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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Urinary Metabolite Profiling Reveals CYP1A2-Mediated Metabolism of NSC686288 (Aminoflavone)

Laboratories of Metabolism (C.C., X.M., K.W.K., F.J.G.) and Molecular Pharmacology (L.M., Y.P.), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland; and Institute of Pharmacology, First Faculty of Medicine, Charles University, Prague, Czech Republic (J.R.I.)

Received for publication March 24, 2006
Accepted June 13, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
NSC686288 [aminoflavone (AF)], a candidate chemotherapeutic agent, possesses a unique antiproliferative profile against tumor cells. Metabolic bioactivation of AF by drug-metabolizing enzymes, especially CYP1A monooxygenases, has been implicated as an underlying mechanism for its selective cytotoxicity in several cell culture-based studies. However, in vivo metabolism of AF has not been investigated in detail. In this study, the structural identities of 13 AF metabolites (12 of which are novel) in mouse urine or from microsomal incubations, including three monohydroxy-AFs, two dihydroxy-AFs and their sulfate and glucuronide conjugates, as well as one N-glucuronide, were determined by accurate mass measurements and liquid chromatography-tandem mass spectrometry fragmentation patterns, and a comprehensive map of the AF metabolic pathways was constructed. Significant differences between wild-type and Cyp1a2-null mice, within the relative composition of urinary metabolites of AF, demonstrated that CYP1A2-mediated regioselective oxidation was a major contributor to the metabolism of AF. Comparisons between wild-type and CYP1A2-humanized mice further revealed interspecies differences in CYP1A2-mediated catalytic activity. Incubation of AF with liver microsomes from all three mouse lines and with pooled human liver microsomes confirmed the observations from urinary metabolite profiling. Results from enzyme kinetic analysis further indicated that in addition to CYP1A P450s, CYP2C P450s may also play some role in the metabolism of AF.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Structure of AF. AF structure is annotated following standard nomenclature of the flavonoids.

To date, information on the identities of AF metabolites was limited to the detection of one N⁴'-acetylated metabolite in plasma (Phillips et al., 2000) and one N⁴'-hydroxylated metabolite after incubations with microsomal membranes (Kuffel et al., 2002). It should also be noted that current knowledge on the bioactivation of AF was mainly derived from in vitro incubation and cell line-based experiments. In human and animal studies, liver, kidney, intestine, and other extrahepatic tissues will likely participate in the absorption, distribution, metabolism, and excretion of AF. Indeed, it is well known that the major circulating forms of chemotherapeutic agents that are destined for uptake into tumors are not the parental compounds in the most cases, and the expression levels of major drug-metabolizing enzymes in tumor tissues are significantly different from those in liver. For example, CYP1A1 may be the major AF-metabolizing enzyme in tumor cells, but in liver, its expression level is far lower than CYP1A2 (Shimada et al., 1996). The literature is replete with examples illustrating significant differences between cell culture models and human and animals studies with regard to the metabolism of specific drugs.

To understand the in vivo metabolism of AF and the role of CYP1A2, AF metabolites were identified and their structures elucidated, and urinary metabolites of AF from wild-type, Cyp1a2-null, and CYP1A2-humanized mice were profiled after oral dosing of AF. The regioselectivity of CYP1A2-mediated oxidation and interspecies differences in the metabolism of AF were confirmed after microsomal incubation, and the contribution of major P450s to the metabolism of AF was determined by incubations with recombinant human P450s and enzyme kinetics.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals and Reagents. Aminoflavone was provided by the Developmental Therapeutics Program, NCI. -Naphthoflavone, sulfaphenazole, mephenytoin, high-performance liquid chromatography-grade acetonitrile, methanol, and formic acid as well as -NADPH were purchased from Sigma (St. Louis, MO). Pooled human liver microsomes (HLMs) and recombinant human P450s (CYP1A1; 1A2; 1B1; 2A6; 2B6; 2C9; 2C19; 2D6; 2E1; 3A4) from baculovirus-infected insect cells were purchased from BD Biosciences (San Jose, CA).

Animals. Both the Cyp1a2-null mouse line (mCyp1a2-/-) and CYP1A2-humanized mouse line on a Cyp1a2-null background (hCYP1A2+/+, mCyp1a2-/-) were described previously (Pineau et al., 1995; Cheung et al., 2005). Wild-type mice on a 129/Sv strain background were used in this study. All animals were maintained in the NCI's animal facilities under a standard 12-h light/dark cycle with food and water ad libitum. Handling and treatment procedures were in accordance with animal study protocols approved by the NCI Animal Care and Use Committee.

