Drug Metabolism and Disposition Fast Forward
First published on April 2, 2007; DOI: 10.1124/dmd.106.014597
0090-9556/07/3507-1042-1049$20.00
DMD 35:1042-1049, 2007
Biotransformation of 6-Methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole (BPR0L075), a Novel Antimicrotubule Agent, by Mouse, Rat, Dog, and Human Liver Microsomes
Hsien-Tsung Yao,
Yu-Shan Wu,
Yi-Wei Chang,
Hsing-Pang Hsieh,
Wei-Cheng Chen,
Shih-Jung Lan,
Chiung-Tong Chen,
Yu-Sheng Chao,
Ling Chang,
Hsu-Yi Sun, and
Teng-Kuang Yeh
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan, Republic of China
(Received January 4, 2007;
accepted March 28, 2007)
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Abstract
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6-Methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole (BPR0L075) is a novel synthetic indole compound with microtubule binding activity. Incubation of BPR0L075 with mouse, rat, dog, and human liver microsomes in the presence of NADPH resulted in the formation of six metabolites. Liquid chromatography-tandem mass spectrometry and comparison with the synthetic reference standards identified two metabolites (M1 and M5) as the products derived from hydroxylation on the indole moiety of the molecule. M3 was also identified as a product derived from hydroxylation, but the structure of this metabolite was not identified because of the lack of a reference standard. M2, M4, and M6 were identified as the products derived from O-demethylation. M2, 6-desmethyl-BPR0L075, was the major metabolite formed by the liver microsomes of the four species. No qualitative species difference in the metabolism of BPR0L075 was observed. There was quantitative species difference in the metabolism of BPR0L075 among the four species. Whereas mouse and rat liver microsomes metabolized BPR0L075 predominantly via O-demethylation, dog liver microsomes metabolized BPR0L075 by O-demethylation and hydroxylation to about the same extent. The rank order of intrinsic clearance rates for the conversion of BPR0L075 to 6-desmethyl-BPR0L075 was mouse > rat > human > dog. Incubation of BPR0L075 with baculovirus-insect cell-expressed human cytochrome P450 (P450) isozymes showed that CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the O-demethylation and hydroxylation of BPR0L075 but to a different degree. Among the six P450 isozymes tested, CYP1A2 and 2D6 were most active on catalyzing the metabolism of BPR0L075. CYP1A2 catalyzed mainly the formation of M1, M2, and M3. M2 was the predominant metabolite formed by CYP2D6.
6-Methoxy-3-(3',4',5'-trimethoxybenzoyl)-1H-indole (BPR0L075) is a new synthetic heterocyclic analog of combretastatin A4 that is a naturally occurring stilbene derived from the South African tree Combretum caffrum. It exerts potent antitumor and antimitotic activities. The mechanism of action of BPR0L075 is known to inhibit tubulin polymerization by binding to tubulin at the colchicine-binding site. BPR0L075 showed broad spectrum in vitro antitumor activity against a variety of human tumor cell lines including leukemia, glioblastoma, breast, gastric, colorectal, and liver cancer cells. It also demonstrated potent activity against the growth of human cervical carcinoma KB and human gastric carcinoma MKN-45 xenografts (Kuo et al., 2004
). BPR0L075 is currently undergoing preclinical investigation for the treatment of cancers.
In general, one of the essential components of the drug discovery stage is to optimize the absorption, distribution, metabolism, excretion, and toxicity properties of the investigational compounds to reduce the attrition rate due to poor pharmacokinetic properties (Kerns, 2001). Knowledge of the metabolic fate of a compound is important because it affects clearance, half-life, and oral bioavailability and governs the outcome of pharmacodynamic and toxicological observations (Hop et al., 2002). Thus, one of the primary roles in early preclinical investigations is to compare the metabolic fate of the compound among different species to provide information vital for lead optimization as well as for toxicological assessment. The purpose of this study was to investigate the metabolic fate of BPR0L075 in mouse, rat, dog, and human liver microsomes.
