Introduction

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1-(3,4-Dichlorobenzyl)-5-octylbiguanide monohydrochloride hemihydrate (OPB-2045)¹ is a newly synthesized antimicrobial agent (Tsubouchi et al., 1997) that has potent microbicidal activity toward fungi (yeast), Gram-negative, and Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) at low concentrations (Ohguro et al., 1997; Sakagami et al., 1999).

The absorption, distribution, metabolism, and excretion of OPB-2045 have been previously reported in rats and dogs after s.c. administration (Kudo et al., 1998a,b,c,d). OPB-2045 is eliminated principally by metabolism in rats and dogs (Kudo et al., 1998c,d), and four metabolites have been structurally identified in dog urine as 6-[5-(3,4-dichlorobenzyl)-1-biguanidino] hexanoic acid (DM-210), 4-[5-(3,4-dichlorobenzyl)-1-biguanidino] butanoic acid (DM-212), 5-[5-(3,4-dichlorobenzyl)-1-biguanidino] pentanoic acid (DM-213), and 3,4-dichlorobenzoic acid (Fig. 1) (Kudo et al., 1998c). These metabolites, except 3,4-dichlorobenzoic acid, are each a methylene chain shorter with a carboxy end, and are predominant metabolites of OPB-2045 in both rats (Kudo et al., 1998d) and dogs (Kudo et al., 1998c). OPB-2045 is not metabolized by skin/tissues at the injection site in rats, but rather by other organ(s) (Kudo et al., 1998d).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Chemical structures of OPB-2045 and metabolites.

It has been considered that the degraded products of OPB-2045 were produced by C-C bond cleavage followed by subtraction of carbon fragments, with possible involvement of the cytochrome P450 system (Kudo et al., 1998c). However, the production mechanism(s) of these C-C bond cleavage metabolites remains unclear.

In this study, examination was made of the in vitro metabolism of OPB-2045 using rat and dog liver preparations to elucidate various metabolic pathways of OPB-2045 to the degraded products. Metabolites in the incubation mixtures were characterized by ¹H NMR, fast atom bombardment (FAB)/mass spectrometry (MS), and liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). All metabolites identified were biotransformed at the octyl side chain of OPB-2045, i.e., monohydroxylates (2-, 3-, and 4-octanol), a ketone derivative, and dihydroxylates (1,2-octandiol, threo-2,3-octandiol, erythro-2,3-octandiol). The metabolites were all novel and possibly intermediates for in vivo end products. The metabolic pathways leading to in vivo end product formation were further studied using in vitro metabolic intermediates as substrates.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Chemicals. OPB-2045, DM-210, M-1 (DM-215), M-2 (DM-218), M-3 (DM-217), M-4 (DM-219), M-5 (DM-221), M-6 (DM-222), and M-7 (DM-220) were provided by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). -NADPH, -NADH, -NAD, -NADP, and -naphthoflavone were obtained from Sigma Chemical Co. (St. Louis, MO); glucose 6-phosphate and glucose 6-phosphate dehydrogenase (approx. 350 U/mg, 1 mg/ml) were purchased from Boehringer Mannheim GmbH (Mannheim, Germany); metyrapone was obtained from Aldrich Chemical Co. (Milwaukee, WI); SKF 525-A hydrochloride was obtained from Research Biochemicals Inc. (Natick, MA); sodium phenobarbital and n-octylamine were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). All other reagents and solvents were of analytical grade.

Animal Treatments and Preparation of 9000g Supernatant Fraction (S9) and Microsomes. Ten male Sprague-Dawley rats from Charles River Japan (Yokohama, Japan) were acclimatized in a controlled area maintained at 23 ± 2°C for temperature and 60 ± 10% for relative humidity during 12-h light/dark cycles. The animals were fed laboratory chow mouse food (MF) (Oriental Yeast Co., Ltd., Japan) and water was available ad libitum during acclimatization. Rats 12 weeks of age weighing 432 to 507 g were used in the experiments. Sodium phenobarbital was dissolved in saline and administered i.p. once daily for four consecutive days at a dose of 80 mg/kg/ml. The animals were fasted on the final day of administration and sacrificed on the following day by decapitation and exsanguination. Laparotomy was immediately performed. The livers were resected, washed, and perfused with ice-cold saline and homogenized in 3 volume/liver weight of 1.15% KCl solution with a Teflon homogenizer. The homogenate was centrifuged at 9000g for 30 min at 4°C, and the supernatant (S9) thus obtained was used for the enzyme preparation.