In Vivo Metabolism of AF in Mice. Four female mice from each genotype group, age 8 to 12 weeks, were used for urinary metabolite profiling. Urine samples were collected by housing mice for 24 h in glass metabolic bowls (Jencons, Leighton Buzzard, UK), and stored at -80°C before usage. Control urine samples were collected 2 days before AF treatment, and then a 50 mg/kg dose of AF (suspended in corn oil) was administrated by oral gavage. After urine collection, mice were killed, and livers were harvested and snap-frozen in liquid nitrogen before storage at -80°C. Samples for ultraperformance liquid chromatography (UPLC)-time-of-flight mass spectrometry (TOFMS) analysis were prepared by mixing 50 µl of urine with 200 µl of 50% acetonitrile and centrifuging at 18,000g for 5 min to remove protein and particulates. A 200-µl aliquot of the supernatant was transferred to an autosampler vial for further analysis.

Preparation of Mouse Liver Microsomes. Livers from untreated wild-type (mCyp1a2+/+), Cyp1a2-null (mCyp1a2-/-), and transgenic (hCYP1A2+/+, mCyp1a2-/-) mice were homogenized, and microsomes (MLMs) were prepared as described previously (Yu et al., 2003).

In Vitro Metabolism of AF in MLM and HLM. Microsomal incubations were carried out in 20 mM phosphate-buffered saline, pH 7.4, containing 0.5 mg/ml microsomal protein, 2 mM MgCl₂,20 µM AF, and 1 mg/ml freshly prepared -NADPH in a final volume of 200 µl. After a 30-min incubation at 37°C, the reaction mix was directly loaded into a pretreated Oasis column (Waters, Milford, MA), and metabolites were eluted with 1 ml of methanol. The extract was dried by N₂ flow and reconstituted in 50% aqueous acetonitrile for LC-MS/MS analysis.

In Vitro Metabolism of AF by Recombinant Human P450s. Reactions were carried out in 20 mM phosphate-buffered saline, pH 7.4, containing 20 nM recombinant human P450s (CYP1A1; 1A2; 1B1; 2A6; 2B6; 2C9; 2C19; 2D6; 2E1; 3A4), 2 mM MgCl₂,20 µM AF, and 1 mg/ml freshly prepared -NADPH in a final volume of 200 µl. After 30 min of incubation at 37°C, the reaction mix was directly loaded into a pretreated Oasis column (Waters), and metabolites were eluted with 1 ml of methanol. The extract was dried by N₂ flow and reconstituted in 50% aqueous acetonitrile for LC-MS/MS analysis.

Enzyme Kinetics of Recombinant Human CYP1A2, CYP2C9, CYP2C19, and CYP3A4. AF ranging from 200 nM to 80 µM was incubated with recombinant human CYP1A2, CYP2C9, CYP2C19, and CYP3A4 for 20 min under the same condition as described above. Kinetic parameters for three major monohydroxylated metabolites, K_m and relative V_max (represented as the percentage of the highest activity detected in all reactions), were evaluated by nonlinear regression (Prism GraphPad 2.0; GraphPad Software Inc., San Diego, CA).

Chemical Inhibition of HLM-Mediated AF Metabolism. Different from the aforementioned condition for microsomal incubations, chemical inhibitors of CYP1A2, CYP2C9, and CYP2C19 were preincubated with HLMs for 5 min before adding AF and NADPH. The reactions were terminated at 20 min, and samples were processed under the same conditions as described above.

LC-Electrospray Ionization-MS/MS Analysis of AF Metabolites. For LC-MS/MS analysis, a 5-µl aliquot of samples from mouse urine or in vitro incubation was injected into a Waters UPLC-QT-OFMS system. An Acquity UPLC BEH C₁₈ column (Waters) was used to separate AF and its metabolites at 30°C. Flow rate of mobile phase was 0.6 ml/min with a gradient ranging from water to 95% aqueous acetonitrile containing 0.1% formic acid in a 10-min run. The QTOF Premier mass spectrometer was operated in the positive electrospray ionization mode. Capillary voltage and cone voltage was maintained at 3 kV and 40 V, respectively. Source temperature and desolvation temperature were set at 120 and 350°C, respectively. Nitrogen was applied as the cone (50 l/h) and desolvation gas (600 l/h) and argon as collision gas. For accurate mass measurement, the QTOFMS was calibrated with sodium formate solution (range m/z, 100-1000) and monitored by the intermittent injection of the lock mass leucine enkephalin ([M + H]⁺ = 556.2771 m/z) in real time. Mass chromatograms and mass spectral data were acquired by MassLynx software (Waters) in centroid format and were further processed by MetaboLynx software (Waters) to screen and identify potential AF metabolites through the mass difference from AF ion (m/z, 321.0851⁺). The structure of each AF metabolite was elucidated by tandem mass spectrometry (MS²) fragmentation with collision energy ranging from 10 to 40 eV.

