Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Metabolism of pravastatin sodium in isolated rat hepatocytes. I. Glutathione conjugate formation reaction S. Muramatsu, K. Miyaguchi, H. Iwabuchi, Y. Matsushita, T. Nakamura, T. Kinoshita, M. Tanaka & H. Takahagi To cite this article: S. Muramatsu, K. Miyaguchi, H. Iwabuchi, Y. Matsushita, T. Nakamura, T. Kinoshita, M. Tanaka & H. Takahagi (1992) Metabolism of pravastatin sodium in isolated rat hepatocytes. I. Glutathione conjugate formation reaction, Xenobiotica, 22:5, 487-498 To link to this article: http://dx.doi.org/10.3109/00498259209053111
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XENOBIOTICA,
1992, VOL. 22,
NO.
5, 487-498
Metabolism of pravastatin sodium in isolated rat hepatocytes. I. Glutathione conjugate formation reaction S. MURAMATSU, K. MIYAGUCHI, H. IWABUCHI, Y. MATSUSHITA, T. NAKAMURA, T. KINOSHITA, M. TANAKA and H. TAKAHAGI
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Analytical and Metabolic Research Laboratories, Sankyo Co. Ltd., 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan
Received 16 January 1990; accepted 22 January 1992
1. T h e metabolic fate of pravastatin sodium (sodium ( +)-(3R, 5R)-3,5-dihydroxy-7-
{ (1’S,2’S,6‘S,8’S,8’aR)-6‘-hydroxy-2’methyl-8’-[(S)-~-methylbutyryloxy]-l’,~,6‘,7’,8‘, 8’a-hexahydro-l‘-naphthyl}heptanoate) was studied in isolated rat hepatocytes. 2. Two polar metabolites were isolated and identified as a glutathione conjugate and a dihydrodiol. 3. Both metabolites were formed via an epoxide which has been identified as the 4‘ap,S’P-epoxide on the decalin moiety. 4. Formation of the glutathione conjugate was enzymic, while the dihydrodiol was formed by non-enzymic hydrolysis of the epoxide accompanied by the intramolecular migration of the double bond.
Introduction Pravastatin sodium (sodium (+)-(3R,5R)-3,5-dihydroxy-7-{(l’S,2’S,6’S,8’S, 8’aR)-6‘-hydroxy-2’-methyl-8’- [( S)-2”-methylbutyryloxy]- 1’,2’,6’,7’,8’,8’a-hexahydro-1’-naphthy1)heptanoate) is a tissue-selective inhibitor of 3-hydroxy-3methylglutaryl coenzyme A reductase, a key enzyme in cholesterol biosynthesis (Haruyama et al. 1986, Tsujita et al. 1986). When l4C-labe1led pravastatin sodium was administered to animals, several metabolites were detected in plasma and bile (Arai et al. 1988, Komai et al. 1988). For elucidation of the structure of these metabolites, as well as of their mechanism of formation, a metabolism study was undertaken in isolated rat hepatocytes and in a cell-free system of rat liver. This paper describes the isolation and structure of the glutathione conjugate and the dihydrodiol, the major metabolites in the cell system, their mechanism of formation, and the metabolic profile of pravastatin sodium in the isolated hepatocytes. Experimental Radiolabelled pravastatin sodium 14C-labelled mevastatin, the precursor of pravastation was prepared by fermentation with P. citrinum using sodium I4C-acetate as the starting material. This compound was converted biologically by S. carbophylus (Arai et al. 1988) into 14C-pravastatin lactone (23.4pCi/mg, 98.0% pure) which was hydrolysed with equimolar NaOH in ethanol (T. Komai, unpublished).
Animals Male Sprague-Dawley (SD) strain rats weighing 200-250 g were pre-treated daily with an i.p. dose of 3-methylcholanthrene (MC, Wako Chemical Industries, Osaka, Japan, 80 mg/kg per day, in rape oil) and 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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phenobarbital (PB, Wako Chemical Industries, 80mg/kg per day, in aqueous solution) for 4 days to induce the cytochromes P-450 in the liver. Preparation of isolated rat hepatocytes Isolated rat hepatocytes were prepared according to the perfusion method of Moldbus et al. (1978). Collagenase was purchased from Boehringer-Mannheim (Clostridium hzstolyticum, 0 1 5 U/mg).
