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Of Dimethyl-4 , 4 *-dimethoxy-5 , 6 , 5 * , 6 *-dimethylene Dioxybiphenyl-2 , 2 *-dicarboxylate ( Ddb ) by Human Liver Microsomes : Characterization of Metabolic Pathways and of Cytochrome P 450 Isoforms Involved
| Content Provider | Semantic Scholar |
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| Author | Baek, Min-Sun Kim, Jiyeon Myung, Seung-Woon Yim, Yong Hyeon Jeong, Jin-Hyun Kim, Dong-Hyun |
| Copyright Year | 2001 |
| Abstract | Metabolic fate of DDB and identification of P450 isozymes involved in the metabolism of DDB were investigated in human liver microsomes. DDB was rapidly metabolized to five different metabolites, and the structures of each metabolite were characterized based on UV, mass, and NMR spectral analyses. The major metabolic pathways of DDB in human liver microsomes were identified as Odemethylation of the carboxymethyl moiety (M4) and demethylenation of the methylenedioxyphenyl group (M2). The intramolecular lactonization between the hydroxyl group at the C6 and carboxymethyl group at the C2* of M2 resulted in the generation of M5, which was either hydrolyzed to its hydrolyzed derivative (M1) or further metabolized to the O-demethylated derivative (M3). The interconversion of M1, M2, and M5 took place nonenzymatically depending on the solvent condition. M5 was predominantly detected at the acidic condition, whereas M1 was preferentially detected at the basic environment. Cytochrome P450 (P450) isoform(s) involved in the metabolism of DDB was identified using several in vitro approaches. Chemical inhibition using isoformselective P450 inhibitors, correlation of DDB metabolites formation with several isoform-specific P450 activities in a panel of liver microsomes, metabolism by microsomes derived from P450 cDNA-expressed B-lymphoblastoid cells, and immunoinhibition by isoform-specific anti-P450 antibodies collectively indicated that CYP1A2, CYP2C9, and CYP3A4 are responsible for the metabolism of DDB. O-Dealkylation of the carboxymethyl group was preferentially catalyzed by CYP1A2, whereas demethylenation of the methylenedioxyphenyl moiety was catalyzed by CYP3A4 and CYP2C9. Dimethyl-4,49-dimethoxy-5,6,59,69-dimethylene dioxybiphenyl2,29-dicarboxylate (DDB) is a synthetic compound derived from Schizandrin C, a component of Fructus schizandrae. DDB protects liver against carbon tetrachloride-, D-galactosamine-, thioacetamide-, and prednisolone-induced hepatic injury in experimental animals, although the exact mechanism is not well characterized (Liu et al., 1979, 1982). This compound has also been reported to be effective in improving liver functions of patients with chronic hepatitis (Lee et al., 1991). Currently, DDB is the most widely used remedy for patients with chronic viral hepatitis B in Asia. Plasma concentration of DDB after oral administration was relatively low (Dr. Y. Lee, Chunman University, Kwang-Ju, Korea, personal communication), and the halflife of this compound was 2 to 3 h, suggesting that metabolism of DDB plays a role in clearing the drug from the body. DDB was reported to modulate cytochrome P450 (P450) activities and glutathioneS-transferase. Several investigators reported that DDB alone or in combination with garlic oil induced CYP2B1/2 levels in rats (Liu et al., 1981; Kim et al., 1995). However, few studies have dealt with the metabolism and pharmacokinetics of DDB. The information on the ability of DDB to modulate drug-metabolizing enzymes led us to hypothesize that P450 enzymes in the liver could metabolize DDB. In this study, we investigated the in vitro metabolism of DDB in the presence of human liver microsomes. Five metabolites were identified using mass and NMR spectral analysis. We then identified the P450 isoforms mainly involved in the metabolism of DDB using several approaches: 1) the effect of selective chemical inhibitors on DDB metabolism, 2) determination of DDB metabolism by microsomes from cells expressing recombinant P450 isoforms, and 3) study of the correlation of DDB metabolism with marker activities of each P450 in different human liver microsomes. Experimental Procedures Materials. DDB (Fig. 1) was synthesized at the Kyung-Dong Pharmaceutical Co. (Kyungkido, Korea). Glucose 6-phosphate, b-NADP, glucose-6phosphate dehydrogenase, troleandomycin, diethyldithiocarbamate, and tolbutamide were purchased from Sigma Chemical Co. (St. Louis, MO). This work was supported by Grant N20910 from the Korean Ministry of Science and Technology and Grants (HMP-98-D-5-0047) from the Korean Ministry of Health and Welfare. 