Statistics. Experimental values are expressed as mean ± S.D. Statistical analysis was performed with independent Student's t tests, with P < 0.01 considered as statistically significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Identification and Structural Elucidation of Phase I Metabolites of AF by Mouse Liver Microsomes. Previously, a number of potential AF metabolites were identified from high-performance liquid chromatography chromatograms of extracted samples derived from microsomal incubations, but only one phase I metabolite of AF was structurally elucidated (Kuffel et al., 2002). To obtain comprehensive structural information on the phase I metabolites of AF and set a basis for further in vivo metabolism studies, AF was incubated with MLMs from untreated mice. Extracted samples containing AF and its metabolites were separated by the UPLC system, and their accurate masses were measured by a time of flight mass spectrometer. By comparing chromatographic differences between control (without NADPH) and normal (with NADPH) samples and by examining the mass difference between AF and new ions generated by the MLM incubation, three monohydroxylated and two dihydroxylated metabolites were identified (Table 1). Their structures were further elucidated by MS² fragmentation (Fig. 2).

View larger version (30K): [in this window] [in a new window]   Fig. 2. LC-MS/MS structural elucidation of phase I AF metabolites generated by microsomal incubation. Conditions for LC-MS/MS analysis and microsomal incubation were described under Materials and Methods.MS2 fragmentation was conducted with collision energy ramping from 10 to 40 eV, except a fixed collision energy of 35 eV for inlaid spectrum in D. A, MS2 fragmentation of AF (I). B, MS2 fragmentation of N5-hydroxy-AF (II). C, MS2 fragmentation of N4'-hydroxy-AF (III). D, MS2 fragmentation of 3-hydroxy-AF (IV). E, MS2 fragmentation of N5,N4'-dihydroxy-AF (V). F, MS2 fragmentation of 3,N5-dihydroxy-AF (VI). Major daughter ions from fragmentation were interpreted in the inlaid structural diagrams.

As the reference compound for the structural elucidation of AF metabolites, the fragmentation pattern of the parent AF ion (I) was examined first to show the formation of major fragment ions (186⁺ and 136⁺) via a retro Diels-Alder (rDA) reaction in C-ring (Fig. 2A). Further detachment of the carbonyl group (-C = O) from ion 186⁺ generated ion 158⁺. In addition, the breaking of the C-C bond between the B-ring and C-ring yielded ion 110⁺ (C₆H₆NF⁺ by accurate mass measurement) from the B-ring.

AF metabolite II was a monohydroxy derivative of AF based on accurate mass measurement and the loss of free radical HO· (-17). The presence of ion 202⁺ instead of ion 186⁺ indicated that the oxidation had occurred in the A-ring (Fig. 2B). Therefore, the identity of this metabolite was assigned as N⁵-hydroxy-AF (N⁵-OH-AF). However, it is recognized that hydroxylation might also have occurred on the 7-methyl group, but this was deemed unlikely due to the ready loss of HO· (from the pseudomolecular ion, a known characteristic of N-hydroxy compounds (Saito et al., 1985).

The structure of AF metabolite III was previously reported as N⁴'-hydroxy-AF (N⁴'-OH-AF) (Kuffel et al., 2002). As shown in Fig. 2C, both the presence of ions 186⁺ and 158⁺ provided evidence that the oxidation did not occurred in the A-ring, and loss of the -CH₂NO group from the parent ion (-44 from 337⁺ to 293⁺) supported this structure.

Generation of ions 186⁺ and 152⁺ by fragmenting the AF metabolite IV ion excluded the possibility of oxidation in the A-ring (Fig. 2D). The high abundance of fragment ion 110⁺ (C₆H₆NF⁺) as a result of breaking of the C-C bond between the B- and C-rings also excluded the presence of a hydroxyl group in the B-ring (inlaid spectrum in Fig. 2D). Therefore, the only possible oxidation site is the carbon-3 in the C-ring, which confirms the structure of metabolite IV as 3-hydroxy-AF (3-OH-AF). In this connection, it should be noted that both quercetin and kaempferol, the two most abundant natural flavonols in human diet, have the 3-hydroxy group in their structure (Havsteen, 2002).

Both AF metabolites V and VI are the dihydroxylated AFs based on their accurate masses (Table 1). Similar to metabolite II, the fragmentation of metabolite V yielded the characteristic ion 202⁺, which indicated a N⁵-hydroxylation reaction. Moreover, neutral loss of mass 44, similar to metabolite III, proved the existence of N⁴'-hydroxy group (Fig. 2E). By combining the aforementioned evidence, the structure of metabolite V was designated as N⁵,N⁴'-dihydroxy-AF. As for metabolite VI, the presence of ions 202⁺ and 152⁺ resembled the MS² spectra of metabolites II and IV (Fig. 2F). Therefore, the structure of metabolite VI was designated as 3,N⁵-dihydroxy-AF.

