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Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.-W.Z., G.-B.G., Y.L., X.-B.L., Y.-Y.Z., W.L., L.Y.); Laboratory of Medical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (H.-B.X.); The Second Affiliated Hospital of Dalian Medical University, Dalian, China (L.-M.W., J.S.); Shanghai University of Traditional Chinese Medicine, Shanghai, China (Y.-Q.H., Z.-T.W.); and Graduate School of Chinese Academy of Sciences, Beijing, China (J.-W.Z., G.-B.G., Y.-Y.Z., W.L.)
(Received August 16, 2007; accepted November 21, 2007)
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Abstract |
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(M2) were proposed as the possible hydroxylation sites, and the structure of M1 was confirmed by 1H NMR. Chemical inhibition studies and assays with recombinant human cytochromes P450 (P450s) indicated that 4''-hydroxycephalomannine was generated predominantly by CYP3A4 and 6-hydroxycephalomannine by CYP2C8. The overall biotransformation rate between paclitaxel and cephalomannine differed slightly (184 vs. 145 pmol/min/mg), but the average ratio of metabolites hydroxylated at the C13 side chain to C6 for paclitaxel and cephalomannine varied significantly (15:85 vs. 64:36) in five human liver samples. Compared with paclitaxel, the major hydroxylation site transferred from C6 to C4'', and the main metabolizing P450 changed from CYP2C8 to CYP3A4 for cephalomannine. In the incubation system with rat or minipig liver microsomes, only 4''-hydroxycephalomannine was detected, and its formation was inhibited by CYP3A inhibitors. Molecular docking by AutoDock suggested that cephalomannine adopted an orientation in favor of 4''-hydroxylation, whereas paclitaxel adopted an orientation favoring 3'-p-hydroxylation. Kinetic studies showed that CYP3A4 catalyzed cephalomannine more efficiently than paclitaxel due to an increased Vm. Our results demonstrate that relatively minor modification of taxane at C3' has major consequence on the metabolism.
). The superior antitumor activity of paclitaxel, however, is undermined by limitations such as multidrug resistance, poor aqueous solubility, poor oral bioavailability, and toxicities (Frapolli et al., 2006). It is generally recognized that poor oral bioavailability of paclitaxel results from its poor solubility (Singla et al., 2002) and P-glycoprotein efflux (Sparreboom et al., 1997). To overcome these shortcomings, numerous novel taxanes superior to paclitaxel were synthesized (Ojima et al., 1996; Mastalerz et al., 2003; Barboni et al., 2005; Lockhart et al., 2007), and some of them such as BAY59-8862 (Sano et al., 2006), BMS-275183 (Broker et al., 2007), and MAC-321 (Lockhart et al., 2007) entered clinical trials. Many of these novel taxanes had modified substituents at C3' of the C13 side chain, where two phenyls were replaced by other functional groups (Sano et al., 2006; Broker et al., 2007; Lockhart et al., 2007). These structural modifications made the novel taxanes effective against paclitaxel-resistant tumor cells, more soluble, and/or more potent. It is still not clear how these structural changes at C3' affect taxane's metabolism property, which is crucial to efficacy, toxicity, route of administration, pharmacokinetics, and pharmacodynamics (Taniguchi et al., 2005; Spratlin and Sawyer, 2007).
P450-mediated oxidative metabolism is the major elimination routine for taxanes (Anderson et al., 1995; Cresteil et al., 2002), which differ strikingly from one another in metabolism despite their structural similarity. For example, when benzamide at C3' in paclitaxel was replaced by the N--tert-butyloxycarbonyl amino group in docetaxel, the principal hydroxylation site transferred from C6 of the taxane ring to the tert-butyl of the side chain (Cresteil et al., 2002; Taniguchi et al., 2005). Meanwhile, the primary monooxygenases changed from CYP2C8 to CYP3A4 (Cresteil et al., 2002; Taniguchi et al., 2005). Interestingly, when both phenyls at C3' of C13 side chain were replaced by the alkyl or alkenyl group, the major hydroxylation site was again moved from tert-butyl back to the isobutyl or isobutenyl group, such as SB-T-1102, SB-T-1214, SB-T-1216 (Gut et al., 2006), and IDN5390 (Frapolli et al., 2006). All of these results suggested that substituents at C3' determined taxane metabolism with regard to the site of hydroxylation and the P450 isoform implicated.
