotes
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Send reprint requests to: Larry C. Wienkers, Drug Metabolism and Disposition Research, Pharmacia & Upjohn, 7265–300-313, 301 Henrietta Street, Kalamazoo, MI 49007.
- Abbreviations used are::
- CYP or P450
- cytochrome P450
- HPLC
- high-performance liquid chromatography
- mEH
- microsomal epoxide hydrolase
- Received January 21, 1998.
- Accepted June 2, 1998.
- The American Society for Pharmacology and Experimental Therapeutics