Introduction

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It has been reported that the novel compound NO-1886¹ ([4-(4-bromo-2-cyano-phenylcarbamoyl) benzyl]-phosphoric acid diethyl ester; Fig. 1) increases lipoprotein lipase (LPL) activity, resulting in a reduction in plasma triglycerides and a concomitant increase in high-density lipoprotein cholesterol in experimental animals, including rats, hamsters, and rabbits (Tsutsumi et al., 1993, 1995, 1997). It was also demonstrated that long-term administration of NO-1886 significantly prevented the development of atherosclerosis in cholesterol-fed rats (Tsutsumi et al., 1993) and rabbits (Chiba et al., 1997). As shown in Fig. 1, the metabolic pathways of NO-1886 in rats have been identified as 1) O-deethylation of the phosphoric acid ester, 2) hydrolysis of the amide bond, 3) hydroxylation of the amino compound (M-1) produced by hydrolysis of the amide bond, and 4) sulfation following hydroxylation of M-1 (Morioka et al., 1996). NO-1886 was almost completely excreted in the urine (28%) and feces (64%) as metabolites within 24 h of postdosing in rats that were maintained in metabolic cages. The major metabolite was monoethyl phosphonate (M-2), which accounted for 70% of all metabolites in rats (Morioka et al., 1996). None of these metabolites, including the major metabolite (the monoester form), increases the activity of LPL (Morioka et al., 1996). Therefore, the metabolic activity in humans is an important factor in determining the duration of administration in clinical use. In the present study, the metabolic disposition of NO-1886 in human liver S9 and microsomes was investigated and the major enzyme involved in metabolism was identified.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Chemical structures of NO-1886 and its metabolites.

	Materials and Methods

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Chemicals and Biochemicals. NO-1886 and its metabolite (M-2) were synthesized at Otsuka Pharmaceutical Factory (Tokushima, Japan). Triacetyloleandomycin, 13-cis-retinoic acid, quinidine, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma Chemical (St. Louis, MO). -NADPH was obtained from Oriental Yeast (Tokyo, Japan). 4-Methylpyrazole and coumarin were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Furafylline, ketoconazole, rabbit anti-human CYP1A1/1A2, CYP2D6, and CYP3A4 sera, and goat anti-human CYP2C serum were purchased from Daiichi Pure Chemicals (Tokyo, Japan). Other chemicals were of the highest grade commercially available. A mixed pool of human liver S9 and microsomes from 15 donors was obtained from In Vitro Technologies, Inc. (lot 1001; Baltimore, MD). Recombinant P450 enzymes expressed in microsomes of baculovirus-infected insect cells and human lymphoblastoid cells were obtained from GENTEST (Woburn, MA).

Incubation Conditions. All incubations related to the liver microsomes were carried out at a protein concentration of 0.5 mg/ml in 100 mM potassium phosphate buffer, pH 7.4, including 0.1 mM EDTA and an NADPH-generating system (1.25 mM NADP⁺, 2.5 mM glucose 6-phosphate, 6 mM MgCl₂, and 0.75-unit/ml glucose-6-phosphate dehydrogenase) at 37°C. In enzyme kinetic experiments, 0.2 to 110 µM NO-1886 was incubated at a final volume of 0.5 ml for 60 min. The reaction was initiated by the addition of the sample solution after 5-min preincubation at 37°C. After incubation, the reaction was terminated by the addition of 1 ml of ice-cold acetonitrile containing 1-µg/ml p-hydroxybenzoic acid butyl ester as an internal standard. After the reaction mixture was centrifuged at 3000 rpm for 10 min at 4°C, the supernatant was evaporated to dryness, and the residue was dissolved in 250 µl of a solution consisting of acetonitrile and sodium phosphate buffer (10 mM, pH 6.4) in a ratio of 1:4 (v/v). A 100-µl aliquot of the sample was subjected to high-performance liquid chromatography (HPLC). Incubation with the liver S9 was carried out for 120 min at a protein concentration of 12 mg/ml under the same conditions as those for liver microsomes. The linearity of reaction with protein concentration and incubation time was confirmed under these experimental conditions.

Chemical Inhibition. Inhibition experiments were carried out with 4.4 and 110 µM NO-1886 in a final volume of 0.5 ml for 60 min. The stock solutions of the inhibitors were added immediately before the addition of NO-1886. For the mechanism-based inhibitors furafylline and triacetyloleandomycin, a mixture of the inhibitor, microsomes, and the NADPH-generating system was preincubated for 10 min at 37°C before the addition of NO-1886. M-2 formation in the presence of inhibitor was compared against appropriate controls and the results were calculated as a percentage of the uninhibited rate.