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). It differs from paclitaxel only in the amide portion; the substituent at the C3' position is tigloylamide instead of benzamide (Fig. 1). Due to structural similarity, cephalomannine shows antitumor activity comparable to paclitaxel toward several different cell lines (Helson and Ainsworth, 1997; Shi et al., 2001). It is especially effective against leukemia and certain neural tumor cells (Helson and Ainsworth, 1997). Despite its promising antitumor activity, relatively little is known about the metabolism of cephalomannine. In this study, we chose cephalomannine, an analog of paclitaxel with slight modifications at C3', to study the effect of structural change on metabolism. LC/MS/MS and 1H NMR were recruited to identify the structures of the observed metabolites. Incubations were conducted with P450 isoform-specific inhibitors and recombinant human P450 isoforms to ascribe individual biotransformation to a single P450 isoform. To compare species difference, the metabolism of cephalomannine in rat and minipig liver microsomes was also examined.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-Hydroxypaclitaxel was obtained from BD Gentest (Woburn, MA). Quercetin was purchased from Acros Organics (Geel, Belgium). Cephalomannine (96%) was purchased from Shanghai Jinhe Bio-Technology Co. (Shanghai, China). All other reagents were of HPLC-grade or of the highest grade commercially available. cDNA-expressed recombinant CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 derived from baculovirus-infected insect cells coexpressing NADPH-P450 reductase were obtained from BD Gentest. cDNA-expressed CYP2C8 and CYP2C19 in Escherichia coli coexpressing NADPH-P450 reductase were purchased from New England Biolabs (Beijing, China). Preparation and Characterization of Liver Microsomes. Human livers were obtained from autopsy samples (n = 5, male Chinese, ages 27–48) from Dalian Medical University (Dalian, China), with the approval of the ethics committee of Dalian Medical University. The medication history of the donors was not known. Research involving human subjects was done under full compliance with government policies and the Helsinki Declaration.
Procedures involving animals complied with the Laboratory Animal Management Principles of China. Sprague Dawley rats (n = 10, male, weight 180–220 g) were purchased from Dalian Medical University. The animals had free access to tap water and pellet diet. P450 was induced by phenobarbital (80 mg/kg i.p.) in 24-h intervals for 3 days. The rats were euthanized by decapitation, and livers were rapidly excised and pooled for preparation of microsomes.
Colony-bred Chinese Bama minipigs weighing 10 to 12 kg (n = 6, male, age 6 months) were obtained from the Department of Animal Science, Third Military Medical University, Chongqing, China. The animals used are descendants of sows and boars obtained from the original stock at Bama County, Guangxi Province, China. The animals were euthanized by intravenous injection of pentobarbital sodium (150 mg/kg b.wt.); tissue samples were taken from the left medial lobe of the liver within 5 min after death. Liver samples were pooled together to prepare microsomes.
Liver specimens were stored in liquid nitrogen until preparation of microsomes. Microsomes were prepared from liver tissue by differential ultracentrifugation as described previously (Li et al., 2006). Protein concentration was determined by using bovine serum albumin as standards (Lowry et al., 1951). Total P450 concentration was determined according to Omura and Sato (1964) with the use of molar extinction coefficient 91 mM–1 · cm–1. Liver microsomes were diluted to 10 mg/ml and were stored at –80 °C. P450 concentration was 0.63, 0.52, and 0.23 to 0.39 nmol/mg in minipig, rat, and human liver microsomes, respectively.