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Materials and Methods
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Chemicals, Microsomes, and Human P450 Isozymes. BPR0L075 and five putative metabolites, 5-hydroxyl-6-methoxy-3-(3',4',5'-trimethoxybenzoyl)indole (5-hydroxyl-BPR0L075), 6-hydroxy-3-(3',4',5'-trimethoxybenzoyl)indole (6-desmethyl-BPR0L075; BPR0L082), 6-methoxy-3-(4'-hydroxy-3',5'-methoxybenzoyl)indole (4'-desmethyl-BPR0L075), 7-hydroxyl-6-methoxy-3-(3',4',5'-trimethoxybenzoyl)indole (7-hydroxy-BPR0L075), 6-methoxy-3-(3'-hydroxy-4',5'-methoxybenzoyl)indole (3'-desmethyl-BPR0L075), and 6-ethoxy-3-(3',4',5'-trimethoxy-benzoyl)-7-azaindole (BPR0L187) were synthesized in The Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Miaoli County, Taiwan, ROC. NADPH was obtained from Sigma Chemical Co. (St. Louis, MO). Pooled liver microsomes from humans (male and female), male mice, male rats, and male dogs and human P450 enzymes (Supersomes) from baculovirus-insect cell-expressed human CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 containing cytochrome P450 reductase were purchased from BD Gentest (Woburn, MA). All other chemicals were of reagent grade and were obtained commercially.
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FIG. 1. The total ion (TIC) and selectively extracted ion chromatograms (EIC) of the acetonitrile extract of BPR0L075 incubated with human liver microsomes in the presence of NADPH.
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Incubation of BPR0L075 with Mouse, Rat, Dog, and Human Liver Microsomes. The incubation mixture, in 84 mM potassium phosphate buffer (pH 7.4), consisted of 3 mM magnesium chloride, 1 mg/ml liver microsomal proteins, 3 mM NADPH, and 20 µM BPR0L075. The concentration of methanol (used for dissolving the substrate) was 1% (v/v). Total volume of incubation was 500 µl. Incubation was carried out aerobically at 37°C with constant shaking in a test tube placed on a temperature-controlled heating block. Reaction was started by the addition of NADPH after preincubating the reaction mixture (without NADPH) for 5 min at 37°C. The incubation mixture without NADPH was used as the blank control. At 60 min after the start of reaction, the incubation mixture was mixed with 1 ml of ice-cold acetonitrile to terminate the reaction. The sample was then transferred to a centrifuge tube, vortexed, and centrifuged at 20,800g for 20 min at room temperature. The supernatant was transferred to a separate tube and evaporated to dryness under nitrogen. The residues were dissolved in 0.1 ml of 40% acetonitrile in water. An aliquot (10 µl) of the reconstituted solution was then analyzed by LC-MS and LC-MS/MS for BPR0L075 and its metabolites.
Incubation of BPR0L075 with Baculovirus-Insect Cell-Expressed Human P450 Isozymes. The incubation system was the same as those applied to the liver microsomal incubations with the exception that microsomes were replaced by baculovirus-insect cell-expressed human CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4. The enzyme concentration in the incubation system was 100 pmol/ml. The total volume of incubation was 200 µl. After 60 min of incubation, the incubation mixture was mixed with 200 µl of ice-cold acetonitrile to terminate the reaction. The sample was then vortexed and centrifuged at 20,800g for 20 min at room temperature. An aliquot (20 µl) of the supernatant was then analyzed by LC-MS and LC-MS/MS for BPR0L075 and its metabolites.
