Introduction

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Dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'- dicarboxylate (DDB¹; Fig. 1) is an intermediate process of synthesizing Schizandrin C, a natural compound isolated from Fructus schizandrae chinensis. Clinical experience has shown that DDB is effective in lowering the serum glutamic pyruvic transaminase activity in patients with chronic viral hepatitis (Lee et al., 1991). DDB protects against carbon tetrachloride-, D-galactosamine-, and thioacetamide-induced hepatic injury (Liu and Lesca, 1982; Yu et al., 1987). Moreover, the enhanced effectiveness in combination with garlic oil (Kim et al., 1995) and the protective effect of DDB against ethanol-induced fatty liver (Kim et al., 1999) were reported. Currently, this drug is widely used in Asian countries.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structure of DDB.

Enzyme inactivation often causes an accumulation of drugs during chronic dosing and an increase of the apparent half-life with dose, and potential interactions with coadministered drugs can cause serious side effects in clinical aspects. One of the mechanisms of enzyme inactivation involves a quasi-irreversible coordination of a reactive intermediate(s) to the P450 (Murray and Reidy, 1990). Macrolide antibiotics, including oleandomycin, triacetyloleandomycin, erythromycin, roxithromycin, and clarithromycin, are major compounds to form metabolite-P450 complexes (Babany et al., 1988; Franklin, 1991; Bensoussan et al., 1995). In addition, methylenedioxyphenyl derivatives, such as 1,3-dioxazole and human immunodeficiency virus protease inhibitors, also undergo P450-catalyzed oxidation to form intermediates that coordinate tightly to the prosthetic heme iron (Philpot and Hodgson, 1972; Hodgson and Philpot, 1974; Chiba et al., 1998). DDB possesses the methylenedioxyphenyl moiety, and P450s were demonstrated to be the primary enzymes responsible for the metabolism of DDB in human liver microsomes (Baek et al., 2001).

The objectives of the present investigation were 1) to examine the potential for inhibition of human P450 by DDB, followed by the determination of the inhibition constants in human liver microsomes; and 2) to gain insight into the mechanism of inhibition for the P450 most potentially inhibited by DDB.

	Materials and Methods

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Chemicals. DDB was supplied by Choongwae Pharma Corp. (Kyungki, Korea). Testosterone, glucose 6-phosphate, -NADP⁺, -NADPH, glucose-6-phosphate dehydrogenase, chlorzoxazone, caffeine, coumarin, diclofenac, and 7-hydroxycoumarin were purchased from Sigma Chemical Co. (St. Louis, MO). 6-Hydroxychlorzoxazone and ketoconazole were obtained from Sigma/RBI (Natick, MA). 6-Hydroxytestosterone, 4-hydroxydiclofenac, dextromethorphan, dextrorphan, and 4-hydroxymephenytoin were purchased from Ultrafine (Manchester, UK). All other reagents used were highest grade commercially available.

Microsomal Preparation. Rats were pretreated with a daily intraperitoneal injection of pregnenolone-16-carbonitrile (PCN; 50 mg/kg in corn oil) for 3 consecutive days. Animals were killed 24 h after the last treatment. The livers were excised quickly and perfused with ice-cold 1.15% KCl solution. Human liver tissues were generously donated by Dr. Guengerich (Vanderbilt University, TN). Microsomal fraction was prepared according to the method described elsewhere (Guengerich et al., 1986), and the final pellets were resuspended in 10 mM Tris acetate buffer, pH 7.4, containing 1 mM EDTA and 20% (w/v) glycerol.

Microsomal Incubation. For incubation studies designed to determine the effects of DDB on various P450 isozyme activities, the incubation mixture contained 0.5 mg/ml microsomal protein, NADPH-generating system (5 mM glucose 6-phosphate, 1 mM -NADP⁺, and 1 U/ml glucose-6-phosphate dehydrogenase), DDB (0.25-50 µM), and various P450 isoform-specific substrates in 0.1 M phosphate buffer, pH 7.4. Substrate concentrations were used at approximately their respective K_m values: 1 mM for caffeine, 5 µM for diclofenac, 20 µM for chlorzoxazone, 0.4 µM for coumarin, 40 µM for testosterone, 5 µM for dextromethorphan, and 80 µM for mephenytoin. DDB was dissolved in dimethyl sulfoxide, and other substrates were dissolved in methanol. The solvent was evaporated under a stream of nitrogen gas before adding the incubation mixture, and the final concentration of dimethyl sulfoxide did not exceed 0.5%. For the determination of the K_i value toward testosterone, testosterone concentrations of 10, 20, and 40 µM were used.