Incubation System. The incubation mixture, with a total volume of 200 µl, consisted of 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, and 4 mM MgCl2), liver microsomes (0.5 mg/ml), and 10 µM cephalomannine or paclitaxel. Taxanes were previously dissolved in methanol, with a final methanol concentration below 1% (v/v) in the reaction mixture. After 3-min preincubation at 37°C, the reaction was initiated by adding the NADPH-generating system. After incubation for 30 min in a shaking water bath, the reaction was terminated by the addition of methanol (100 µl). The mixture was kept on ice until it was centrifuged at 20,000g for 10 min at 4 °C. Aliquots of supernatants were transferred for HPLC analysis. Control incubations without NADPH or without substrate or without microsomes were carried out to ensure that metabolite formation was microsome- and NADPH-dependent. The metabolites of cephalomannine were quantified based on the calibration curves of cephalomannine, assuming similar molar extinction coefficients. The metabolites of paclitaxel were quantified by comparing the calibration curves of 6-hydroxypaclitaxel and 3'-p-hydroxypaclitaxel.
HPLC/MS Method. The HPLC system (Shimadzu, Kyoto, Japan) consisted of an SCL-10A system controller, two LC-10AT pumps, an SIL-10A autoinjector, and an SPD-10AVP UV detector; a Shim-pack (Shimadzu) C18 column (4.6 x 150 mm, 5 µ) was used to separate taxanes and its metabolites. The mobile phase was 65% methanol in water. The eluent was monitored at 230 nm with a flow rate of 1.0 ml/min.
LC/MS was used to characterize the structures of cephalomannine metabolites. The HPLC eluent from detector was introduced into the mass spectrometer via a 1:4 split. The mass spectrometer was a TSQ triple quadrupole (Thermo Fisher Scientific, Waltham, MA) equipped with an electrospray ionization interface. The spray voltage was 4.5 kV, and the capillary temperature was 300°C. Nitrogen was used as nebulizing and auxiliary gas. The nebulizing gas backpressure was set at 40 psi, and the auxiliary gas was set at 20 arbitrary units. For MS/MS, argon at a pressure of 3 mTorr was used as the collision gas. The collision energy was set at 15 to 30 V. Initially, the mass spectrometer was programmed to perform full scans between m/z 200 and 1000 to observe the [M+H]+ and [M-H]– signals.
Metabolite Purification. Rat liver microsomes (RLMs) catalyzed cephalomannine efficiently to one of its metabolites (M1), which allowed for the preparation of sufficient quantities of M1 for 1H NMR analysis. The incubation system was scaled up to 250 ml. Cephalomannine (50 µM) was incubated with RLMs (1.0 mg/ml) and the NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, and 4 mM MgCl2) for 90 min at 37 °C. Under these conditions, approximately 20% of cephalomannine was converted to M1. The reaction mixture was extracted with 50% ethyl acetate, and the organic layer was separated after the reaction mixture was centrifuged at 9000g for 10 min. The extraction procedure was repeated three times, and the organic layer was combined. The organic phase was dried in vacuo, and the residue was dissolved in methanol (1.5 ml). Cephalomannine and its metabolite were separated by HPLC, and the eluent containing the metabolite was collected and dried in vacuo. The purity of M1 was approximately 92% (HPLC).
NMR Spectroscopy. Proton NMR spectra were obtained at 400 MHz on a Bruker AV-400 spectrometer (Bruker, Newark, DE). Compounds were dissolved in CDCl3, and experiments were conducted at 21°C. Chemical shifts are reported in parts per million with reference to tetramethylsilane.
Chemical Inhibition Studies. Chemical inhibition studies were performed by adding different human P450 inhibitors to the incubation mixture of cephalomannine (20 µM) before the addition of the NADPH-generating system. Information concerning the plasma concentration of cephalomannine was lacking. Selection of a 20-µM concentration was based on the plasma concentration of paclitaxel (Cmax, 14 µM) (Bhalla et al., 1999). The selective inhibitors (or substrates) of eight major P450s and their concentrations were as follows (Bjornsson et al., 2003; Huang et al., 2007): furafylline (10 µM) for CYP1A2, 8-methoxypsoralen (2.5 µM) for CYP2A6, quercetin (20 µM) for CYP2C8 (Harris et al., 1994), sulfaphenazole (10 µM) for CYP2C9, S-mephenytoin (100 µM) for CYP2C19, quinidine (10 µM) for CYP2D6, clomethiazole (50 µM) for CYP2E1, and ketoconazole (1 µM) for CYP3A4. 8-Methoxypsoralen is known as a mechanism-based inhibitor, so it was preincubated with human liver microsomes (HLMs), buffer, and the NADPH-generating system at 37°C for 10 min, and the reaction was initiated by the addition of cephalomannine.