Kinetic Analysis of BPR0L075 Metabolism. In the kinetic experiments, eight concentrations of BPR0L075 (2–250 µM) were incubated in triplicate with mouse, rat, dog, and human liver microsomes. The incubation conditions were the same as those described above except that the microsomal protein concentration was 0.5 mg/ml and the total volume of incubation was 200 µl. The reaction was started by the addition of 20 µl of 30 mM NADPH in phosphate buffer. At 10 min after the start of reaction, 100 µl of each incubation mixture was taken and mixed with 100 µl of ice-cold acetonitrile containing the internal standard (BPR0L187; 200 ng/ml) to stop the reaction. The sample was then vortexed and centrifuged at 20,800g for 20 min at room temperature. An aliquot (10 µl) of the supernatant was used for analysis of the formation of 6-desmethyl-BPR0L075 (BPR0L082) by LC-MS. The amount of formed 6-desmethyl-BPR0L075 was determined by comparing the peak area ratio of the testing samples with that of a sample containing a known concentration of the metabolite (BPR0L082) in microsomes incubated in the absence of NADPH.
LC-MS and LC-MS/MS Analyses. LC-MS was carried out with an Agilent 1100 Series LC System equipped with a UV detector (Agilent Technologies, Palo Alto, CA). An Agilent Extend-C18 reverse-phase column (5 µm, 250 x 4.6 mm) was used. Mobile phase consisted of 10 mM ammonium acetate containing 0.1% formic acid (solvent A) and acetonitrile (solvent B). The flow rate was 0.5 ml/min. Total running time was 60 min. The gradient system used to separate BPR0L075 and its metabolites was 30% B (0–5 min), 30% B to 70% B (5–45 min), 70% B (45–50 min), and 70% B to 30% B (50–60 min). The column temperature was 25°C. The effluent was monitored using a UV detector set at 286 nm and a mass spectrometer operating in the positive ion mode over the m/z range 200 to 600.
For the determination of 6-desmethyl-BPR0L075 (BPR0L082) formation in the kinetic experiment, LC-MS was carried out with an Agilent Zorbax Eclipse XDB-C8 reverse-phase column (5 µm, 150 x 3.0 mm). Mobile phase (solvent A and B), flow rate, and column temperature were the same as those described above. The gradient system used to separate 6-desmethyl-BPR0L075 and BPR0L187 (internal standard) was 30% B to 98% B (0–5 min), 98% B (5–7 min), 98% B to 30% B (7–10 min), and 30% B (10–14 min). The retention times of 6-desmethyl-BPR0L075/BPR0L187 were 4.8/6.4 min. Ions representing the [M + H]+ species for both the metabolite and internal standard were selected and the peak areas were measured.
LC-MS/MS was carried out with an Agilent 1100 Series LC System and an AB Sciex API 3000 tandem mass spectrometer equipped with an electrospray ion in the positive ion mode (Applied Biosystems, Foster City, CA). The source and desolvation temperatures were held at 400°C. The molecular ions of the analytes were extracted and fragmented by collision-induced dissociation. Nitrogen was used as the collision gas. The collision energy was 33 V. Product ions were scanned in the m/z range 100 to 400. The analytical data were processed by Analyst software (version 1.4; Applied Biosystems).
Data Analysis. Nonlinear regression analysis, V = Vmax[S]/(Km + [S]), was used to describe the kinetics of biotransformation of BPR0L075 to 6-desmethyl-BPR0L075 in liver microsomes of the four species and was fitted to the untransformed data (Enzyme Kinetics, version 1.1; SPSS Science, Chicago, IL). V is the velocity of the reaction at substrate concentration [S], Vmax is the maximum velocity, and Km is the substrate concentration at which the reaction velocity is 50% of Vmax. The intrinsic clearance (CLint) was calculated as Vmax/Km.
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Results and Discussion
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Identification of Metabolites in Human Liver Microsomal Incubation. Figure 1 shows the total ion and representative extracted ion chromatograms of the acetonitrile extract of BPR0L075 incubated with human liver microsomes in the presence of NADPH. In addition to the unchanged BPR0L075, six metabolites were detected. Three of the six metabolites (M1, M3, and M5) showed the [M + H]+ ion at m/z 358, which was 16 amu higher than that of the parent compound (342 amu), suggesting the addition of an oxygen atom to the molecule. The other three metabolites (M2, M4, and M6) showed the [M + H]+ ion at m/z 328, which was 14 amu lower than that of the parent compound, suggesting the loss of a CH2 group from the molecule. Thus, metabolites M1, M3, and M5 were tentatively identified as the metabolites derived from hydroxylation of BPR0L075 and metabolites M2, M4, and M6 as the metabolites derived from demethylation of BPR0L075.