Identification and Structural Elucidation of Urinary Metabolites of AF. The observation of five major phase I metabolites after MLM incubation suggested that AF might also be extensively metabolized in vivo. To test this hypothesis, urine samples from the AF-treated mice (50 mg/kg) were analyzed under the same chromatographic and spectroscopic conditions as extracts from in vitro incubation. To achieve an exhaustive detection and comprehensive coverage of major urinary metabolites, a software-assisted metabolite screening and detection (as described under Materials and Methods) was conducted based on the data from accurate mass measurements of all urinary ions and the theoretical mass changes caused by the common phase I- and II-metabolizing reactions. The results revealed the presence of AF parent ion (I), N⁵-OH-AF (II), and eight new conjugated metabolites (VII-XIV) in mouse urine samples (Table 2). Structural identities of those urinary metabolites were further elucidated by MS² analysis. Their major fragment ions are shown in Table 2, and MS² spectra of the six most abundant phase II conjugates (VIII-XII and XIV) are presented in Fig. 3, A to F.

View larger version (30K): [in this window] [in a new window]   Fig. 3. LC-MS/MS structural elucidation of major phase II metabolites of AF in urine. Urine samples from wild-type mice were collected for 24 h after oral dosing with 50 mg/kg AF. Chromatographic and spectroscopic conditions for LC-MS/MS were described under Materials and Methods.MS2 fragmentation was conducted with collision energy ramping from 10 to 40 eV. A, MS2 fragmentation of 3-hydroxy-AF sulfate (VIII). B, MS2 fragmentation of 3,N5-dihydroxy-AF sulfate (IX). C, MS2 fragmentation of AF-N5-glucuronide (X). D, MS2 fragmentation of N5-hydroxy-AF-N5-glucuronide (XI) with inlaid spectrum of deconjugated N5-hydroxy-AF ion. E, MS2 fragmentation of N4'-hydroxy-AF-N4'-glucuronide (XII). F, MS2 fragmentation of 3,N5-dihydroxy-AF-N5-glucuronide (XIV). Major daughter ions from fragmentation were interpreted in the inlaid structural diagrams.

AF metabolite VII as a sulfate conjugate was confirmed by the neutral loss of mass 80 (SO₃). Appearance of fragment ions 202⁺ and 136⁺ matched the fragmentation pattern of N⁵-OH-AF (II) (Table 2). Therefore, it was designated as N⁵-hydroxy-AF sulfate.

Metabolite VIII was also a sulfate conjugate of monohydroxylated AF because of its accurate mass and neutral loss of mass 80. Fragment ions 186⁺ and 152⁺ indicated its resemblance to 3-OH-AF (III) (Fig. 3A), so its chemical identity was designated as 3-hydroxy-AF sulfate (VIII).

Formation of ion 353⁺ after neutral loss of sulfate group (-80) from metabolite IX indicated a dihydroxylated AF moiety (Fig. 3B). The presence of ion 202⁺ and 152⁺ validated the structure as 3,N⁵-dihydroxy-AF sulfate. Since the retention time (RT) shift caused by sulfation at the 3-OH group (0.19 min) was far less than that derived from sulfation at N⁵-OH group (0.90 min) (Table 1 and 2), and RT shift of metabolite IX from its parent compound 3,N⁵-dihydroxy-AF (V) was 0.11 min, the sulfate group of metabolite IX was putatively conjugated to the 3-OH group.

Metabolite X was a direct glucuronidation product of AF because of neutral loss of mass 176 (glucuronic acid moiety) and characteristic fragment ions of 186⁺ and 136⁺ from deconjugated AF (Fig. 3C). Furthermore, the presence of ion 363⁺ from potential rDA reaction revealed that glucuronidation occurred on the A-ring, in which the N⁵ atom is the only possible conjugation site. Therefore, metabolite X was designated as AF-N⁵-glucuronide.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Representative chromatograms of major AF metabolites in urine samples of wild-type (mCyp1a2+/+), Cyp1a2-null (mCyp1a2-/-), and CYP1A2-humanized (hCYP1A2+/+, mCyp1a2-/-) mice. Chromatographic and spectroscopic conditions for LC-MS analysis were described under Materials and Methods. Ions within the 20 ppm range of theoretical accurate mass ([M + H]+) of AF and its metabolites (321.0851, 337.0800, 417.0368, 433.0317, 497.1172, 513.1121, and 529.1070) were extracted from each 10-min mass scan on the urine samples from three mouse lines. The identities of metabolites (I, II, and VII-XIV) are presented in Table 2.