Troleandomycin (Anzenbacher et al., 1998), ketoconazole (Li et al., 2006), sulfaphenazole (Kobayashi et al., 2003), and furafylline (Kobayashi et al., 2003) were found to be inhibitors of rat or minipig CYP3A, CYP2C, and CYP1A, respectively. Therefore, inhibitory effects of troleandomycin (25 µM), ketoconazole (1 µM), sulfaphenazole (10 µM), and furafylline (10 µM) toward cephalomannine (10 µM) metabolism in RLMs and minipig liver microsomes (PLMs) were examined. Troleandomycin was preincubated with liver microsomes, buffer, and the NADPH-generating system at 37°C for 10 min, and the reaction was initiated by the addition of cephalomannine.
Assay with Recombinant Human P450s. cDNA-expressed recombinant human P450 isoforms coexpressing NADPH-P450 reductase either from insect cells (CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4) or from E. coli (CYP2C8 and CYP2C19) were used. The incubations were carried out as described for the human liver microsomal study. To examine the contribution of each P450 isoform, cephalomannine (60 µM) was incubated with each of the recombinant P450s (40–80 pmol of P450 per milliliter) at 37°C for 60 min. HPLC with UV detection was used to monitor possible metabolites. Relative high substrate concentration was selected so that adequate metabolites were generated for the convenience of detection.
Kinetic Assays. To estimate kinetic parameters, cephalomannine (4.0–70 µM) or paclitaxel (2.0–50 µM) was incubated with liver microsomes (0.5 mg/ml) or recombinant P450s (40 nM) for 30 min. For RLMs, the incubation time was reduced to 10 min because of extensive metabolism of cephalomannine. The apparent Vm and Km values were calculated from nonlinear regression analysis of experimental data according to the Michaelis-Menten equation, and the results were graphically represented by double-reciprocal plots of velocities versus concentrations of substrate. The H1 human liver microsomal sample was used in kinetics and inhibition studies because it exhibited high activity of CYP3A4 and CYP2C8 and enabled analysis of both metabolites at low substrate concentrations. Preliminary experiments were carried out to make sure that the formation of metabolites was in the linear range of both reaction time and the concentration of liver microsomes. All incubations were carried out in duplicate with S.D. values generally below 10%.
Molecular Docking. AutoDock 4.0 (Scripps Research Institute, La Jolla, CA) (Morris et al., 1998) was used to dock paclitaxel and cephalomannine to the active site CYP3A4 (PDB entry 2j0d) (Ekroos and Sjogren, 2006). The cocrystallized ligand erythromycin was removed from the protein structure before docking. Three-dimensional structures of paclitaxel and cephalomannine were obtained from the National Cancer Institute database of small molecules (http://129.43.27.140/ncidb2). AutoDockTools 1.4.5 (Scripps Research Institute) (Sanner, 1999) was used to prepare all docking parameter files for both taxanes and CYP3A4. A 60- x 60- x 60-point grid with a spacing of 0.375 Å centered at –15.0, –17.0, and 25.0 was used for the protein model. For other parameters, the default values were selected. The poses with the lowest docking energy were selected for analysis.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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LC/MS/MS was employed to elucidate the structures of cephalomannine metabolites. Both positive-ion mode and negative-ion mode were examined in the LC/MS. In the positive-ion mode, the following fragments were observed for the cephalomannine (Table 1): m/z 832.7, [M+H]+; m/z 854.9, [M+Na]+; m/z 870.7, [M+K]+; m/z 509.6, i.e., consecutive loss of C13 side chain and acetic acid; m/z 309.8, i.e., consecutive loss of C2 side chain, acetic acid, and water on m/z 509.6; m/z 264.6, i.e., C13 side chain; and m/z 246.5, i.e., water loss on m/z 264.6. These fragments were in agreement with Vivekanandan et al. (2006). Characteristic fragment ions of metabolites were 16 mass units greater than that of cephalomannine, indicating that both metabolites were monohydroxylated (Table 1). For M1, m/z 509.3 and 280.2 indicated that the hydroxylation site was in the C13 side chain but not in the taxane core ring (Table 1). For M2, m/z 525.3, 325.3, and 264.3 implied that the hydroxylation site was in the taxane core ring but not in the C2 or C13 side chain (Table 1).