P450 Probe Substrate Assays. Testosterone 6-hydroxylation activity, an index of CYP3A activity, was done as described elsewhere with minor modification (Lee et al., 1994). The reaction was initiated, following a 3-min preincubation at 37°C, by addition of NADPH-generating system and allowed to proceed for 20 min. Reactions were terminated by addition of 2 volumes of methylene chloride, and corticosterone (10 µl of 200 µg/ml) was added as an internal standard. The organic layer was taken after vortexing for 2 min and evaporated to dryness under nitrogen stream. The residue was then dissolved in 100 µl of mobile phase. Sample aliquots (50 µl) were injected on a reverse phase C₁₈ high-pressure liquid chromatography column (Ultrasphere ODS; 4.6 × 150 mm, 5 µm; Beckman Coulter, Inc., Fullerton, CA), using a Waters M600 liquid chromatography system (Waters, Milford, MA). The mobile phase consisted of 60% methanol in water, and the flow rate was 1.0 ml/min. The eluent was monitored at 254 nm, and the amount of 6-hydroxytestosterone was determined by comparison to a standard curve.

Dialysis Experiments. Pooled human liver microsomes were incubated with DDB in the presence or absence of NADPH for 20 min. Samples were immediately placed in 6000 to 8000 molecular weight cutoff dialysis tubing (Spectral Medical Industries, Los Angeles, CA) and dialyzed for 18 h at 4°C against 2000 ml of 0.1 M potassium phosphate buffer, pH 7.4, containing 5 mM EDTA. Protein content was subsequently measured, and testosterone 6-hydroxylase activity was assayed after 20-min incubation.

Inactivation Studies. To characterize the time-dependent inhibition of CYP3A4 activity by DDB, human liver microsomes (2 mg of protein/ml) were incubated with varying concentrations of DDB. The reaction mixture was preincubated at 37°C for 3 min before initiating the reaction by addition of NADPH (1 mM final concentration). At 0, 2, 5, 10, and 20 min after the incubation, a 25-µl aliquot of each incubation was taken from the primary reaction mixture and added to a secondary reaction mixture containing 200 µM testosterone and NADPH-generating system in 0.1 M potassium phosphate buffer, pH 7.4, in a final volume of 0.5 ml. The reaction was allowed to proceed for a further 20 min, and 6-hydroxytestosterone formed in the reaction mixture was determined by high-pressure liquid chromatography analysis.

Spectral Analysis. Metabolite-P450 complex formation was measured with liver microsomes obtained from humans and PCN-treated rats. The reaction mixture contained 50 mM Tris-HCl, pH 7.4, 150 mM KCl, 10 mM MgCl₂, and 2 mg of microsomal protein in a final volume of 1 ml. DDB (dissolved in dimethyl sulfoxide) was added to the test cuvette, whereas the same volume of dimethyl sulfoxide (final concentration 0.5%) was added to the reference cuvette. After the baseline was corrected from 400 to 500 nm, the reactions were initiated by addition of NADPH (1 mM) to both cuvette at 37°C. The spectral changes between 400 and 500 nm due to metabolite-P450 complex formation were recorded using a JASCO V-550 UV/VIS spectrophotometer (JASCO, Tokyo, Japan).

1

Data Analysis. The IC₅₀ values were obtained from a plot of percentage activity remaining (relative to solvent alone incubate) versus log₁₀ of DDB concentration (0.05-10 µM). Inhibition data were graphically represented by Lineweaver-Burk (one per initial rate versus one per substrate concentration) and Dixon (one per initial rate versus inhibitor concentration) plots, and type of inhibition was determined using both plots. Estimates of inhibition constant (K_i) were determined using secondary plot of Lineweaver-Burk (slope versus inhibitor concentration) and mean value of three independent experiments was presented.

	Results

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Selectivity of DDB on the Inhibition of P450 Isoforms. The inhibition of P450 isoforms by DDB was investigated by using various P450 isoform-specific probe substrates (Table 1). DDB did not significantly inhibit the CYP2A6-, CYP2C9-, and CYP2C19-mediated reactions up to 50 µM DDB. Caffeine N₃-demethylase (CYP1A2), dextromethorphan O-demethylation (CYP2D6), and chlorzoxazone 6-hydroxylase (CYP2E1) were inhibited by DDB, but the extent of inhibition was less than 50% even at the highest concentration of DDB. Testosterone 6-hydroxylation, a marker activity of CYP3A4, was markedly inhibited in the presence of DDB with the IC₅₀ value of 0.38 µM. Figure 2 shows a double reciprocal (Lineweaver-Burk) plot of substrate concentration versus testosterone 6-hydroxylation and a Dixon plot of inhibitor concentration versus testosterone 6-hydroxylation by HL114 microsomes. The Lineweaver-Burk plot tended to intersect on the y-axis, and Dixon plot intersected above the x-axis, indicating that DDB inhibited the reaction in a competitive mode over the substrate range of 10 to 40 µM testosterone. The apparent K_i value, obtained by secondary plot of Lineweaver-Burk, was estimated to be 0.27 ± 0.21 µM in human liver microsomes (Table 2).