Incubation of OPB-2045 with Rat Liver S9. A NADPH-generating system [750 ml, 0.1 M Tris-HCl buffer, pH 7.4, containing 1 mM -NADPH, 2 mM -NADP, 16.9 mM glucose 6-phosphate, 0.75 ml of glucose 6-phosphate dehydrogenase (approx. 263 U), and 10 mM MgCl₂] and 5 ml of OPB-2045 in methanol solution at a concentration of 104 mg/ml (1.27 mM) were added to 250 ml of prepared rat liver S9. The mixture was incubated for 60 min at 37°C. This procedure was carried out in duplicate and the reaction mixtures were combined and stored frozen at 80°C until use.

Incubation of OPB-2045 with Dog Liver S9. The reaction mixtures contained 0.1 M phosphate buffer (pH 7.4), 10 mM -NADPH, 245 µM OPB-2045, and 250 µl of dog S9 protein in a final incubation volume of 0.5 ml. OPB-2045 was dissolved in methanol. Reactions were carried out in air at 37°C in a shaking water bath for 45 min. The reaction was stopped by addition of 1 ml of methanol. The mixture was centrifuged at 140g for 10 min and the supernatant was evaporated to dryness. The residue was dissolved in 200 µl of methanol, 30 µl of which was analyzed by HPLC as described below.

Incubation of M-1 with Dog Liver S9. A NADPH-generating system [500 ml, 0.1 M Tris-HCl buffer, pH 7.4, containing 0.5 mM -NADPH, 2 mM -NADP, 20 mM glucose 6-phosphate, 1 ml of glucose 6-phosphate dehydrogenase (approx. 350 U), and 10 mM MgCl₂] and 0.5 ml of M-1 in methanol solution at a concentration of 200 mg/ml (362 µM) were added to 150 ml of prepared dog liver S9. The mixture was incubated for 3 h at 37°C. This procedure was carried out in duplicate and the reaction mixtures were combined and stored frozen at 80°C until use.

Isolation Procedure of In Vitro Metabolites. M-1, M-2, and M-3 were isolated from the reaction mixture with rat liver S9 and OPB-2045 by liquid-liquid extraction, solid-phase extraction, and HPLC. The reaction mixture was extracted by first adding 3 volumes of methanol. The methanol-soluble extract was evaporated to dryness and the residue was dissolved in 350 ml of methanol. Methylene chloride was added to the methanol solution to obtain a methylene chloride/methanol (9:1, v/v) solution, which was then applied to 96 Sep-Pak Vac silica cartridges (20cc; Waters Associates, Milford, MA). The cartridges were washed with the methylene chloride and methanol solution (9:1, v/v), and eluted with the methylene chloride and methanol solution (1:1, v/v). The eluent was dried under reduced pressure. The residue was suspended in 50 ml of acetic acid. Water was added to make a 10% aqueous acetic acid solution, which was applied to 48 Sep-Pak Vac C₁₈ cartridges (20cc; Waters). Elution was performed in a stepwise fashion using 1% acetic acid aqueous solution, 25% methanol in water containing 1% acetic acid, 50% methanol in water containing 1% acetic acid, 60% methanol in water containing 1% acetic acid, and 70% methanol in water containing 1% acetic acid. The resulting 70% methanol elution fraction was evaporated to dryness and dissolved in 1 ml of methanol to further purify the metabolites by HPLC. This fraction contained metabolites M-1, M-2, and M-3.

HPLC. The HPLC system consisted of two model 510 high-pressure HPLC pumps (Waters), a model 717 automatic sample processor (Waters), a model U6K universal injector (Waters), model 484 or 486 UV detectors (Waters), a model 680 automatic gradient controller (Waters), and model C-R3A, C-R6A, or C-R7A Chromatopacs (Shimadzu, Kyoto, Japan).

¹H NMR and Mass Spectral Analysis. The ¹H NMR spectra were measured with a Bruker AC-200 NMR spectrometer (Karlsruhe, Germany) with tetramethylsilane as the internal standard. All samples were dissolved in DMSO-d₆. The FAB mass spectrum was measured with a JMS-SX102A mass spectrometer and LMA-DA 6000 data system (JEOL, Tokyo, Japan), with a direct inlet using glycerol as the matrix.