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Consistent with Song et al. (2005), the negative-ion mode provided better sensitivity than the positive-ion mode; therefore, negative-ion mode was adopted for MS/MS analysis in this investigation. MS spectra were dominated by [M+AcO]– in these conditions; therefore, [M+AcO]– was selected as a precursor ion to be further fragmented. Fragment ions of cephalomannine (Table 2) included m/z 890.7, [M+AcO]–; m/z 830.7, [M-H]–; m/z 770.6, [M-AcOH-H]–; m/z 525.5 (base peak), [M-Ac-C13-H]–, i.e., consecutive loss of hydrogen, acetyl, and C13 side-chain-breaking C13 ester bond; m/z 373.4, [525.5-BzOH-HCHO]–, i.e., loss of C2 benzoic acid and formaldehyde on m/z 525.5; and m/z 262.5, i.e., C13 side chain. For M1, m/z 278.4 and m/z 525.5 implied that the hydroxylation site was in the C13 side chain and not in the taxane ring, and the m/z shift from 525.5 to 373.2 excluded the benzoate ring at C2 as the hydroxylation site (Table 2). Similarly, for M2, m/z 541.4, 389.6, and 262.3 suggested that the hydroxylation site was in the taxane ring but not in the C13 side chain or C2 benzoate ring (Table 2). The hydroxylation sites deduced from the negative-ion mode were in agreement with that from the positive-ion mode.
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The 1H NMR spectra of cephalomannine and its metabolite (M1) indicated that the phenyls in the C13 side chain were intact (Table 3). In agreement with previous reports (Miller et al., 1981; Chen et al., 2001), the most distinct spectra changes were involved in the tigloylamide group. The 4''-methyl proton signal at 1.73 ppm (3H, d, J = 6.8 Hz) was replaced by a signal at 4.25 ppm (2H, t, J = 5.6 Hz). The corresponding 3''-proton signal changed from 6.43 ppm (1H, q, J = 7.2 Hz) to 6.36 ppm (1H, t, J = 6.0 Hz). These observations suggested that the hydroxylation site was in 4''-methyl group. Another proton signal that appeared at 3.70 ppm (1H, t, J = 4.8 Hz) was assigned to 4''-OH (Table 3). The structure of M2 (RT, 10.9 min) could not be ascertained from the fragment ions. In parallel with hydroxylation sites of paclitaxel and some other taxanes (Cresteil et al., 2002; Frapolli et al., 2006), we propose C6 as the possible hydroxylation site for M2 (Fig. 1).
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Different Metabolism between Cephalomannine and Paclitaxel. To compare the metabolism between cephalomannine and paclitaxel, we studied the metabolism of two taxanes in five individual human liver samples (H1, H2, H3, H4, and H5); the results are shown in Fig. 3. For comparison, substrate concentration (10 µM), microsomal concentration (0.5 mg/ml), and reaction time (30 min) were all kept the same. Under these conditions, there was little difference between cephalomannine and paclitaxel in the overall biotransformation rate (145 vs. 184 pmol/min/mg). For paclitaxel, 6-hydroxypaclitaxel by CYP2C8 was always the dominant product in five liver samples, and the average ratio of the two metabolites hydroxylated at 6 to C3' was 85:15 (Fig. 3). For cephalomannine, however, 4''-hydroxycephalomannine instead of 6-hydroxycephalomannine was the main metabolite in four of the five liver samples, and H3 produced approximately the same amount of the two metabolites (Fig. 3). The average ratio of the two metabolites hydroxylated at 6 to C4'' was 36:64.
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-Hydroxycephalomannine was absent in both liver microsomal incubations. For paclitaxel, only 3'-p-hydroxypaclitaxel was detected after paclitaxel (50 µM) was incubated with RLMs (0.5 mg/ml) for 30 min (Table 4). Neither 3'-p-hydroxypaclitaxel nor 6-hydroxypaclitaxel was detected after paclitaxel (50 µM) was incubated with minipig liver microsomes (0.5 mg/ml) for 30 min (Table 4), although the minipig liver microsomes sample showed high testosterone 6β-hydroxylation activity (Li et al., 2006).