1 Abbreviations used are: DDB, dimethyl-4,49-dimethoxy-5,6,59,69-dimethylene dioxybiphenyl-2,29-dicarboxylate; HPLC, high-performance liquid chromatography; MS, mass spectrometry; ESI, electrospray ionization; EI, electron impact ionization; P450, cytochrome P450. Send reprint requests to: Dong-Hyun Kim, Ph.D., Bioanalysis and Biotransformation Research Center, KIST, P.O. Box 131, Chungryang, Seoul 136-791, Korea. E-mail: dhkim@kist.re.kr 0090-9556/01/2904-381–388$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 4, Part 1 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 245/891538 DMD 29:381–388, 2001 Printed in U.S.A. 381 at A PE T Jornals on N ovem er 6, 2017 dm d.aspurnals.org D ow nladed from Sulfaphenazole and ketoconazole were obtained from RBI/Sigma (Natick, MA). Methyl iodide, d3-methyl iodide, andN,O-bis(trimethylsilyl)-trifluoroacetamide were purchased from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were of the highest commercially available grade. Microsomes. Frozen human liver samples were kindly donated by Dr. Guengerich (Vanderbilt University, Nashville, TN). The microsomal fraction was prepared according to the method described elsewhere (Guengerich et al., 1986). Human B-lymphoblastoid-derived P450 microsomes were purchased from GENTEST Corp. (Woburn, MA). For the correlation experiment, 10 different human liver microsomes were purchased from Human Biologics Inc. (Phoenix, AZ). Microsomal Incubations. Individual incubations (final volume5 0.5 ml) consisted of 1.0 mg/ml microsomal protein in 100 mM phosphate buffer (pH 7.4) with final concentrations of 5 mM glucose 6-phosphate, 1 mM b-NADP, and 1 U/ml glucose-6-phosphate dehydrogenase. DDB, buffer, and microsomes were mixed and kept at 37°C for 3 min, and the reaction was started by adding an NADPH-generating system. Incubations were conducted at 37°C and stopped by adding 0.1 ml 1 N HCl and 1 ml chloroform/isopropanol (85:15, v/v). Organic phase was taken after vortexing for 2 min and dried under nitrogen stream. Residue was reconstituted in a high-performance liquid chromatography (HPLC) mobile phase and injected into a HPLC column. HPLC. All analyses were performed using a Waters M600 liquid chromatography system (Waters Corp., Milford, MA) consisting of M 600 quaternary pump, M717 autosampler, and M486 UV detector operated at 280 nm. Analyses were done at ambient temperature on a Capcell-pak C 8 column (4.63 250 mm, 5mm, Shiseido, Japan). The flow rate was 1.0 ml/min. Separations were conducted using a 25-min gradient: 45% B for 1 min, followed by a linear increase to 70% B over 12 min, then maintained for 10 more min. Solvent A consisted of 10 mM ammonium phosphate adjusted with 1N HCl solution to pH 3.0; solvent B consisted of methanol/water (95:5, v/v). Mass Spectroscopy (MS) and Electrospray MS (ESI-MS).The mass spectrometry system was based on a HP 5989A MS Engine mass spectrometer with a HP 59987A electrospray MS interface (Hewlett Packard, Palo Alto, CA). Samples dissolved in methanol containing 0.1% formic acid were introduced into the ESI interface by the syringe pump through a heated nebulizer probe (150°C) using nitrogen as a nebulizing gas. CapEx voltage was 100 V. Electron Impact Ionization MS (EI-MS). A Hewlett Packard gas chromatography/mass selective detector (5890/5972) was used in EI mass analysis. A cross-linked Ultra-1 capillary column (18-m3 0.2-mm i.d., 0.33mm film thickness; Hewlett Packard) was directly connected to the ion source. Helium at a flow rate of 0.9 ml/min was used as carrier gas. Initial oven temperature was 150°C; it was held for 1 min and subsequently increased by 20°C/min to 300°C and held there for 5 min. Before analysis, isolated metabolites were either methylated by treatment with 200 ml of CH3I/acetone (10:90, v/v) containing 50 mg of K2CO3 at 60°C for 2 h or trimethylsilylated by treatment with 50 ml of N-methyl-N-trimethylsilyl-trifluoroacetamide/CH 3CN (30:70, v/v) at 80°C for 30 min. The reaction mixtures were directly injected into the column in a split mode. NMR Spectroscopy. H NMR experiments for structural elucidation of metabolites were carried out on a Bruker AMX 300 spectrometer in 5-mm tubes (Bruker Analytik GmbH, Rheinstetten, Germany). The metabolites were dissolved in 0.5 ml of CDCl 3 and H NMR spectra were recorded. Chemical shifts for theH NMR spectra are reported in parts per million relative to trimethylsilyl using the residual solvent (CDCl 3) signal at 7.2 ppm. Metabolism by B-Lymphoblastoid Microsomes.Incubations with B-lymphoblastoid microsomes were conducted as described above with 50 mM DDB and microsomes containing 200 pmol of P450/ml, with an incubation time of 2 h. Correlation Analysis. Correlation of the formation of DDB metabolites with P450 isoform-specific activities in the microsomes prepared from 10 different human livers (HepatoScreen Test Kit, Human Biologics) was studied at a concentration of 10 mM DDB. The analysis was done in duplicate, and the experiments were repeated for the conformation. Chemical Inhibition. The chemical inhibitors used in this study were as follows: furafylline for CYP1A2 (Kunze and Trager, 1993); sulfaphenazole for CYP2C9 (Baldwin et al., 1995); quinidine for CYP2D6 (Harris et al., 1994); diethyldithiocarbamate for CYP2E1 (Guengerich et al., 1991); ketoconazole for CYP3A4; and troleandomycin for CYP3A4 (Rodrigues, 1994). Concentration of DDB in all chemical inhibition experiments was 10 mM. The reaction mixture was preincubated for 10 min at 37°C in the presence of inhibitor and an NADPH-generating system before the addition of DDB in the case of mechanism-based inhibitors. Other inhibitors were added directly to the incubation mixture at the start of each experiment. The experiments were repeated with duplicate determination. Immunoinhibition St |
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| Language | English |
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| Resource Type | Article |