In Vitro Metabolism of M-5 and M-6 with Dog Liver Microsomes. The reaction mixtures contained 0.1 M phosphate buffer (pH 7.4), 5 mM -NADPH, 5 mM -NADH, 210 µM M-5 or M-6, and 250 µl of dog microsomal protein in a final incubation volume of 0.5 ml. M-5 or M-6 was dissolved in methanol. Reactions were carried out in air at 37°C in a shaking water bath for 60 min. The reaction mixtures were extracted by adding 1 ml of methanol, and the extract was evaporated to dryness. The residue was dissolved in 200 µl of methanol, and an aliquot (10-20 µl) was analyzed by HPLC-UV and LC/ESI-MS/MS for characterization of the chemical structures of the metabolites.

HPLC-UV analysis. HPLC analysis was carried out with the same Waters HPLC as above. A TSKgel ODS-80TM column (4.6 mm i.d. × 150 mm; Tosoh) was used at a flow rate of 1 ml/min with detection at 240 nm. The mobile phase used was a solution of 10% acetonitrile in water containing 0.1% acetic acid as solution A and 80% acetonitrile in water containing 0.1% acetic as solution B. The metabolites were analyzed using a linear gradient developed from 0 to 50% solution B over a period of 45 min.

LC/ESI-MS/MS analysis. HPLC analysis was carried out with a Waters HPLC system equipped with a model 600s controller, a model 616 FHU pump, a model 717P automatic sample processor, a model 717HC cooler, a model 486 UV detector, and a model IL-DEGA in-line degasser. A YMC-Pack Pro C18 column (2.0 mm i.d. × 150 mm; YMC Co., Ltd., Kyoto, Japan) was used at a flow rate of 0.2 ml/min. The metabolites were analyzed using a linear gradient developed from 0 to 50% solution B over a period of 45 min.

Assay of DM-210 Formation Activity. The reaction mixtures contained 0.1 M phosphate buffer (pH 7.4), 2.5 mM -NADPH, 10 µM M-5 or M-6, and 1 mg of dog microsomal protein in a final incubation volume of 0.5 ml. M-5 or M-6 was dissolved in methanol. The reaction was started by the addition of the cofactor, and was carried out in air at 37°C in a shaking water bath for 30 min. Four compounds (n-octylamine, SKF 525-A, metyrapone, and -naphthoflavone) were tested for their inhibitory effects on DM-210 formation. Each inhibitor was dissolved in DMSO. For the experiment using CO, the microsomal protein was gassed with a direct flow of CO gas for 1 min and then added to the incubation tubes.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

In Vitro Metabolism of OPB-2045 by Rat Liver S9. A representative HPLC chromatogram of OPB-2045 metabolites produced by rat liver S9 in the presence of NADPH is shown in Fig. 2. Detected UV peaks were primarily found for three types of metabolites in the reaction mixture: M-1, M-2, and M-3. These metabolites were further isolated and purified, and chemical structures were determined from ¹H NMR and FAB/MS spectra. Comparison with prepared authentic standards was made.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   HPLC chromatogram of the extract obtained from the incubation mixture of OPB-2045 with rat liver S9. OPB-2045 (1.27 mM) was incubated at 37°C with rat liver S9 in the NADPH-generating system for 60 min. HPLC was performed with methanol extract from the incubation mixture as described under Materials and Methods.

OPB-2045. The FAB/MS spectrum of OPB-2045 exhibited a protonated molecular ion [M+H]⁺ at m/z 372 (100% abundance). The ¹H NMR ( in ppm) of OPB-2045 in DMSO-d₆ was as follows: 0.85 (t, J = 6.8 Hz, 3H), 1.11 to 1.27 (m, 10H), 1.34 (br. s, 2H), 2.96 (br. s, 2H), 4.33 (br. d, J = 5.9 Hz, 2H), 6.95 (br. s, 4H), 7.29 (br. d, J = 8.3 Hz, 1H), 7.54 (br. s, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.61 (br. s, 1H), and 7.94 (br. s, 1H).