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Chemical Inhibition Studies. Selective inhibitors of the eight major P450s were used to screen the P450 isoforms responsible for cephalomannine metabolism in humans (Fig. 4). Among tested inhibitors, ketoconazole inhibited the activity of cephalomannine 4''-hydroxylation by approximately 90% but did not significantly inhibit the formation of 6-hydroxycephalomannine. Quercetin, on the other hand, inhibited the activity of cephalomannine 6-hydroxylation by approximately 90% but did not remarkably inhibit the formation of 4''-hydroxycephalomannine. Inhibitors of other P450 isoforms did not show significant inhibition (less than 30% inhibition) toward the formation of either metabolite. These results revealed that CYP3A4 was involved in cephalomannine 4''-hydroxylation and CYP2C8 in 6-hydroxylation.
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-hydroxylation was catalyzed by CYP2C8 (1.29 pmol/min/pmol P450). Other P450 isoforms did not produce detectable amount of metabolite (less than 0.01 pmol/min/pmol P450). Therefore, cephalomannine 6-hydroxylation was ascribed to CYP2C8, and cephalomannine 4''-hydroxylation was ascribed to CYP3A4.
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- and 3'-p-hydroxylation were similar (13 vs. 17 µM), but their Vm values showed approximately 3-fold difference (233 vs. 84 pmol/min/mg). The catalytic efficiency of 6-hydroxylation, as measured by Vm/Km, was approximately 3-fold higher than 3'-p-hydroxylation (17 vs. 5). On the other hand, cephalomannine 6- and 4''-hydroxylation showed approximately 3-fold difference in their Km values (52 vs. 16 µM) despite the fact that the Vm values were similar (234 vs. 275 pmol/min/mg). The catalytic efficiency of 6-hydroxylation, as reflected by Vm/Km, was 4-fold lower than 4''-hydroxylation (4 vs. 17).
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In RLMs, the Km values toward cephalomannine and paclitaxel differed only slightly (15 vs. 21 µM; Table 4). The 12-fold difference in their Vm values (1853 vs. 153 pmol/min/mg) made RLMs metabolize cephalomannine much more efficiently than paclitaxel (Vm/Km, 125 vs. 7). In PLMs, the Km and Vm values for cephalomannine 4''-hydroxylaiton were 11 µM and 108 pmol/min/mg (Table 4), and the catalytic efficiency of cephalomannine was approximately 10-fold lower in PLMs than in RLMs (Vm/Km, 10 vs. 125).
Kinetic parameters of cephalomannine and paclitaxel metabolism in recombinant CYP3A4 and CYP2C8 are shown in Table 5. CYP3A4 catalyzed cephalomannine more efficiently than paclitaxel (Vm/Km, 195 vs. 16) mainly by an increased Vm (1600 vs. 91 pmol/min/nmol P450). On the other hand, increased Km (33 vs. 8.5 µM) made CYP2C8 transform cephalomannine less efficiently than paclitaxel (Vm/Km, 150 vs. 221).
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Taxanes are known to be hydrophobic compounds, and the aqueous solubility of paclitaxel is 35 µM (Swindell et al., 1991). The addition of 1% methanol slightly increases the solubility. The solubility limitation makes it impossible to extend the substrate concentration further to cover the range of a 5-fold Km value. Therefore, the Km might be overestimated, and the resulting intrinsic clearance might be inaccurate because of the narrow substrate concentration range.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Cresteil et al., 2002).10-Acetyl and the C13 side chain were found to affect taxane's regioselective oxidation (Cresteil et al., 2002). However, more evidence indicated that the regioselective hydroxylation was determined by the substituents at C3' (Frapolli et al., 2006; Gut et al., 2006). Many of the new-generation taxanes had modified substituents at C3' compared with paclitaxel, such as BAY59-8862 (Sano et al., 2006), IDN5390 (Ferlini et al., 2005), BMS-275183 (Broker et al., 2007), MAC-321 (Lockhart et al., 2007), and SB-T-1102 (Ojima et al., 1996). It would be helpful to synthesize metabolism-stable taxanes if we knew how substituents at C3' affected taxane's metabolism. Therefore, we selected a pair of analogs slightly different only at C3', cephalomannine and paclitaxel, to evaluate the effect of slight structural change on metabolism.