View this table: [in this window] [in a new window]   TABLE 1 Effect of DDB on P450 isoform-specific marker activities in human liver microsomes Percentage of remaining P450 activity was calculated after incubation with various concentrations of DDB and substrates in the presence of 0.5 mg/ml of pooled human liver microsomal protein and NADPH-generating system at 37°C. The uninhibited rates of the reactions were 0.441 nmol/min/mg of protein for coumarin 7-hydroxylation, 92 pmol/min/mg of protein for caffeine N-demethylation, 0.456 nmol/min/mg of protein for chlorzoxazone 6-hydroxylation, 13.6 pmol/min/mg of protein for S-mephenytoin 4'-hydroxylation, 0.521 nmol/min/mg of protein for testosterone 6-hydroxylation, 0.122 nmol/min/mg of protein for diclofenac 4'-hydroxylation, and 18.1 pmol/min/mg of protein for dextromethorphan O-demethylation. Each data represents the mean of two independent experiments.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Lineweaver-Burk (A) and Dixon (B) plot for DDB-induced inhibition of testosterone 6-hydroxylation in human liver microsomes. Human liver microsomes (0.5 mg/ml) from HL114 were incubated with various concentrations of testosterone in the presence of increasing amounts of DDB.

View this table: [in this window] [in a new window]   TABLE 2 Inhibition constants characterizing the inhibition of human liver microsomal CYP3A4 in the presence of DDB CYP3A4 activity was measured by testosterone 6-hydroxylation assay, as described under Materials and Methods. The concentration of testosterone used for the determination of IC50 was 40 µM.

Time-Dependent Inhibition of Testosterone 6-Hydroxylation. As shown in Fig. 3, DDB caused a time- and concentration-dependent inactivation of CYP3A4 with a NADPH-dependent manner. Significant inhibition was observed in the zero-time samples, probably due to the residual DDB since the initial incubation was diluted only 20-fold to retain sufficient sensitivity for testosterone 6-hydroxylation assay. Dialysis experiments were performed to evaluate whether CYP3A4 activity could be restored to that of control after a 20-min incubation with DDB and NADPH. After an 18-h dialysis, microsomes were incubated with testosterone for 20 min, and testosterone 6-hydroxylase activity was measured (Table 3). After dialysis of the samples incubated without NADPH, testosterone 6-hydroxylation activity was restored near to control level. In the case of the samples incubated with NADPH, the activity was partially restored at all concentrations of DDB in comparison with undialyzed samples, suggesting that the inhibition of the activity is partly due to the inactivation of the enzyme by DDB.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Time- and concentration-dependent loss of testosterone 6-hydroxylation activity in human liver microsomes with various concentrations of DDB. The human liver microsomes were preincubated with 0.5 to 5 µM DDB, 1 mM NADPH, and 2 mg/ml microsomal protein. At the indicated time points, 25 µl of the incubation mixture was added to 475 µl of reaction mixture containing 200 µM testosterone and NADPH-generating system, and further incubated for 20 min. Each point is the average of three determinations.

Metabolite-P450 Complex Formation. NADPH-dependent metabolite-P450 complex formation by DDB was measured in rat and human liver microsomes. When DDB was added to liver microsomes in the presence of NADPH, a maximum of 456 nm in the absorbance spectrum developed, indicating the formation of metabolite-P450 complex. No such maximum was obtained in the absence of NADPH. Approximately 40% of the initial P450 gave the reduced CO-P450 spectrum in the presence of dithionate (Fig. 4). The formation of metabolite-P450 complex increased dose dependently in PCN-treated rat microsomes, whereas the formation of metabolite-P450 complex was observed only at a high concentration of DDB in human liver microsomes (Table 4).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Metabolite-P450 complex spectrum in PCN-treated rat liver microsomes. Spectrum a represents the baseline spectrum, and spectrum b was recorded after a 5-min incubation with 100 µM DDB and 1 mM NADPH in PCN-treated rat liver microsomes. Then, the reduced CO-difference P450 spectrum (c) was recorded. Spectrum d represents the reduced CO-difference P450 spectrum obtained before incubation with DDB. Abs, absorbance.