M-1. The FAB/MS spectrum of M-1 exhibited a protonated molecular ion [M+H]⁺ at m/z 388 (100% abundance). The ¹H NMR ( in ppm) of M-1 in DMSO-d₆ was as follows: 1.01 (d, J = 6.0 Hz, 3H), 1.08 to 1.45 (m, 11H), 2.93 (br. s, 2H), 4.28 (br. s, 2H), 7.27 (br. d, J = 8.3 Hz, 1H), 7.52 (br. s, 1H), and 7.57 (d, J = 8.3 Hz, 1H). M-1 produced a pseudomolecular ion at an m/z of 388 [M+H]⁺ in the FAB mass spectrum. This metabolite has a molecular weight of 387, 16 greater than that of the unchanged compound, and thus was considered a monohydroxylated OPB-2045. The ¹H NMR spectrum showed a change from a triplet proton signal ( = 0.85) for the terminal methyl group on the OPB-2045 alkyl chain to a doublet ( = 1.01). This suggests that M-1 is a metabolite in which the alkyl chain of OPB-2045 is hydroxylated at the 2 position. DM-215, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-2-octanol, appeared on the cochromatogram with M-1 in HPLC analysis. FAB mass and ¹H NMR spectra of M-1 were shown identical with those of the prepared standard, DM-215.

M-2 and M-3. The FAB/MS spectrum of M-2 and M-3 each exhibited a protonated molecular ion [M+H]⁺ at m/z 388 (100 and 52% abundance, respectively). The ¹H NMR ( in ppm) of M-2 or M-3 in DMSO-d₆ was as follows: for M-2: 0.83 (t, J = 7.5 Hz, 3H), 1.04 to 1.46 (m, 11H), 2.93 (br. s, 2H), 4.28 (br. s, 2H), 7.27 (br. d, J = 6.3 Hz, 1H), 7.52 (br. s, 1H), and 7.57 (d, J = 8.3 Hz, 1H); for M-3: 0.85 (t, J = 6.6 Hz, 3H), 1.05 to 1.53 (m, 11H), 2.95 (br. s, 2H), 4.29 (br. s, 2H), 7.28 (br. d, J = 8.2 Hz, 1H), 7.53 (br. s, 1H), and 7.57 (d, J = 8.3 Hz, 1H). These M-2 and M-3 each have a molecular weight of 387, and thus may be considered the monohydroxylated form of OPB-2045, as in the case of M-1. The ¹H NMR spectrum proton signals showed chemical shifts and splitting patterns almost identical with those of unchanged OPB-2045, without notable changes. M-2 and M-3 would thus appear to be metabolites, the hydroxylation of which occurs at a carbon atom other than the 1, 2, or 8 position on the octyl side chain of OPB-2045. FAB mass and ¹H NMR spectra of M-2 and M-3 were, respectively, identical with those of the prepared standard, DM-218, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-3-octanol or DM-217, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-4-octanol. DM-218 or DM-217 appeared on the cochromatogram with M-2 or M-3, respectively, in HPLC analysis. M-2 and M-3 were thus identified as DM-218 and DM-217, respectively.

In Vitro Metabolism of OPB-2045 by Dog Liver S9. A representative HPLC chromatogram of OPB-2045 metabolites produced by dog liver S9 in the presence of NADPH is shown in Fig. 3. Detected UV peaks were primarily found for eight types of metabolites in the reaction mixture: M-1, M-2, M-3, M-4, M-5, M-6, M-7, and M-8. M-4, M-5, M-6, M-7, and M-8 were also produced by the incubation of M-1 with dog liver S9 (data not shown). M-4, M-5, M-6, and M-7 were thus isolated from the incubation mixture of M-1 with dog liver S9, and chemical structures were determined from ¹H NMR and FAB/MS spectra. Comparison with prepared authentic standards was made.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   HPLC chromatogram of the extract obtained from the incubation mixture of OPB-2045 with dog liver S9. OPB-2045 (245 µM) was incubated at 37°C with dog liver S9 in the presence of 10 mM NADPH in 0.1 M phosphate buffer (pH 7.4) for 45 min. HPLC was performed with methanol extract from the incubation mixture as described under Materials and Methods.