In the incubation system with HLMs, two cephalomannine metabolites (M1 and M2) were detected, and M1 was identified to be 4''-hydroxycephalomannine. Mass spectra suggested that the hydroxylation site of M2 was in the taxane core ring but not in the C2 or C13 side chain or in the acetyls at C4 or C10. C6 was proposed as the possible hydroxylation site for M2. Chemical inhibition studies and assays with recombinant human P450s indicated that 4''-hydroxycephalomannine was produced predominantly by CYP3A4 and 6-hydroxycephalomannine by CYP2C8. In five different human liver samples, cephalomannine 6-hydroxylation correlated with paclitaxel 6-hydroxylation (CYP2C8, r2 = 0.95), and cephalomannine 4''-hydroxylation correlated with paclitaxel 3'-p-hydroxylation (CYP3A4, r2 = 0.96). Relative poor correlations were found between paclitaxel 6-hydroxylation (CYP2C8) and paclitaxel 3'-p-hydroxylation (CYP3A4, r2 = 0.64) and between cephalomannine 6-hydroxylation and cephalomannine 4''-hydroxylation (r2 = 0.40). These results were in agreement that M1 was produced by CYP3A4 and M2 was produced by CYP2C8.
In humans, paclitaxel was primarily hydroxylated by CYP2C8 at C6 and secondarily by CYP3A4 at C3' (Harris et al., 1994). Although 3'-p-hydroxypaclitaxel could be the dominant metabolite in certain cases (Desai et al., 1998; Taniguchi et al., 2005), 6-hydroxypaclitaxel was generally regarded as the major product, and the ratio of two metabolites hydroxylated at C6 to C3' was approximately 80:20 in HLMs (Venkatakrishnan et al., 2003; Taniguchi et al., 2005). This result was in agreement with the present study, in which the average ratio of paclitaxel metabolites hydroxylated at C6 to C3' in five liver microsomes was 85:15 (Fig. 3). For cephalomannine, on the other hand, the average ratio of the two metabolites hydroxylated at 6 to C4'' was 36:64 in the same panel of liver samples (Fig. 3). Therefore, the main hydroxylation site transferred from C6 to C4'', and the main metabolizing P450 for cephalomannine changed from CYP2C8 to CYP3A4 compared with paclitaxel. In vitro antitumor activity of 4''-hydroxycephalomannine was more than 60-fold less potent than cephalomannine (Chen et al., 2001); therefore, cephalomannine 4''-hydroxylation could be regarded as a detoxification process.
From kinetics of paclitaxel and cephalomannine hydroxylation (Tables 4 and 5), it can be concluded that the presence of a tigloyl group instead of a benzoyl group at C3' reduces the contribution of CYP2C8 by decreasing the affinity for the protein. It probably indicates that the hydrophobic interactions with the CYP2C8 peptide chain as suggested previously (Cresteil et al., 2002) decrease when the benzoyl is replaced by the tigloyl. On the other hand, the replacement of benzamide by tigloylamide increases the overall metabolism by CYP3A via an increased Vm in human, rat, and minipig liver microsomes and recombinant CYP3A4 (Tables 4 and 5). This may imply that alkyl is more susceptible to CYP3A hydroxylation than phenyl in the amide part of taxane. During the P450 catalytic cycle, the target C—H bond would break and form radicals before oxygen was added (Porter and Coon, 1991). The bond dissociation energy (DH) of the C—H bond at C4'' (CH3C = CHCH2—H, DH
88.8 kcal/mol) in cephalomannine is lower than that at 3'-p-phenyl (C6H4—H, DH109 kcal/mol) in paclitaxel (Blanksby and Ellison, 2003), which gives cephalomannine a lower activation energy. When the activation energy decreases, the rate constant would increase according to the Arrhenius equation. Thus, the rate of cephalomannine 4''-hydroxylation is faster than that of paclitaxel 3'-p-hydroxylation.