Effect of DDB on P450 Content in Microsomes. As an indirect evidence for P450 inactivation, P450 content changes were measured after incubation of DDB with human liver microsomes. DDB in the absence of NADPH did not result in a loss of P450 content in microsomes, whereas the addition of NADPH to reaction mixture resulted in a 45% loss of P450 content after 20 min (Fig. 5). NADPH itself also decreased the P450 content by 20%, as previously observed (Chiba et al., 1995). The loss of P450 content was also observed in rat liver microsomes (Table 5). When potassium ferricyanide was added to microsomes, the amount of P450 content was restored to the control level, resulting from the disruption of the metabolite-P450 complex (data not shown).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Time-dependent loss of P450 content in human liver microsomes. Human liver microsomes (1 mg of protein/ml) were incubated in duplicate at 37°C. At the designated time points, the P450 concentrations were measured by reduced CO-difference P450 spectra. , microsome control; , 25 µM DDB; , 1 mM NADPH; , 25 µM DDB + NADPH.

	Discussion

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The human liver microsomal studies described in these experiments showed that only CYP3A4 was inhibited at clinically relevant concentrations of DDB with a K_i value of 0.27 µM. DDB had little or no effects on the activities of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1. All of the IC₅₀ values toward these isoforms were above 50 µM, which is about 50-fold greater than the typical C_max (~1 µM). These findings suggest that DDB drug interactions involving the CYP3A family are possible, whereas clinical interactions of other isoforms are not expected.

Incubations of DDB with human liver microsomes demonstrated that time-dependent inhibition of CYP3A4 was not observed for DDB alone; however, the addition of NADPH to the preincubation mixture containing DDB and human liver microsomes resulted in a time-dependent loss of CYP3A4 activity. Furthermore, the inhibition was only partially restored after the dialysis of incubation mixture. The dialysis of samples incubated in the absence of NADPH restored the activity to near control level, suggesting that the inhibition of the activity of samples incubated in the presence of NADPH after the dialysis was not due to the residual DDB. These results can be attributed to the formation of a metabolite-P450 complex with DDB. Methylenedioxyphenyl compounds have long been recognized as effective in vitro inhibitors of microsomal oxidation, and the ability is widely attributed to their oxidative metabolism to a reactive metabolic intermediate, possibly a carbene, that forms a stable inhibitory complex with ferrocytochrome P450 (Marcus et al., 1985; Murray et al., 1985). DDB also has methylenedioxyphenyl moiety and is metabolized to catechol derivatives through the initial hydroxylation of methylene carbon (Baek et al., 2001). Consequently, DDB can generate a reactive metabolic intermediate that coordinates tightly to the heme of P450, like other methylenedioxyphenyl compounds. The peak at 456 nm due to the complex formation was detected in human liver microsomes when DDB was incubated in the presence of NADPH. The formation of metabolite-P450 complex was increased in a concentration-dependent manner, and the maximum complex formation was estimated to be about 33% at 100 µM. It is suggested that the extent of metabolite-P450 complex formation is dependent on the side chain substituent on the methylenedioxy moiety. Complex formation is increased when the substituent is an electron-donating or a long-alkyl group, which stabilizes the carbene-iron bridges (Mansuy et al., 1979; Chiba et al., 1998). The relatively high extent of metabolite-P450 complex may be due to the presence of the methoxy group on the phenyl ring of methylenedioxyphenyl moiety.

The inhibition of CYP3A4 activity via the formation of a metabolite-P450 complex was further supported by the diminished P450 content. Since P450 that has the heme occupied by a ligand cannot bind carbon monoxide, the amount of metabolite-P450 complex formed can be determined by the loss of carbon monoxide binding. P450 content decreased when DDB was incubated with human liver microsomes or reconstituted CYP3A4 enzyme in the presence of NADPH. The loss of P450 was restored when the incubation mixture was treated with potassium ferricyanide, suggesting that the enzyme-DDB related complex is considered tight and noncovalent in nature. Considering that the activity was only partially regained after dialysis at 4°C, the metabolite-P450 complex was tight enough not to be easily dissociated.

P450 isoform selectivity has been demonstrated in metabolite-P450 complex formation (Bensoussan et al., 1995; Sinal and Bend, 1995). The present study showed that DDB selectively inhibited CYP3A4-mediated testosterone 6-hydroxylase activity in human liver microsomes. Our previous results demonstrated that DDB was metabolized to the catechol derivatives through the demethylation of methylenedioxybiphenyl moiety, and CYP3A4 was mainly involved in this reaction (Baek et al., 2001). These findings suggest that CYP3A4 generates a metabolic intermediate and is inactivated selectively by metabolite-P450 complex formation with DDB in human liver microsomes.

In conclusion, DDB formed a metabolite-P450 complex with P450 upon incubation with human liver microsomes in the presence of NADPH. In vitro studies using human liver microsomes demonstrated that the CYP3A4 activity was selectively inhibited during preincubation with NADPH in microsomes and that the formation of a quasi-irreversible metabolite-P450 complex with DDB was responsible for the time-dependent loss of CYP3A4 catalytic function. This inhibition may modulate the elimination of other coadministered drugs metabolized by CYP3A4.