The compound with a retention time of 28.5 min had the same retention time, [M + H]+ ion at m/z 342, and fragmentation pattern as BPR0L075 standard. It was thus identified as the unchanged parent compound. The LC-MS/MS fragmentation pattern of BPR0L075 showed two prominent diagnostic fragment ions at m/z 195 and 174 (Fig. 2).
M2, the major metabolite, had the [M + H]+ ion at m/z 328, which was 14 amu lower than that of BPR0L075, suggesting the loss of a CH2 group, and was thus identified as a product of O-demethylation. LC-MS/MS of M2 yielded two fragment ions at m/z 195 and 160 (Fig. 2). Compared with BPR0L075, the fragment ion at m/z 195 indicated that the methoxyl groups on the phenyl ring remained intact. The fragment ion at m/z 160 was 14 amu lower than that of the corresponding fragment ion at m/z 174 of BPR0L075 standard, suggesting that the demethylation reaction took place at the methoxyl group on the 6-position of the indole moiety. M2 had the same retention time, [M + H]+ ion, and fragmentation pattern as BPR0L082 standard (6-desmethyl-BPR0L075) and was thus identified as 6-desmethyl-BPR0L075.
M4 and M6 both had the [M + H]+ ions at m/z 328, suggesting that they were also derived from O-demethylation of BPR0L075. Both M4 and M6 yielded the diagnostic fragment ions at m/z 174 and 181 (Fig. 2), suggesting that the methoxyl group at the 6-position of the indole moiety remained intact and that O-demethylation occurred at the methoxyl groups on the phenyl ring. M4 and M6 showed the same retention times, and [M + H]+ and fragment ions as the synthetic 4'-desmethyl-BPR0L075 and 3'-desmethyl-BPR0L075 standards, respectively. Thus, they were identified as the metabolites derived from O-demethylation at the 4'- and 3'-positions, respectively, on the phenyl moiety of the molecule.
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FIG. 3. The total ion (TIC) and selectively extracted ion chromatograms (EIC) of the acetonitrile extract of BPR0L075 incubated with mouse, rat, dog, and human liver microsomes in the presence of NADPH.
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FIG. 4. The high-performance liquid chromatography-UV chromatograms of BPR0L075 and its metabolites in mouse, rat, dog, and human liver microsomal incubations.
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M1, M3, and M5 all had the [M + H]+ ion at m/z 358, which was 16 amu higher than that of the parent compound, suggesting that an oxygen atom was inserted to the molecule of BPR0L075. The LC-MS/MS fragmentation pattern of the three metabolites all yielded the diagnostic fragment ions at m/z 195 and 190, indicating that the oxygen atom was inserted to the indole moiety of the molecule (Fig. 2). M1 and M5 showed the same retention times, [M + H]+ ion, and fragment ions of the synthetic 5-hydroxyl-BPR0L075 and 7-hydroxyl-BPR0L075 standards, respectively. Thus, they were identified as the metabolites derived from hydroxylation at the 5- and 7-positions, respectively, on the indole moiety of the molecule. The LC-MS/MS data showed that M3 also was a hydroxylation product of BPR0L075 with an oxygen atom being inserted to the indole moiety of the molecule. However, the reference standard was not synthesized for technical reasons. Thus, the position of hydroxylation of M3 remains unknown.
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FIG. 6. The total ion (TIC) and selectively extracted ion chromatograms (EIC) of the acetonitrile extract of BPR0L075 incubated with six baculovirus-insect cell-expressed human P450 isozymes.