Ions 513⁺, 337⁺ (loss of glucuronic acid)n and 320⁺ (loss of HO·) in MS² spectra of metabolite XII indicated that it was another glucuronide of monohydroxylated AF (Fig. 3E). The presence of ions 293⁺ and 186⁺ was consistent with the fragmentation pattern of N⁴'-OH-AF (III). Therefore, metabolite XII was putatively designated as N⁴'-OH-AF-N⁴'-glucuronide.

Formation of ion 353⁺ after neutral loss of glucuronic acid group (-176) from metabolite XIII indicated a dihydroxylated AF moiety (Table 2). The presence of ions 309⁺ and 202⁺, which were the characteristic fragments of N⁵,N⁴'-diOH-AF (V), indicated metabolite XIII as N⁵,N⁴'-diOH-AF glucuronide.

Ions 529⁺, 353⁺ (loss of glucuronic acid) and 336⁺ (loss of HO·) in MS² spectra of metabolite XIV indicated it was another glucuronide of dihydroxylated AF (Fig. 3F). The presence of ions 202⁺ and 152⁺ resembled the fragmentation pattern of 3,N⁵-diOH-AF (VI). Since glucuronidation was the preferred conjugation reaction for N⁵-OH-AF and sulfation for 3-OH-AF, as proven by the high abundance of N⁵-OH-AF-N⁵-glucuronide (XI) and 3-OH-AF sulfate (VIII), metabolite XIV was putatively designated as 3,N⁵-diOH-AF-N⁵-glucuronide.

Relative Composition of Urinary AF Metabolites in Wild-Type, Cyp1a2-Null, and CYP1A2-Humanized Mice. Previous studies on the metabolism of AF by using tumor cell lines have shown that CYP1A1 and CYP1A2 contributed to the metabolic bioactivation of AF through an aryl hydrocarbon receptor-mediated pathway (Kuffel et al., 2002; Loaiza-Perez et al., 2004a, b). However, it is well known that CYP1A2 plays a much more prominent role in xenobiotic metabolism in vivo than does CYP1A1 based on its much higher expression level and catalytic activity in liver (Shimada et al., 1996; Turesky et al., 2002). In addition, CYP1A2 is also an important enzyme in the bioactivation of heterocyclic aromatic amines, a chemical class to which AF belongs (Butler et al., 1989). To investigate the role of CYP1A2 in the in vivo metabolism of AF, three mouse lines including wild-type, Cyp1a2-null (mCyp1a2-/-), and CYP1A2-humanized (hCYP1A2+/+, mCyp1a2-/-) mice were used for oral dosing of AF. Although all of the urine samples from wild-type, Cyp1a2-null, and CYP1A2-humanized mice contained AF (I) and its nine urinary metabolites (II, VII-XIV), significant differences in the relative abundance of several metabolites among three mouse lines became evident after pooling all the peak areas of AF and its urinary metabolites and calculating the percentage of each metabolite in this metabolite cluster (Fig. 4; Table 2). In wild-type mice (Fig. 4), AF was mainly converted to the 3,N⁵-diOH-AF sulfate (IX) and N⁵-OH-AF-N⁵-glucuronide (XI), but in the Cyp1a2-null mice (Fig. 4), the major metabolites were N⁵-OH-AF (II) and its glucuronide (XI). Comparing the metabolic profiles of wild-type and Cyp1a2-null mice, it was apparent that the removal of CYP1A2 from the metabolic system diminished the 3-hydroxylation reaction, which subsequently led to significantly decreased levels of the 3-hydroxy metabolites including 3-OH-AF sulfate (VIII), 3,N⁵-di-OH-AF sulfate (IX) and 3,N⁵-diOH-AF-N⁵-glucuronide (XIV), and to the accumulation of N⁵-hydroxy metabolites, such as N⁵-OH-AF (II), N⁵-OH-AF sulfate (VII), and N⁵-OH-AF-N⁵-glucuronide (XI). Therefore, 3-hydroxylation of AF was a selective CYP1A2-mediated reaction. This conclusion was further strengthened by the relative composition of urinary AF metabolites in CYP1A2-humanized mice. Introduction of the human CYP1A2 gene into Cyp1a2-null mice resulted in recovery of the 3-hydroxylation reaction of AF and its downstream metabolites and led to comparable levels of metabolites II, VII, IX, and XIV as found in wild-type mice (Fig. 4). However, there still existed significant differences between wild-type and CYP1A2-humanized mice with regard to the metabolites VIII and XI. The higher level of 3-OH-AF sulfate (VIII) and the lower level of N⁵-OH-AF glucuronide (XI) in CYP1A2-humanized mice implied that 3-hydroxylation of AF was a more preferred reaction for human CYP1A2 than for mouse CYP1A2. Besides the major metabolites from 3-hydroxylation and N⁵-hydroxylation, an AF-N⁵-glucuronide (X) and two N⁴'-hydroxylation products, N⁴'-OH-AF-N⁴'-glucuronide (XII) and N⁵,N⁴'-diOH-AF-N-glucuronide (XIII), were also detected as minor metabolites found at comparable levels in the three mouse lines. It should also be noted that all three mouse lines including Cyp1a2-null mice retained similar levels of the AF parent compound (I) in their urine, which comprised about 10 to 15% of total urinary AF metabolites. This observation indicated that, even without the mouse CYP1A2 enzyme, AF was still extensively metabolized. Therefore, some other P450 enzymes were also involved in the metabolism of AF.