M-4. The FAB/MS spectrum of M-4 exhibited a protonated molecular ion [M+H]⁺ at m/z 386 (100% abundance). The ¹H NMR ( in ppm) of M-4 in DMSO-d₆ was as follows: 1.00 to 1.50 (m, 8H), 2.06 (s, 3H), 2.37 (t, J = 7.1 Hz, 2H), 2.92 (br. s, 2H), 4.28 (br. s, 2H), 7.27 (br. d, J = 8.2 Hz, 1H), 7.52 (br. s, 1H), and 7.57 (d, J = 8.3 Hz, 1H). M-4 produced a pseudomolecular ion at an m/z of 386 [M+H]⁺ in the FAB mass spectrum. This metabolite has a molecular weight of 385, 2 smaller than that of the unchanged compound, and thus was thought to be a ketone derivative of M-1. The ¹H NMR spectrum showed a change from a doublet proton signal ( = 1.01) for the terminal methyl group on the M-1 alkyl chain to a singlet ( = 2.06). This suggests that M-4 is a metabolite in which the hydroxy group of M-1 is oxidized to a ketone. DM-219, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-2-octanone, appeared on the cochromatogram with M-4 in HPLC analysis. FAB mass and ¹H NMR spectra of metabolite were the same as those of the prepared standard, DM-219.

M-5 and M-6. The FAB/MS spectrum of M-5 and M-6 each exhibited a protonated molecular ion [M+H]⁺ at m/z 404 (47 and 100% abundance, respectively). The ¹H NMR ( in ppm) of M-5 or M-6 in DMSO-d₆ was as follows: for M-5: 1.00 (d, J = 6.2 Hz, 3H), 1.07 to 1.53 (m, 10H), 2.94 (br. s, 2H), 4.27 (br. s, 2H), 7.27 (br. d, J = 8.3 Hz, 1H), 7.52 (br. s, 1H), and 7.56 (d, J = 8.3 Hz, 1H); for M-6: 0.96 (d, J = 6.3 Hz, 3H), 1.08 to 1.50 (m, 10H), 2.94 (br. s, 2H), 4.27 (br. s, 2H), 7.27 (br. d, J = 8.3 Hz, 1H), 7.52 (br. s, 1H), and 7.56 (d, J = 8.3 Hz, 1H). M-5 and M-6 each have a molecular weight of 403, which is 16 greater than that of the unchanged compound, and thus were considered the monohydroxylated forms of M-1. The ¹H NMR spectrum proton signals showed chemical shifts and splitting patterns almost identical with those of unchanged M-1, without notable changes. M-5 and M-6 may thus be metabolites in which hydroxylation occurs at a carbon atom other than the 1, 2, or 8 position on the octyl side chain of M-1. FAB mass and ¹H NMR spectra of M-5 and M-6 were, respectively, identical with those of the prepared standard, DM-221, threo-8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-2,3-octandiol or DM-222, erythro-8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-2,3-octandiol. DM-221 or DM-222 appeared on the cochromatogram with M-5 or M-6, respectively, in HPLC analysis. M-5 and M-6 were thus identified as DM-221 and DM-222, respectively.

M-7. The FAB/MS spectrum of M-7 exhibited a protonated molecular ion [M+H]⁺ at m/z 404 (28% abundance). The ¹H NMR ( in ppm) of M-7 in DMSO-d₆ was as follows: 1.04 to 1.52 (m, 13H), 2.93 (br. s, 2H), 4.27 (br. s, 2H), 7.26 (br. d, J = 6.2 Hz, 1H), 7.52 (br. s, 1H), and 7.56 (d, J = 6.2 Hz, 1H). M-7 has a molecular weight of 403, which is 16 greater than that of the unchanged M-1. It was thus thought to be the monohydroxylated form of M-1, as in the case of M-5 or M-6. The ¹H NMR spectrum showed the disappearance of a doublet proton signal ( = 1.01) for the terminal methyl group on the M-1 alkyl chain. This suggests that M-7 is a metabolite in which the alkyl chain of M-1 is hydroxylated at the 8 position. DM-220, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-1,2-octandiol, appeared on the cochromatogram with M-7 in HPLC analysis. FAB mass and ¹H NMR spectra of metabolite were identical with those of the prepared standard, DM-220.