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Metabolism of BPR0L075 in Mouse, Rat, Dog, and Human Liver Microsomes. To compare the metabolic fate of BPR0L075 across the species, BPR0L075 was separately incubated with mouse, rat, and dog liver microsomes under the same conditions applied to human liver microsomes. The incubation mixtures were then extracted and analyzed for metabolites by the same LC-MS and LC-MS/MS methods applied to the human liver microsomal incubation.
Figure 3 shows the total ion and representative extracted ion chromatograms of the acetonitrile extract of BPR0L075 incubated with mouse, rat, dog, and human liver microsomes in the presence of NADPH. Similar to the results obtained from the incubation of human liver microsomes, three hydroxylated metabolites of BPR0L075 (M1, M3, and M5) and three O-demethylated metabolites of BPR0L075 (M2, M4, and M6) were also presented in the incubations of mouse, rat, and dog liver microsomes. Figure 4 shows the high-performance liquid chromatography-UV chromatograms of the acetonitrile extract of BPR0L075 incubated with the liver microsomes of the four species. Table 1 shows the retention time, [M + H]+ and major fragment ions, and the percentage distribution of BPR0L075 and the metabolites in the liver microsomal incubations of the four species. The percentage of metabolite formation was estimated based on the UV absorption and the assumption that all metabolites had the same extinction coefficient as the parent compound at 286 nm. The results showed that BPR0L075 was mainly metabolized by O-demethylation and hydroxylation in the liver microsomes of the four species. All six metabolites identified in the human liver microsomal incubation were also detected in the incubations of mouse, rat, and dog liver microsomes. However, quantitative differences in the metabolism of BPR0L075 in the liver microsomes of the four species were observed. Whereas mouse and rat liver microsomes metabolized BPR0L075 predominantly via O-demethylation, dog liver microsomes metabolized BPR0L075 by O-demethylation and hydroxylation to about the same extent. The metabolic profile of BPR0L075 in human liver microsomes appears to be between those of mouse and rat and dog liver microsomes (Table 1). Figure 5 depicts the proposed metabolic pathways of BPR0L075 in the liver microsomes of the four species.
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TABLE 1 The retention time, [M + H]+ ions, and major fragment ions and the relative percentage distribution of metabolites identified in the incubation mixture of BPR0L075 in mouse, rat, dog, and human liver microsomes
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FIG. 7. Representative Michaelis-Menten plots and Lineweaver-Burk plots (inset) for the biotransformation of BPR0L075 to 6-desmethyl-BPR0L075 in mouse, rat, dog, and human liver microsomes. Each point represents the mean of three determinations.
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Metabolism of BPR0L075 by Individual Human P450 Isozymes. BPR0L075 was separately incubated with baculovirus-insect cell-expressed human CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 to determine the metabolic reactions of BPR0L075 catalyzed by individual P450 isozymes. After the incubation, the incubation mixtures were extracted and analyzed by LC-MS and LC-MS/MS for the formation of metabolites. Figure 6 shows the total ion and selectively extracted ion (at m/z 358 and 328 amu) chromatograms of the incubation of each P450 isozyme. The results showed that all six tested P450 isozymes catalyzed both the hydroxylation and O-demethylation of BPR0L075, but the degree of each metabolite formed by individual isozymes varied. Among the six P450 isozymes tested, CYP1A2 and 2D6 were most active in catalyzing the metabolism of BPR0L075, followed by CYP2C19 and 3A4. CYP2C9 and 2E1 were least active in catalyzing the metabolism of BPR0L075. Although all six isozymes were capable of catalyzing the formation of the six identified metabolites, CYP1A2 catalyzed mainly the formation of M1, M2, and M3, whereas M2 was the major metabolite of CYP2D6, and M2 and M4 were the major metabolites of CYP2C19 and 3A4.