View larger version (25K): [in this window] [in a new window]   Fig. 5. In vitro oxidation of AF by MLMs from wild-type, Cyp1a2-null, and CYP1A2-humanized mice and by pooled HLMs. A, representative chromatograms of monohydroxylated AF (337+) and dihydroxylated AF (353+) ions (II-VI). Ions within the 20 ppm range of theoretical accurate masses (337.0800 and 353.0749) were extracted from each 10-min mass scan on the samples generated by incubating 20 µM AF with 0.5 mg/ml MLMs from three mouse lines and pooled HLMs. B, relative activity of MLMs and HLMs for the generation of phase I AF metabolites (II-VI). Enzymatic activity of the microsome with the highest yield of targeted metabolite was set arbitrarily as 1. Relative activity was presented as mean ± S.D. (n = 3).

Comparison of in Vitro Metabolism of AF by Recombinant P450s. Extensive metabolism of AF in Cyp1a2-null mice implied the potential involvement of other P450s in the oxidation of AF (Table 2). Therefore, 20 µM AF was incubated with a panel of recombinant human P450s, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Chromatograms generated by three most reactive P450s (CYP1A1, CYP1A2, and CYP2C19) are presented in Fig. 6A, and the relative activities of the whole P450 panel on the generation of three monohydroxylated metabolites (II-IV) and two dihydroxylated metabolites (V and VI) are illustrated in Fig. 6, B to F. As shown in Fig. 6, CYP1A2 was more selective in the conversion of AF to 3-OH-AF (IV), whereas CYP1A1 was more active than CYP1A2 in the production of N⁵-OH-AF (II) and N⁴'-OH-AF (III). The catalytic activities of CYP1A1 and CYP1A2 for the formation of N⁵,N⁴'-diOH-AF (V) were comparable, but CYP1A2 was far more active than CYP1A1 for the formation of 3,N⁵-diOH-AF (VI) since it required CYP1A2-mediated 3-hydroxylation. Besides CYP1A P450s, two CYP2C P450s, CYP2C9 and CYP2C19, were also active in the N-hydroxylation of AF to generate metabolites II and III. In addition, both CYP3A4 and CYP2D6 also had low but measurable levels of AF-metabolizing activity (Fig. 6, B-D).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Relative monohydroxylation and dihydroxylation activities by recombinant human P450s. A, representative chromatograms of monohydroxylated AF (337+) and dihydroxylated AF (353+) ions (II-VI) after incubation of 20 µM AF with recombinant human CYP1A1, CYP1A2, and CYP2C19 enzymes. Ions within the 20 ppm range of theoretical accurate masses (337.0800 and 353.0749) were extracted from each 10-min mass scan on the samples generated by Supersome incubation. B, relative activity of P450s for the generation of N5-OH-AF (II). C, relative activity of P450s for the generation of N4'-OH-AF (III). D, relative activity of P450s for the generation of 3-OH-AF (IV). E, relative activity of P450s for the generation of N5,N4'-diOH-AF (V). F, relative activity of P450s for the generation of 3,N5-diOH-AF (VI). Enzymatic activity of the P450s with the highest yield of targeted metabolite was set arbitrarily as 1. Relative activity was presented as mean ± S.D. (n = 3).