In contrast to paclitaxel, C4'' instead of 3'-p-phenyl in cephalomannine is hydroxylated. Molecular docking suggests the regioselective metabolism of paclitaxel and cephalomannine at C3' is determined by the ligand and receptor interaction. Another possible explanation is the difference in the DH values. As discussed earlier, substituent with a lower C—H DH value is more susceptible to P450 oxidation because it needs lower activation energy. The DH of C—H at C4'' is lower than that at 3'-p-phenyl; thus, C4'' instead of 3'-p-phenyl in cephalomannine is hydroxylated. The same rationale can also explain the observations that the tert-butyl [OC(CH3)2CH2—H, DH101 kcal/mol] but not the phenyl (C6H4—H, DH109 kcal/mol) at C3' of docetaxel is hydroxylated by CYP3A4 (Cresteil et al., 2002), and the isobutyl [(CH3)2C—H, DH98.6 kcal/mol] or the isobutenyl (CH = CCH2—H, DH88.8 kcal/mol) instead of the tert-butyl [OC(CH3)2CH2—H, DH101 kcal/mol] is hydroxylated in SB-T-1102, SB-T-1214, SB-T-1216 (Gut et al., 2006), and IDN5390 (Frapolli et al., 2006). It should be noted that we neglected other aspects such as steric hindrance, structural modifications at C10, and the influence of nearby functional groups, which may also contribute to the regioselective metabolism.
Similar to the metabolism of paclitaxel (Vaclavikova et al., 2004), species differences in cephalomannine metabolism were observed, and 6-hydroxycephalomannine was missing in the incubation system of RLMs and PLMs. The main metabolites of paclitaxel varied substantially among different species (Vaclavikova et al., 2004), but for cephalomannine, 4''-hydroxycephalomannine was the main metabolite in HLMs, RLMs, and PLMs. In RLMs and PLMs, the activity of cephalomannine 4''-hydroxylation was strongly inhibited by the CYP3A inhibitors troleandomycin and ketoconazole. These observations suggested that human CYP3A4 orthologs in minipig (CYP3A29) and rat (CYP3A1/2) might play a major role in cephalomannine metabolism; however, detailed examinations with recombinant rat and minipig P450s were needed to ascribe the reaction to particular animal P450 isoforms.
In conclusion, the major hydroxylation site and primary P450 isoform are quite different between cephalomannine and paclitaxel despite their structural similarity. Like paclitaxel, C6 of cephalomannine is still the hydroxylation site, whereas the major hydroxylation site transfers to C4'' in the side chain, and CYP3A4 instead of CYP2C8 becomes the main metabolism enzyme. Our results demonstrate that relative minor modifications of taxane at C3' have major consequences on its metabolism, and taxane's C3' substituent with a lower C—H DH value is more susceptible to P450 oxidation.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: P450, cytochrome P450; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; HPLC, high-performance liquid chromatrography; RLM, rat liver microsome; HLM, human liver microsome; PLM, minipig liver microsome; PDB, protein data bank; RT, retention time; DH, bond dissociation energy; BAY59-8862, 13-(N-tert-butoxycarbonyl-β-isobutylisoserinyl)-14-hydroxybaccatin-1,14-carbonate; BMS-275183, 3'-dephenyl-3'-tert-butyl-4-deacetyl-4-O-methoxycarbonyl-10-acetyldocetaxel; MAC-321, 3'-dephenyl-3'-furanyl-7-propionyl-docetaxel; SB-T-1102, 10-acetyl-3'-dephenyl-3'-(2-methylpropyl)docetaxel; SB-T-1214, 10-(cyclopropylcarbonyl)-3'-dephenyl-3'-(2-methylpropenyl)docetaxel; SB-T-1216, 3'-dephenyl-10-(N,N-dimethylcarbamoyl)-3'-(2-methylpropenyl)docetaxel; IDN5390, 13-(N-Boc-3-i-butylisoserinoyl)-C-7,8-seco-10-deacetylbaccatin III.
Address correspondence to: Dr. Ling Yang, Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023 China. E-mail: yling{at}dicp.ac.cn
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