Immunoinhibition by Anti-P450 Antiserum. Various concentrations of anti-P450 antiserum and microsomes (0.5 mg/ml) were incubated for 30 min at room temperature. The NADPH-generating system was then added, and the mixture was maintained at 37°C for 5 min. The reaction (60 min) was initiated by the addition of NO-1886 (4.4 µM final concentration). The results were calculated as a percentage of the duplicate control measurements.

Metabolism of NO-1886 by Recombinant P450 Enzymes. This experiment involved the use of microsomes from two kinds of cells (baculovirus-infected insect cells and human lymphoblastoid cells), which expressed CYP1A2, 2A6, 2B6, 2C8, 2C9-Arg144, 2C9-Cys144, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5. Control microsomes were obtained from cells of the same type that had not been transfected. All P450s used were coexpressed (from cDNA) with NADPH-P450 reductase. In addition, CYP3A4 and CYP2E1 were also coexpressed with cytochrome b₅. The final concentrations of microsomes and NO-1886 were 50 to 150 pmol of P450/ml and 0.2 to 440 µM, respectively. After 5-min preincubation of microsomes containing the NADPH-generating system, a 60-min reaction at 37°C was initiated by adding NO-1886.

HPLC Analysis. NO-1886 and M-2 levels were determined using a Shimadzu (Tokyo, Japan) HPLC system comprising an LC-6A pump, SPD-6A UV detector and CR-4A Chromato-integrator, SIL-6A autosampler, CTO-6A column oven, and SCL-6AV system controller. An aliquot of the sample was injected onto a TSKgel ODS-120A column (5 µm, 4.6 × 250-mm i.d.; Tosoh, Tokyo, Japan) and eluted at a flow rate of 1.2 ml/min with 50 mM phosphate buffer, pH 2.2/acetonitrile according to the following gradient schedule: 20% acetonitrile for the first 20 min; a linear gradient from 20 to 24% over the next 20 min; a linear gradient from 24 to 35% over 10 min; 35% for 20 min; and a linear gradient from 35 to 70% over 10 min, which was then maintained at 70% for 10 min. The temperature of the column was maintained at 45°C. UV detection was performed at 260 nm and the detection limit for M-2 was 5 nM in incubation samples.

Data Analysis. Experimental reaction velocity measurements were combined to obtain mean ± S.D. values. The parameters K_m and V_max were calculated by fitting the Michaelis-Menten equation to the data by nonlinear regression analysis (MULTI; Yamaoka et al., 1981) with weighted data (1/y).

	Results

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Metabolism of NO-1886 in Human Liver. The metabolism of NO-1886 was investigated in pooled human liver S9 fractions and microsomes. Figure 2 shows the HPLC chromatogram of the human liver S9 fraction after reaction with NO-1886 for 60 min. The formation rates of metabolites in the S9 fraction are listed in Table 1. It is clear that the cleavage of diethyl phosphonate is the major metabolic pathway of NO-1886 in human liver as well as in the rat and that the amide bond is scarcely hydrolyzed in human liver. The M-2 formation rate per protein content was 15 times greater in microsomes than in the S9 fraction. The lower activity of S9 than microsomes might be explained by the microsomal protein content in the S9 fraction (Duve et al., 1955). M-2 formation was detected at 9.77 pmol/min/mg of protein in human liver microsomes without the NADPH-generating system (Table 1), suggesting that A-esterase and other esterases were involved in the formation of M-2 from NO-1886. In contrast, hydrolysis of the amide bond of NO-1886 occurred in human liver S9 with and without the NADPH-generating system but not in microsomes. These data suggest that the amide bond of NO-1886 is hydrolyzed by cytosolic esterases.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Representative HPLC chromatograms of M-2 formed in human liver S9 with and without the NADPH-generating system. A control mixture of human liver S9 (12 mg/ml) was mixed with n-butyl p-hydroxybenzoic acid after incubation under the same conditions as described for the incubation of samples (A); a mixture of human liver S9 (12 mg/ml) was incubated with 110 µM NO-1886 for 60 min with the NADPH-generating system (B); a mixture of human liver S9 (12 mg/ml) was incubated with 110 µM NO-1886 for 60 min without the NADPH-generating system (C).