In Vitro Metabolism of M-5 and M-6 by Dog Liver Microsomes. M-5 was incubated with dog liver microsomes in the presence of NADPH and NADH, and the methanol extract was analyzed by HPLC-UV and LC/ESI-MS/MS. In the reaction mixture, three metabolites were observed: M-9, M-10, and M-11 (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   HPLC chromatogram of the extract obtained from the incubation mixture of M-5 with dog liver microsomes. M-5 (210 µM) was incubated at 37°C with dog liver microsomes in the presence of 5 mM NADH and 5 mM NADPH in 0.1 M phosphate buffer (pH 7.4) for 60 min. HPLC was performed with methanol extract from the incubation mixture as described under Materials and Methods.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   ESI product ion mass spectrum of [M+H]+ of metabolites M-9 at m/z 404 (A) and M-10 at m/z 374 (B). LC/ESI-MS/MS conditions are described under Materials and Methods.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   ESI product ion mass spectrum of [M+H]+ of metabolite M-11 at m/z 402.  LC/ESI-MS/MS conditions are described under Materials and Methods.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study has demonstrated the structural characterization of metabolites produced by rat and dog liver preparations in vitro and elucidated the metabolic pathways of OPB-2045 to the degraded products. All metabolites identified were biotransformed at the octyl side chain of OPB-2045, indicating the octyl side chain to be predominantly metabolized in vitro as well as in vivo.

M-1, M-2, and M-3, produced by the incubation of OPB-2045 with rat liver S9 in vitro, were all monohydroxylated forms, and the hydroxylation occurred at the 2-, 3-, or 4-position of the octyl group of OPB-2045, producing respective 2-, 3-, and 4-octanols. These metabolites were primarily found in the reaction mixture, although there were also several minor metabolites (Fig. 2). M-1, M-2, and M-3 were also produced by the incubation of OPB-2045 with dog liver S9 in vitro. M-4, M-5, M-6, M-7, and M-8 were produced by the incubation of OPB-2045 or M-1 with dog liver S9. M-4, M-5, M-6, and M-7 were isolated and identified as a ketone derivative, threo-2,3-octandiol, erythro-2,3-octandiol, and 1,2-octandiol, respectively. In vitro metabolites were all novel and have not been seen in vivo. These oxidation reactions required NADPH as a cofactor, implicating cytochrome P450 as an enzyme involved in the metabolite formation. The reduction of M-4 to M-1 was also observed in the incubation mixture (data not shown), and possibly may be catalyzed by ketone reductase.

M-5 and M-6 were further biotransformed to a ketol derivative and DM-210 (hexanoic acid derivative), an in vivo end product, in the incubation with dog liver microsomes. The same results were obtained in the incubation of these compounds with rat liver microsomes (data not shown). The DM-210 formation required NADPH as a cofactor, and it was inhibited by the cytochrome P450 inhibitors; this indicates that this reaction is catalyzed by the cytochrome P450 system. The ketol may possibly be M-8 found in the incubation of OPB-2045 or M-1 with dog liver S9, although this remains to be confirmed. In the reaction mixture, the conversion of M-5 to M-6 or vice versa was observed. These conversions may occur via a ketol derivative and may be catalyzed by ketone reductase located in the microsomes. Thus, these experiments have shown that degraded products of OPB-2045 can arise from C-C bond cleavage after sequential oxidative reactions at the octyl side chain of OPB-2045.

There are two possible mechanisms for C-C bond cleavage in biotransformation: one is -oxidation after hydroxylation and oxidation at the terminal carbon atom of the chain, and the other is oxidative cleavage by cytochrome P450 (Shikita and Hall, 1973a,b; Watabe and Akamatsu, 1975; Takikawa et al., 1978; Kashiwagi et al., 1980; Nakajin et al., 1981; Nakajin and Hall, 1981; Akhtar et al., 1993).

-Oxidation, as well known in fatty acid metabolism, conducts C-C bond cleavage of successive two-carbon fragments starting from the carboxy-terminal, leading to fatty acids with an even number of carbon atoms in the methylene chain. In contrast, OPB-2045 metabolites biotransformed at the octyl side chain have both odd- and even-carbon chains, and a metabolite of a compound with octanoic acid cannot be detected in the excreta of rats and dogs (data not shown). In addition, 1-octanol was not observed in the incubation of OPB-2045 with rat or dog liver S9. These evidences indicate that -oxidation is not responsible for the metabolic degradation of OPB-2045.