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Preparation of rat liver cytosol Rat liver was obtained after carotid bleeding under ether anaesthesia. The liver was homogenized in a Polytron (Kinematica GmbH, Switzerland) in 3 vol. of 1.15%KC1/10mM potassium phosphate buffer, pH 7.4. T h e homogenate was centrifuged at 105 OOOg for 60min at 4°C and the supernatant was stored at - 70°C (cytosol fraction). Incubation of prawastatin sodium with isolated rat hepatocytes Hepatocytes were diluted to 2 x lo6 cells/ml in Krebs-Henseleit buffer p H 7.4 supplemented with 1OmM HEPES, after preincubation at 37°C for 5min. ‘‘C-pravastatin sodium was added ( 0 1 mM final conc. 0.5 pCi/mg) and incubated in a round-bottomed flask which was rotated (30 rpm) in an incubation bath at 37°C. During incubation, carbogen gas (95% 0,-5%CO,) was applied continuously to the surface of the incubation medium. Aliquots of the incubation media were taken at regular intervals, and substrate and metabolite concentrations were determined by h.p.1.c. T h e cells were kept at 4°C before incubation. Cell viability was 88-96%, on the basis of trypan blue staining method, throughout the experiment. Incubation of pravastatin sodium was also carried out in the P-450-induced rat hepatocytes prepared from PB- and MC-treated rats and inhibition of P-450 was carried out in the presence of SKF-525A (a gift of Smith Kline & Franch Labs. Ltd, 0.2 mM final conc.) in hepatocytes before adding substrate. Isolation of the major metabolites from the isolated hepatocytes T h e two major polar metabolites (CM-1 and CM-2) obtained from the isolated rat hepatocytes were isolated and purified for structure analysis. All the supernatant solution (250 ml) of the medium incubated for 2 h with hepatocytes was adjusted to p H 3 with formic acid and charged onto 30 Bond-Elut C,, cartridges (Analytichem International, CA, USA) for clean-up procedure. After washing with water (twice), one metabolite, CM-1, was eluted with 300ml of water-methanol (80: 20v/v), and the other metabolite, CM-2, was eluted with 100mlof water-methanol (50 : 50 v/v). The CM-1 fraction was further purified by two procedures of h.p.l.c., as shown; 1. Column: YMC-ODs, S-343-15 (20mm x 250mm, Yamamura Kagaku, Osaka, Japan); mobile phase: 0.05% aq. trifluoroacetic acid-acetonitrile (80 : 20 v/v); flow rate 4 ml/min; detection U.V. (210 nm). 2. Column: YMC-ODS, A-312 (6mm x 150mm, Yamamura Kagaku, Osaka, Japan); mobile phase same as (1); flow rate 1ml/min; detection same as (1). The eluates at around 8min for CM-1 and at 12.5 min for CM-2 with h.p.1.c. condition (2) were collected and evaporated to dryness under reduced pressure. Purified CM-1 (0.9mg) and CM-2 (1.4mg) were obtained from 60mg (3 pCi) of pravastatin sodium. Incubation of the various epoxides in rat liver cytosol Rat liver cytosol(4OOpl of 25% w/v) was incubated at 37°C with 50pl of 1-16 mM epoxide and 50mM glutathione (Sigma Chem. Co., USA) in 1OmM phosphate buffer (pH 7.4) containing 1.15% KCI. These reactions were also carried out in cytosol after dialysis using cellulose tubing (UC 20-32-100, Viscase Corp., USA) for 4 h, and in medium without cytosol. After 15 min incubation the reaction was stopped by adding 4vol. of methanol, and the substrate epoxides and products were determined by h.p.1.c. as described in figure 4. Preparation of metabolites and their intermediates 1. Synthesis of 4‘ag, S’/l-epoxypravastatin sodium (epoxide (1)) (1) 4’a/l, 5‘B-epoxypravastation lactone. T o an ice-cold solution of pravastatin lactone in lOml of ethyl acetate were added 2ml of 5%aq. NaHCO, and 431 mg of m-chloroperbenzoic acid, successively. After stirring for 1 h under cooling by ice, lOml of ethyl acetate was added to extract the product, the organic layer was separated, washed with water, 2% aq. Na,CO, and 2% aq. Na,SO, and water, successively and dried over anhydr. Na,SO,. After removal of the solvent the crude product was purified by column chromatography on alumina (Xlumina B, activity grade IV, ICN Biomedicals, dichloromethane-methanol(95 : 5, v/v) as eluent) to afford 260mg of the 4’ag,S’fi-epoxy derivative. ‘H-n.m.r. (CDCI,, 6ppm): 0.90 (3H, t, J=7.7Hz, 4”-CH,), 0.99 (3H, d, J=7.3 Hz, 2’-CH,), 1.14 (3H, d, J=7.0Hz, 2”-CH3),2.61 ( l H , ddd, J=1.5, 3.7, 17.6Hz, 2-CHH), 2.74 ( l H , dd, J=4.8, 17.6Hz, 2-CHH), 3.36 ( l H , br, 5’-CH),4,14(1H,m,6‘-CH), 4.38(1H,m, 3-CH), 4.60(1H, m, 5-CH), 5.08 ( l H , d, J=9.5Hz, 4‘-CH=), 5.22 ( l H , m, 8’-CH), 6.28 ( l H , dd, J=6.2, 9.9Hz, 3’-CH=).