Phosphonate O-deethylation of NO-1886 to M-2 in Human Liver Microsomes. Figure 3 shows an Eadie-Hofstee plot of the formation of M-2 from NO-1886 in pooled human liver microsomes. The biphasic nature of the plot indicates that multiple enzymes contribute to the phosphate O-deethylation. The calculated K_m and V_max values for the low-K_m component (K_m1 and V_max1) were 3.56 ± 0.01 µM and 14.2 ± 1.2 pmol/min/mg, respectively. For the high-K_m component, the K_m2 was 172.5 ± 37.1 µM and the V_max2 was 334.2 ± 56.3 pmol/min/mg.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Eadie-Hofstee plot for NADPH-dependent M-2 formation from NO-1886 by pooled human liver microsomes. Human liver microsomes (0.5 mg/ml) were incubated with NO-1886 (0.4-110 µM) for 60 min, and the rates of formation of M-2 were determined by HPLC. Data are expressed as the mean of three determinations.

Inhibition Analysis. To identify the specific P450 isozyme(s) involved in the biotransformation of NO-1886 to M-2, incubation was performed using anti-human P450 antibodies. As shown in Fig. 4, anti-human CYP3A4 antibody and anti-human CYP2C antibody inhibited 40 to 50% of M-2 formation from 4.4 µM NO-1886 with the addition of 160 µl of antisera. In contrast, anti-human CYP1A1/1A2, CYP2A6, CYP2D6, and CYP2E1 antibodies showed no effect on the hydrolysis of NO-1886.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Immunoinhibition by anti-human P450 sera of phosphonate O-deethylation of NO-1886 by human liver microsomes. NO-1886 (4.4 µM) was incubated with human liver microsomes (0.5 mg/ml) in the presence of anti-human P450 antibodies for 60 min at 37°C after preincubation of each antibody with liver microsomes for 30 min at room temperature. Values represent the mean of two measurements as a percentage of two control measurements, 6.2 pmol/min/mg.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Inhibition of phosphonate O-deethylation of NO-1886 by P450 inhibitors in human liver microsomes at NO-1886 concentrations of 4.4 µM () and 110 µM (). The following inhibitors were used: 50 µM furafylline, 50 µM coumarin, 50 µM triacetyloleandomycin, 25 µM ketoconazole, 50 µM orphenadrine, 50 µM 13-cis-retinoic acid, 50 µM sulfaphenazole, 50 µM S-(+)-mephenytoin, 10 µM quinidine, and 50 µM 4-methylpyrazole. Each column represents the mean of two determinations.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Inhibition of phosphonate O-deethylation of NO-1886 by CYP2C and CYP3A4 inhibitors in human liver microsomes at an NO-1886 concentration of 4.4 µM. Symbols are represented as follows: , triacetyloleandomycin; , ketoconazole; , sulfaphenazole; , 13-cis-retinoic acid. Data are expressed as the mean (±S.D.) of two determinations.

Metabolism of NO-1886 to M-2 by Recombinant P450 Enzymes. The metabolism of NO-1886 by microsomes from baculovirus-infected insect cells expressing human P450s was compared with that from human lymphoblastoid cells (Table 2). The activities of CYP3A4 and CYP2C8 were significantly higher than those of other P450 isoforms in both baculovirus-infected insect cells and human lymphoblastoid cells. In addition, the activity of CYP3A4 in the phosphonate O-deethylation of NO-1886 was increased 7-fold by coexpression with cytochrome b₅. Recombinant CYP3A5 showed much lower activity than recombinant CYP 3A4 coexpressed with and without cytochrome b₅.

	Discussion

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A number of studies have shown a significant inverse relationship between high-density lipoprotein (HDL) cholesterol and coronary heart disease, and HDL cholesterol is now known to be a strong protector against coronary artery sclerosis (Gorden and Rifking, 1989). Plasma HDL originates from three sources: the liver, small intestine, and lipoprotein lipase-mediated lipolysis of chylomicrons and very low-density lipoproteins (Eisenberg, 1984). Because manipulation of physiological processes in the organs will undoubtedly involve the perturbation of complicated biological systems, up-regulation of lipoprotein lipase activity appears to be a more promising strategy in the development of new therapeutic agents. The novel compound NO-1886 was shown to increase lipoprotein lipase activity and to induce an elevation of HDL cholesterol in a few studies (Tsutsumi et al., 1993, 1995, 1997). The O-deethylation of the phosphoric acid ester of diethyl phosphonate and the hydrolysis of amide bonds have been reported to be the metabolic pathways of NO-1886, and the metabolites of NO-1886 do not increase lipoprotein lipase activity (Morioka et al., 1996). Because the disposition of NO-1886 has been investigated only in the rat (Morioka et al., 1996), it is necessary to clarify the metabolism of this new drug in humans. The enzymes involved in the metabolism of NO-1886 were therefore identified to obtain information to avoid drug-drug interactions in the clinical use of NO-1886.