Kinetic Analysis of BPR0L075 Metabolism. M2 (6-desmethyl-BPR0L075), the major metabolite of BPR0L075 identified in the mouse, rat, dog, and human liver microsomal incubations, has also been shown to have cytotoxic activities against several human cancer cell lines but was less potent than BPR0L075 (Liou et al., 2004). Thus, it is of importance to determine the extent and the rate of BPR0L075 conversion to M2 in the liver microsomes of the four species. Table 2 and Fig. 7 show the kinetic data of BPR0L075 to M2 metabolism in the liver microsomes of the four species. The Km and Vmax values for the metabolism of BPR0L075 to M2 were evaluated by Michaelis-Menten and Lineweaver-Burk plots (Fig. 7). The calculated Km values were 3.4, 14.8, 50.1, and 26.7 µM, respectively, for mouse, rat, dog, and human liver microsomes. The corresponding values for Vmax were 465, 512, 295, and 415 pmol/mg protein/min, respectively. The intrinsic clearance rates (Vmax/Km) for the conversion of BPR0L075 to M2 were 137, 34.6, 5.9, and 19.3 µl/mg protein/min, respectively, for mouse, rat, dog, and human liver microsomes. The rank order of intrinsic clearance was mouse > rat > human > dog.
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Conclusion
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Six metabolites of BRP0L075 in the liver microsomal incubations of mouse, rat, dog, and human were tentatively identified by LC-MS/MS as the products derived from hydroxylation on the indole ring (M1, M3, and M5) and O-demethylation (M2, M4, and M6). The structures of five metabolites were confirmed by comparing with the synthetic standards. The structure of one metabolite (M3) had not been identified due to the lack of a reference standard. Metabolite M2 (6-desmethyl-BPR0L075) was the major metabolite in all four species. No qualitative species difference in the metabolism of BPR0L075 was observed. There was quantitative species difference in the metabolism of BPR0L075 among the four species. The rank order of intrinsic clearance rates for the conversion of BPR0L075 to 6-desmethyl-BPR0L075 was mouse > rat > human > dog. Incubation of BPR0L075 with baculovirus-insect cell-expressed human P450 isozymes showed that CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the O-demethylation and hydroxylation of BPR0L075. CYP1A2 and 2D6 were most active in catalyzing the metabolism of BPR0L075. CYP1A2 catalyzed mainly the formation of M1, M2, and M3. CYP2D6 catalyzed predominantly the formation of M2.
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Footnotes
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The authors are grateful to the National Health Research Institutes, Taiwan, Republic of China, for financial support.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.106.014597.
ABBREVIATIONS: BPR0L075, 6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole; BPR0L082, 6-hydroxy-3-(3',4',5'-trimethoxybenzoyl)indole; BPR0L187, 6-ethoxy-3-(3',4',5'-trimethoxy-benzoyl)-7-azaindole; P450, cytochrome P450; LC-MS, liquid chromatography-mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; amu, atomic mass units.
Address correspondence to: Dr. Teng-Kuang Yeh, Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County, Taiwan. E-mail: tkyeh{at}nhri.org.tw
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References
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Kuo CC, Hsieh HP, Pan WY, Chen CP, Liou JP, Lee SJ, Chang YL, Chen LT, Chen CT, and Chang JY (2004) BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res 64: 4621–4628.[Abstract/Free Full Text]
Kerns EH (2001) High throughput physicochemical profiling for drug discovery. J Pharm Sci 90: 1838–1858.[CrossRef][Medline]
Hop CE, Tiller PR, and Romanyshyn L (2002) In vitro metabolite identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry. Rapid Commun Mass Spectrom 16: 212–219.[CrossRef][Medline]
Liou JP, Chang YL, Kuo FM, Chang CW, Tseng HY, Wang CC, Yang YN, Chang JY, Lee SJ, and Hsieh HP (2004) Concise synthesis and structure-activity relationships of combretastatin A-4 analogues, 1-aroylindoles and 3-aroylindoles, as novel classes of potent antitubulin agents. J Med Chem 47: 4247–4257.[CrossRef][Medline]
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Abstract
Materials and Methods