View larger version (31K): [in this window] [in a new window]   Fig. 7. Relative contribution of CYP1A2, CYP2C, and CYP3A P450s to AF metabolism. A to C, enzyme kinetics of recombinant human CYP1A2 (), CYP2C9 (), CYP2C19 (), and CYP3A4 () isozymes on the formation of N5-OH-AF (II), N4'-OH-AF (III), and 3-OH-AF (IV) were evaluated after the incubations with AF ranging from 200 nM to 80 µM. Relative activity was calculated as the percentage of the highest activity detected in all kinetic reactions. D, effect of specific CYP inhibitors on HLM-mediated phase I metabolism. Ten micromolar -naphthoflavone (-NF), 100 µM sulfaphenazole (SUL), or 100 µM mephenytoin (MEPH) was preincubated with HLMs before adding 20 µM AF. Catalytic activity of HLMs for the formation of each phase I metabolite (II-IV) was set arbitrarily as 1. Relative activity was presented as mean ± S.D. (n = 3).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Structure-activity relationship protocols guided the development of AF as a chemotherapeutic agent. Based on the fact that natural or synthetic flavonoids can elicit antiestrogenic and/or estrogenic activities, AF was derived from the structure of flavone (Havsteen, 2002). By adding two amino groups as electron donors and three fluoride atoms plus a methyl group for blocking metabolism, AF possesses much higher cytotoxicity against the estrogen receptor-positive cell line MCF-7 than its parent compounds (Akama et al., 1996, 1997). Recent results from the NCI 60 cell line screenings indicated that the bioactivity of AF may surpass the original expectation of developing an antiestrogen for breast cancer therapy (http://www.dtp.nci.nih.gov/docs/dtp_search.html). Interestingly, although one of the original medicinal chemistry strategies for developing this compound was to decrease the potential metabolism in the flavone moiety (Akama et al., 1997), which was thought to have a deactivating effect on its bioactivity, the results from this study clearly demonstrated that AF was still extensively metabolized since multiple metabolites were detected after in vitro incubation and oral administration, and the parent compound only accounted for about 10 to 15% of all detected urinary metabolites in the three mouse lines used in this study.

To identify and profile of phase I and phase II metabolites of AF, the UPLC system coupled with a TOFMS were adopted to achieve high-resolution chromatographic separation and accurate mass measurements (in the 5-10 ppm range of real mass). By combining the information on the retention time and the mass of each ion, computer-assisted analysis can automatically interrogate the mass chromatograms and spectra for identifying the potential AF metabolites. Compared with a manual search, this strategy was more efficient and exhaustive. It should be noted that in-source fragmentation occurred for several sulfate and glucuronide metabolites, presumably because of weak chemical bonds between the parent compound and the conjugated moiety. With careful examination and adjustment of conditions on the mass spectrometer, the identities of those fragments can be unambiguously resolved. Structures of major AF metabolites were elucidated through analyzing the fragmentation pattern recorded by tandem mass spectrometry. Like typical flavone compounds, the rDA reaction contributes to the formation of major daughter ions of AF and its metabolites by breaking the C-ring (Mullen et al., 2002). Examining the product ions from the rDA reaction and the breaking of the C-C bond between the B- and C-rings can pinpoint whether the phase I reaction happens on the A-, B-, or C-rings. As for phase II metabolites, the conjugation sites of sulfation and glucuronidation were putatively assigned based on the common rules of drug metabolism and previous knowledge of the N-glucuronidation of hydroxylamine (Kadlubar et al., 1977; Kaderlik et al., 1994).

As mentioned above, previous studies on the AF bioactivation and metabolism have focused on the CYP1A P450s, CYP1A1 and CYP1A2 (Kuffel et al., 2002; Loaiza-Perez et al., 2004b). Results from urinary metabolite profiling (Table 2), in vitro incubation (Figs. 5 and 6), and chemical inhibition by -naphthoflavone (Fig. 7D) in this study also support the view that CYP1A2 plays an important role in AF metabolism, especially for the 3-hydroxylation reaction. However, as shown in Table 2, mice without CYP1A2 can still extensively metabolize AF, which suggests that CYP1A2 was not the sole enzyme responsible for AF metabolism in vivo. Indeed, incubation of AF with a panel of recombinant human cytochromes P450 revealed that CYP2C9 and CYP2C19 have significant N-hydroxylation activities (Fig. 6), and enzyme kinetic analysis further indicated that CYP2C19 has a pharmacologically relevant K_m value for the formation of hydroxylamines (II and III) (Fig. 7). Although mephenytoin, a CYP2C19 substrate and inhibitor, did not inhibit the HLM-mediated metabolism, which is possibly attributed to the ineffective competitive inhibition mechanism, a preliminary inhibition experiment showed that anti-rat CYP2C11 antibody can significantly inhibit the N-hydroxylation reactions catalyzed by MLMs (data not shown). It is known that there is some degree of overlap in the substrate specificity of CYP1A and CYP2C enzymes (Venkatakrishnan et al., 1999; Otake and Walle, 2002). Because of their prominence in hepatic xenobiotic metabolism system, CYP2C enzymes could contribute to the N-hydroxylation of AF, although the 3-hydroxylation reaction is highly CYP1A2-selective. Further in vivo and in vitro studies are required to understand the role of CYP2C P450s in AF metabolism and the potential interspecies difference.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Major in vivo AF metabolism pathways. Both phase I- and II-metabolizing enzymes can convert AF (I), collectively or separately, into more hydrophilic metabolites. P450-mediated oxidation reactions at C3, N5, and N4' positions transform AF to three monohydroxylated metabolites (II-IV), which can be further metabolized to two dihydroxylated metabolites (V and VI). Among these reactions, 3-hydroxylation is CYP1A2-selective. Besides direct N-conjugation of AF (I) to metabolite X, UDP-glucuronosyltransferase (UGT) can also convert phase I metabolites (II, III, V, and VII) into glucuronides (XI-XIV), respectively. Furthermore, cytosolic SULTs can metabolize phase I metabolites (II, IV, and VI) to sulfates (VII-IX), respectively. Solid line, phase I oxidation reaction; dashed line, phase II conjugation reaction. Thicker line, pathway to major urinary metabolites in oneor all mouse lines in this study; thinner line, pathway to minor urinary metabolites.