Incubation of NO-1886 in the human liver S9 fraction and microsomes demonstrated the major metabolic pathway of NO-1886 to be conversion to monoethyl phosphonate (M-2), with the hydrolysis of the amide bond as the minor pathway (Fig. 2; Table 1). Most esterases are capable of catalyzing hydrolytic reactions of several types of bonds such as those of carboxylester, caboxyamide, carboxythioester, and phosphoric acid esters. Among these esterases, carboxylesterase has been found to be frequently involved in the detoxification process of several types of ester compounds. In the case that some organophosphates are poor substrates for carboxylesterase, A-esterase is normally the enzyme that catalyzes the hydrolytic reactions (Walker and Mackness, 1983; Tang and Chambers, 1999). Therefore, it has been thought that A-esterase and/or carboxylesterase catalyzes the biotransformation from diethyl phosphonate to the monoethyl phosphonate of NO-1886. The esterases in S9 and microsomes generally catalyze hydrolysis in the absence of NADPH. However, M-2 formation without NADPH was only 9.77 pmol/min/mg of protein in human liver microsomes. The results of the present study show that more than 90% of the biotransformation of NO-1886 to M-2 requires NADPH. Meanwhile, there have been several reports concerning the NADPH- and oxygen-dependent hydrolysis of phosphoric acid ester (Søderlund et al., 1979; Dunkov et al., 1997). For example, tris-(2,3-dibromopropyl)phosphate is converted to bis(2,3-dibromopropyl)phosphate after oxidation of the 2,3-dibromopropyl group by cytochrome P450 (Søderlund et al., 1984). Therefore, the phosphonate bond of NO-1886 is probably cleaved after oxidation of the ethyl group by P450. In contrast, the hydrolysis of the amide bond of NO-1886 might be catalyzed by cytosolic esterases. The hydrolysis of the amide bond of NO-1886 is the second major metabolic pathway in rats (Morioka et al., 1996), and it mainly proceeds in rat plasma (data not shown). However, the amide cleavage might occur at very low levels in humans, because the amide hydrolysis of NO-1886 was not observed in 30-min incubation in human plasma (data not shown). The existence of this phenomenon is suggested by the fact that esterase such as carboxylesterase is hardly expressed in human plasma as opposed to abundant expression of esterase in rat plasma (Satoh and Hosokawa, 1998).

To identify the cytochrome P450 isoform involved in the phosphonate O-deethylation of NO-1886, further experiments were performed in human liver microsomes. Eadie-Hofstee plots indicated that at least two cytochrome P450 enzymes in human liver microsomes might catalyze M-2 formation, as shown in Fig. 3. The results of inhibition studies with highly specific anti-P450 antibodies and chemical inhibitors suggested that the phosphonate O-deethylation of NO-1886 was catalyzed by CYP3A4 and CYP2C8. At the concentration of NO-1886 (4.4 µM) used in chemical and immuno-inhibition studies, the metabolic rates of human liver microsomal formation of M-2 are calculated to be 8.34 and 7.74 pmol/min/mg of protein for the high- and low-K_m components, respectively, by using the observed K_m and V_max values. Therefore, it is expected that chemical inhibitors and anti-P450 antibody incompletely inhibit the formation of M-2 from NO-1886 in human liver microsomes. The maximum inhibition of the human liver microsomal formation of M-2 by triacetyloleandomycin, a selective CYP3A inhibitor, was only 50%. Anti-CYP3A inhibited the human liver microsomal M-2 formation by a maximum of about 40% (Fig. 4). The close similarity between the inhibition of human liver microsomal M-2 formation by anti-CYP2C (a maximum of about 55%; Fig. 4) and 13-cis-retinoic acid, a selective inhibitor of CYP 2C8, suggests that 50 to 55% of the human liver microsomal M-2 formation from 4.4 µM NO-1886 is catalyzed by CYP2C8 isoforms. Thus, the inhibition data for the formation of M-2 from 4.4 µM NO-1886 by human liver microsomes suggest that the contribution of CYP2C8 and CYP3A isoforms is similar.