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(2) 4‘a8, 5’8-epoxy pravastatin sodium T o a solution of 260 mg of the above epoxy derivative in 3 ml of acetonitrile was added 6.2 ml of 0-1 MNaOH under cooling by ice. After stirring for 30min, 300ml of acetonitrile was added and the solution was evaporated in vucuo, to afford 285 mg o f the title compound as white powder. Retention time by h.p.1.c.: 13.15 min (condition; column: Cosmosil 5C18 6 m m x 150mm, eluent: 50mM potassium phosphate buffer (pH 7,4)-acetonitrile (8 : 2 v/v), flow rate l.Oml/rnin, detection u.v., 210nm). ‘H-n.m.r. (D,O, 6ppm): 0 8 8 (3H, t, J = 7 . 0 H z , 4”-CH,), 0.95 (3H, d, J = 7 . 0 H z , 2’-CH,), 1.12 (3H, d , J=6.8Hz, 2”-CH,), 1.27-1.63 (9H, m), 1.86 ( l H , m, 1’-CH), 2.14 ( l H , m, 7’-CHH), 2.20 ( l H , dd, 5=4.0,12.2Hz,8’a-CH), 2.31 (lH,dd,J=8.0,14.8Hz, 2-CHH),2.37(1H,dd,J=5.4,14.8Hz, 2-CHH), 2.45 (2H, m, 2‘7-CH), 3.50 ( l H , br, 5’-CH), 3.70 ( l H , ddt, J=3.3, 5.0, 7,7Hz, 5-CH), 4.08 ( l H , tt, J = 5-5,8.0 Hz, 3-CH), 4.25 (ZH, ddd, J=2.0,5.5,11.5 Hz, 6‘-CH), 5.1 1 ( l H , d, J=9.7 Hz, 4’-CH=), 5.23 ( l H , tbr, J = 3 , 9 H z , 8’-CH), 6.45 ( l H , dd, J=6.4, 7,8Hz, 3’-CH=). 2. Synthesis of 3’a,4a-epoxypravastatin sodium (epoxide (2)) (1) 3,6’-bis-t-Butyldimethylsilylpravastatinlactone T o a solution of 2.03 g of the pravastatin lactone in 10 ml of dimethylformamide were added 0.82 g of imidazole and 1.81 g of t-butyldimethylsilylchloride,successively. The whole mixture was stirred for 17 h at room temp. Ethyl acetate was added and washed with water and dried over anhydr. Na,SO,. After removal of the solvent the crude products were purified by silica gel column chromatography (ethyl acetate-hexane (2 : 8 v/v) as eluent) to afford 2.67 g of the bis-silyl derivative. ‘H-n.m.r. (CDCI,, Spprn): 0.8M.94 (24H, t-butyl x 2, CH, x 2), 1.12 (3H, d , J=7.0Hz, 2”-CH,), 4.28 ( l H , m, 3-CH), 4.43 ( l H , m, 6’-CH), 4.58 ( l H , m, 5-CH), 5.37 ( l H , m, 8’-CH), 5.48 ( l H , br, 5’-CH=), 5.85 ( l H , dd, 5 ~ 5 . 89.5H2, , 3’-CH=), 5.99 ( l H , d, 5=9.5Hz, 4’-CH=) (2) 3’,6‘-bis-t-Butyldimethylsilyl-3‘a,4‘a-epoxypravastatin lactone T o a solution of 2.1 3 g of the above bis-silyl derivative in 25 ml of ethyl acetate were added 10 ml of 5% aq. NaHCO, and 1.16g of m-chloroperbenzoic acid, successively. After stirring at room temp. for 20min, 30ml of ethyl acetate was added and the organic layer was separated and washed with 2%aq. Na,CO,, 2% aq. Na,SO, and water, successively, and dried over anhyd. Na,SO,. After removal of the solvent, the crude products were separated by silica gel column chromatography (hexane-ethyl acetate (75 : 25 v/v) as eluent) to give 1.Og of the major product of and 0.59 g of the minor one with R, values of 0 3 7 and 045, respectively, by t.1.c. on silica gel (hexane-ethyl acetate (75 : 25 v/v) as developing solvent). T h e major product was confirmed to be the 3’a,4’a-epoxide