One of the drawbacks of using mouse urine samples for metabolism studies, especially for the purpose of quantitation, is the high degree of variance in urine volume and quality caused by dosing, water intake, evaporation, and contamination from diet and feces. By calculating the relative percentage of each metabolite in the whole metabolite cluster, the aforementioned variability problem among the samples can be significantly reduced since the concentrations of all urinary components change proportionally with the alteration in experimental conditions. As shown in Table 2, the results from this study demonstrated that urinary metabolite profiling based on the relative composition was able to establish a data set with small variance; therefore, it can function as an efficient tool to investigate the influence of genetic modification on drug metabolism. This strategy may also be applicable to drug-drug interaction studies.

The use of genetically modified animal models, including gene knockout and transgenic mice, provides a powerful tool for studying xenobiotics metabolism. Cyp1a2-null mice have been used to demonstrate the important role of CYP1A2 on the pharmacokinetics and metabolism of caffeine (Buters et al., 1996) and CYP1A2-humanized mice to the interspecies difference in metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Cheung et al., 2005). This study further proved the value of both animal models for studying the metabolism of CYP1A2 substrates as well as interspecies differences between human and mouse. Besides their application to drug metabolism, these animal models can also be used to determine the role of drug-metabolizing enzymes in the metabolism of endogenous chemicals and the modulation of normal homeostasis and for understanding the role of a candidate enzyme in the carcinogenicity of known carcinogens (Gonzalez, 2003).

In summary, by combining the accurate mass measurement and the software-assisted global metabolite profiling strategy, 13 AF metabolites, from CYP-mediated oxidation and phase II glucuronidation and sulfation, were identified, and a comprehensive in vivo metabolic pathway of AF was constructed (Fig. 8). Besides one known metabolite, 12 novel metabolites were structurally elucidated by LC-MS/MS analysis. Significant differences in the relative composition of urinary metabolites among three mouse lines demonstrated that CYP1A2 plays an important role in the AF metabolism, although it is not the sole enzyme capable of oxidizing AF; 3-hydroxylation is a CYP1A2-selective reaction; and there is a clear interspecies difference between mouse and human CYP1A2 in the metabolism of AF. In vitro incubations of AF with microsomes and recombinant P450s as well as enzyme kinetics confirmed these observations and further indicated that CYP2C enzymes might also contribute to AF metabolism.

	Acknowledgements

 
We thank the NCI Developmental Therapeutics Program for providing aminoflavone.

	Footnotes

 
doi:10.1124/jpet.106.105213.

This work was supported by the NCI Intramural Research Program of the National Institutes of Health and by the U.S. Smokeless Tobacco Company (grant for collaborative research to J.R.I.).

ABBREVIATIONS: NSC686288, 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methylchromen-4-one; AF, aminoflavone; NCI, National Cancer Institute; SULT, sulfotransferase; HLM, human liver microsome; UPLC, ultraperformance liquid chromatography; TOFMS, time-of-flight mass spectrometry; MLM, mouse liver microsome; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MS², tandem mass spectrometry; rDA, retro Diels-Alder reaction; N⁵-OH-AF, N⁵-hydroxy-AF; N⁴'-OH-AF, N⁴'-hydroxy-AF; 3-OH-AF, 3-hydroxy-AF; RT, retention time.

Address correspondence to: Dr. Frank J. Gonzalez, Laboratory of Metabolism, National Cancer Institute, 37 Convent Drive, Bethesda, MD 20892. E-mail: fjgonz{at}helix.nih.gov

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