In addition, the M-2 formation by recombinant P450s suggested that CYP3A4 might be responsible for the high-K_m component, whereas CYP2C8 might be responsible for the low-K_m component for phosphonate O-deethylation of NO-1886 in human liver microsomes. This assumption is supported by the chemical inhibition of the human liver microsomal M-2 formation at a high concentration of NO-1886 (110 µM; Fig. 5). However, the observed K_m value for M-2 formation by recombinant P450 isoforms is different from the K_m value in human liver microsomes, possibly due to several reasons, e.g., binding of NO-1886 to intracellular and/or microsomal proteins, or a conformational change of the P450 molecule by binding with endogenous compounds.

The human liver microsomal CYP2C subfamily is known to comprise at least four members: CYP2C8, CYP2C9, CYP2C18, and CYP2C19. CYP2C19 is considered to be the polymorphically expressed (S)-mephenytoin 4'-hydrolase that is involved in the metabolism of omeprazole, diazepam, imipramine, propranolol, and proguanil (Goldstein and de Morais, 1994; Goldstein et al., 1994). On the other hand, CYP2C8 exhibits selectivity for retinol (hydroxylation), Taxol (6-hydroxylation), and arachidonic acid (epoxidation) (Leo et al., 1989; Daikh et al., 1994; Rahman et al., 1994). CYP2C has been reported to constitute approximately 25% of the P450 isoforms expressed in human liver microsomes (Shimada et al., 1994). In addition, 60% of the CYP2C cDNA clones isolated from a human liver library were CYP2C9. Only 1% was CYP2C19, whereas CYP2C8 and CYP2C18 content was much lower than that of CYP2C19 (Inoue et al., 1997). Among the recombinant CYP2C enzymes, only CYP2C8 showed activity for the phosphonate O-deethylation of NO-1886 with a low K_m value. Considering the relative quantities of CYP2C8 and CYP3A4 in the human liver, it is suggested that CYP2C8 may be saturated in the early stage of metabolism of NO-1886 in the liver. The fact that the phosphonate O-deethylation of NO-1886 is partly mediated by CYP2C8 in a high-affinity manner suggests that drug-drug interactions may occur with CYP2C8 cosubstrates. Because NO-1886 may be administered for prolonged periods in patients with hypertriglyceridemia, drugs that are selectively metabolized by CYP2C8 should be carefully monitored or should be avoided as much as possible.

CYP3A in the human liver has been reported to be approximately 29% of the total P450 content in Japanese and Caucasian people (Shimada et al., 1994). The CYP3A4 level in some individuals is more than 60% of the total P450 content, probably due to induction by various chemical agents (Guengerich, 1995). Because NO-1886 might be administered at relatively high doses in clinical use according to its pharmacological activity in phase I trials, CYP3A4 will become an important isozyme for the elimination of NO-1886. The O-deethylation of NO-1886 mediated by CYP3A4 may not occur only in the liver but also in other CYP3A4-rich sites such as the intestine. Therefore, individual differences in the plasma concentration of NO-1886, which is influenced by the CYP3A content in the liver and intestine, may be observed in the clinical setting.

In addition, the hepatic clearance for the NADPH-dependent M-2 formation from NO-1886 was estimated using the K_m and V_max values for the low- and high-K_m components of human liver microsomes and the average protein content of microsomes (51 mg/g of liver) in the human liver (21 g of liver/kg of body weight). The intrinsic hepatic clearance was calculated to be 6.34 ml/min/kg, and hepatic clearance was then roughly calculated to be 0.42 ml/min/kg by using 21 ml/min/kg of hepatic blood flow rate and 0.067 of the free fraction of NO-1886 in human plasma (Y. Morioka, M. Harada, S. Naito, T. Imai, unpublished data). The estimated hepatic clearance is much lower than the hepatic blood flow rate, suggesting that NO-1886 is slowly eliminated from the body, with almost no first-pass metabolism in the liver.

In conclusion, the results of the present study suggest that both CYP2C8 and CYP3A4 are major P450 enzymes involved in the phosphonate O-deethylation of NO-1886 in the human liver. The roles of these two enzymes vary depending on the amounts available in the liver. CYP3A is the major P450 subfamily in the human liver and intestine and is involved in the metabolism of a variety of pharmaceutical agents that are metabolized by P450. CYP3A enzymes have been reported to be involved in interactions with several drugs such as macrolides, ketoconazole, and cyclosporin (Pichard et al., 1990; Periti et al., 1992). In addition, CYP2C8 that catalyzes the oxidative reaction of selective substrates plays an important role as the low-K_m component in the human liver. To further confirm the clinical safety of NO-1886, enzymatic inhibition studies focusing on drug-drug interactions related to metabolism by P450 enzymes are currently underway.