isomer by n.m.r. ‘H-n.m.r. (CDCl,, 6 ppm) 085-095 (24H, t-butyl x 2, CH, x 2), 1.12 (3H, d , J = 7.0 Hz, 2”-CH,), 3.29 ( l H , m, 3’-CH), 3.42 ( l H , d ,J=3.7Hz,4’-CH),4.28(1H,m,3-CH),4.42(1H,m, 6‘-CH),4,55(1H,rn, 5-CH), 5.26(1H, br, 8’CH), 5.94 ( l H , br, 5’-CH=). T h e minor product with R, value of 0.45 was confirmed to be the 4’a/?,S’j-epoxide isomer by n.m.r. ‘H-n.m.r. (CDCI,, Gppm): 0.85-0.95 (21H, t-butyl x 2, CH,), 0.97 (3H, d, J = 7 . 0 Hz, 2’-CH3), 1.41 (3H, d, J=7.0Hz, 2”-CH3), 2.5C2.67 (2H, m, 2-CH2), 3.21 ( l H , br, 5’-CH), 4 1 8 4 . 3 3 (2H, m, 6’,3-CH), 4.58 ( l H , m, 5-CH), 5.07 ( l H , d, 5=9.9Hz, 4‘-CH=), 5.18 ( l H , m, 8’-CH), 6.24 ( l H , dd, J=6.6, 9.9Hz, 3‘-CH=). (3) 3’a, 4’a-Epoxypravastatin lactone T o a solution of 326 mg of bis-silyl-3’a, 4’a-epoxide in 5 ml of tetrahydrofuran was added 5 ml of 1 Mtetrabutyl-ammonium fluoride in tetrahydrofuran and 0 6 3 ml of acetic acid at 0°C. After stirring at room temperature for 17 h, 10 ml of ethyl acetate was added, the organic layer was separated, washed with water and dried over anhydr. Na,SO,. After removal of the solvent, the crude products were separated by column chromatography with silica gel (dichloromethane-methanol(95.5v/v) as eluent) to give 139mg of the 3’a, 4’a-epoxypravastatin lactone. T.1.c.: R, = 0.21 (silica gel, ethyl acetate as developing solvent). ‘H-n.m.r. (CDCl,, Sppm) 0.88 (3H, t, J = 7 . 7 H z , 4”-CH3), 0.94 (3H, d, J = 7 . 3 H z , 2’-CH,), 1.11 17.6Hz, 2-CHH),273(1H,dd,J=5.1, 17.6Hz2(3H, d,J=7~0Hz,2“-Me),2.62(1H,ddd,J=1.1,4.0, CHH), 3.39 ( l H , m, 4’-CH), 3.46 ( l H , d , J=3.7 Hz, 4‘-CH), 4.35 ( l H , m, 3-CH), 4.41 ( l H , m, 6‘-CH), 4.58 ( l H , m, 5-CH), 5.31 ( l H , br, 8’-CH), 6.04 ( l H , br, 5’-CH=). (4) 3’a,4’t~-Epoxypravastatinsodium To a solution of 42.2 mg of 3’a,4’a-epoxypravastatin lactone in 0.5 ml of acetonitrile was added 0.1 M NaOH solution under cooling by ice. After stirring at room temperature for 1 h, the whole mixture was purified by column chromatography (CHP-20P (MCI Gel, 75-1 50pm, cu. 20m1, Mitsubishi Chemical Industries, Tokyo, Japan), stepwisegradient with water(100 ml), water-acetone (95 : 5 v/v, SOml), wateracetone (90: 1Ov/v, 50 ml), water-acetone (85 : 15 v/v, 50ml), and water-acetone (80: 20v/v, SOml), successively) to afford 32 mg of the desired product. Retention time by h.p.1.c.: 6-23min (column: Cosmosil 5C18, 6 m m x 150mm. eluent: 50mM potassium phosphate buffer (pH 7,4)-acetonitrile (8 : 2 v/v), flow rate l.Oml/rnin, detection U.V. 210nm).
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'H-n.m.r. (D,O, 6ppm): 086 (3H, t, J=7.5Hz, 4"-CH3), 0.94 (3H, d, J = 6 6 H z , 2'-CH,), 1.10 (3H, d, J=7.0Hz, 2"-CH), 1.27-1.69(10H,m), 2.3&2.50(6H,m), 3.50(1H, tbr,J=3-4Hz, 3'-CH), 3.65(1H,d, J=4.0Hz, 4'-CH), 3.71 (lH, m, 5-CH), 4.08 (lH, tt, J=60, 7.3 Hz, 3-CH), 440 (lH, ddt, J = 105, 6.6, 3.3 Hz, 6'-CH), 5.31 (lH, m, 8'-CH), 6.08 ( l H , br, 5'-CH).