Some cytochrome P450 systems have been reported to have catalytic activity not only in conventional hydroxylation reactions but also in the oxidation of an alcohol to a carbonyl compound (Tyndale et al., 1991) and is also involved in C-C bond cleavage (Akhtar et al., 1993). Removal of the pregnenolone side chain to produce dehydro-iso-androsterone is mediated by the cytochrome P450 system (Corina et al., 1991; Miller et al., 1991). Watabe and Akamatsu (1975) have shown that sequential steps of the oxidative reaction by rabbit liver microsomes are involved in the cleavage of ethylenic double bonds of stilbene. Stilbenes are converted by rabbit liver microsomes to glycols via epoxides and the resultant glycols are oxidized to benzoin ketol and benzil diketone. C-C bonds of the ketol or diketone are enzymatically cleaved with benzoic acid as an end product. Cytochrome P450 in microsomes may possibly be involved in the metabolism of stilbene because NADPH is essential for metabolic conversion of the compound (Watabe and Akamatsu, 1972, 1974, 1975).

DM-210 was found to be produced by sequential oxidative reactions, indicating a C-C bond cleavage mechanism similar to that in stilbene metabolism to likely be involved in DM-210 production (Fig. 7). Namely, OPB-2045 is initially monhydroxylated to M-1 and then oxidized to the ketol (M-8) via formation of the diol (M-5 or M-6). The ketol is biotransformed to the end product, DM-210, by C-C bond cleavage. These sequential oxidative reactions may be considered involved in the production of DM-212 and DM-213 in vivo (Fig. 7). The enzymes in this process are considered to be cytochrome P450s. CYP4A, CYP1A, CYP2C, CYP2E, and CYP3A isoforms, which catalyze the metabolism of fatty acids (Kimura et al., 1989; Falck et al., 1990; Tanaka et al., 1990; Yokotani et al., 1991; Laethem et al., 1993), may be involved in the oxidation. Further investigation concerning cytochrome P450 isoform(s) involved in C-C bond cleavage of the octyl side chain of OPB-2045 is now in progress.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Presumed metabolic pathways on the formation of in vivo end products, DM-210, DM-212, and DM-213 from OPB-2045. Pathways of metabolism are hypothetical and compounds shown in square brackets were not observed in this study.

The mechanism of C-C bond cleavage by cytochrome P450 has not been fully determined. Akhtar et al. (1993) propose that C-C bond cleavage reaction occurs through the participation of the Fe^III-O-OH species that is produced as an intermediate in cytochrome P450 reactions and is trapped by the electrophilicity of the carbonyl compound to afford a peroxide adduct that fragments with consequent acyl-carbon cleavage. The same mechanism may be applicable to C-C bond cleavage of the octyl side chain of OPB-2045, because the carbonyl compound was observed here in the reaction mixture.

As with the metabolic pathway of OPB-2045, there are compounds whose metabolites contain side chains, in which odd and even numbers of carbon fragments have been removed by metabolism. The pentyl side chain of cannabidiol and its derivatives is reduced to two, three, or four carbon atoms by removal of even and odd numbers of carbon atoms (Harvey and Leuschner, 1985; Harvey, 1989, 1990; Harvey and Mechoulam, 1990; Samara et al., 1990). Sinz et al. (1997) have shown that CI-976, a specific acyl coenzyme A/cholesterol acyltransferase inhibitor, is metabolized to a 5- and 6-carbon cleavage metabolite in rats after oral administration. The metabolic route of these compounds to the degraded metabolites with removal of an even number of carbon fragments has been shown to be -oxidation after hydroxylation and oxidation at the terminal carbon atom of the chain (Harvey and Leuschner, 1985; Sinz et al., 1997). On the other hand, the mechanism for removal of an odd number of carbon fragments remains only partially characterized, but the metabolites of these compounds may possibly arise from pathways other than classical -oxidation and involve intermediates derived from hydroxylation of the side chain (Harvey, 1989; Samara et al., 1990; Woolf et al., 1990). Oxidative C-C bond cleavage by sequential oxidative reactions may thus be essential for removal of an odd number of carbon fragments.

In summary, this study demonstrates that the degraded products of OPB-2045 are produced by C-C bond cleavage after sequential oxidative reactions, but not -oxidation, with possible involvement of cytochrome P450 systems. Such aliphatic C-C bond cleavage by sequential oxidative reactions could play an important role in the metabolism of other drugs or endogenous compounds containing aliphatic chains.