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3. Preparation of the glutathione conjugate (CM-1) with rat liver cytosol To 45 ml of rat liver cytosol, 20mg of the synthetic epoxide (1) was added and incubated at 37°C for 30min as described previously. The reaction was stopped by addition of 2201-1-11 of methanol, and the reaction mixture filtered through celite (Hyflo Super-Cel, John-Manville/Iwai Kagaku Co., Tokyo, Japan). The filtrate was concentrated to about 20ml under reduced pressure, adjusted to p H 4 with 2 M-HCI and purified by column chromatography on porous polymer CHP-2OP (ca. 20 ml) by gradient elution with water (250ml), water-acetone (95 : 5 v/v, 200ml), water acetone (90: lOv/v, 200ml), and water-acetone (80:20v/v, 200 ml), successively. The glutathione conjugate was obtained from the 5% and 10% aqueous acetone fractions after concentration under reduced pressure; 7.7 mg of purified conjugate (CM-1) was obtained from 20mg of epoxide (1). Structure of the conjugate was shown to be pravastatin 4'aa glutathione conjugate by n.m.r. and mass spectrometry, as described elsewhere (Nakamura et al. 1991). Identification of glutathione conjugate by specific enzyme reaction The isolated cell metabolite, the glutathione conjugate, CM-1, was incubated with y-glutamyl transpeptidase (y-GTP, 20 units, Sigma, USA) in 2ml of 5 0 m Tris ~ buffer (pH 7-4) containing 5 0 m ~ glycylglycine and 1OmM MgCI, at 37°C for 1h. A part of the reaction mixture was further incubated after addition of 10 units of dehydropeptidase-I (DHP-I, Hirota et al. 1985) at 37°C for 30min. The enzymically cleaved products with p G T P alone and p G T P followed with DHP-I were charged onto Bond-Elut C,, cartridges and each product was eluted with water-methanol (90: lOv/v) and (85 : 15 v/v), respectively, after washing with water. A part of the hydrolysis product with DHP-I was acetylated by addition of a drop of acetic anhydrideacetone (30 : 70v/v) in an ice bath. The enzymic cleavage process and acetylated product thus obtained were separated by t.1.c. (conditions, see analytical procedures, t.1.c. (2)). After development, the t.1.c. plate was contacted with X-ray film SB (Kodak Co., USA) in the dark. Identification of the glutathione conjugate by mass spectrometry The structure of the glutathione conjugate, CM-1, was determined by secondary ion mass spectrometry (M-80A, Hitachi, Tokyo, Japan). Glycerol was used as a matrix.
T.1.c. of the metabolites T h e hepatocyte incubation mixture was centrifuged, and the supernatant was freeze-dried, dissolved in 0.41~11of water-methanol (20 : 80 v/v) and 10 jd aliquots of the sample solution were analysed by t.1.c. as follows: (1) silica gel 60 F254 (250p-1, E. Merck, Germany) in chloroform-methanol-acetic acid (9 : 1 : 1 by vol.), (2) silica Gel 60 F254 in n-butanol-acetic acid-water (4 : 1 : 1 by vol.). After development, the concentration of the individual metabolites were determined by Bio-Image Analyzer BA-100 (Fuji Photo Film Co. Ltd, Tokyo, Japan).
Results Identification of major polar metabolites by specific enzyme reaction and mass spectrometry The major polar metabolite CM-1 was analysed by secondary ion mass spectrometry after separation and purification by two procedures of h.p.1.c. From the mass spectra of CM-1 shown in figure 1, CM-1 had intense ions at m/z748 (M H)+ and m / z 770 (M Na)+ in the positive spectrum and at m / z 746 (M-H)and m / z 768 (M-2H Na)- in the negative spectrum, indicating that the molecular weight of CM-1 was 747. T h e fact that the molecular weight of the metabolite was 323 higher than that of the parent compound (mol. wt, 424) indicated the formation of a glutathione conjugate. T h e metabolite CM-1 was then subjected to enzyme cleavage reaction with y-GTP followed by DHP-I, and the reaction product was analysed by secondary ion mass spectrometry after monitoring the reaction products by t.1.c. and h.p.1.c. and peak fractionation. y-GTP is an enzyme which hydrolyses glutathione, liberating glutamic acid. DHP-I hydrolyses the cysteinylglycine moiety acting on the hydrolysate of y-GTP. T h e hydrolysis product with y-GTP showed an intense peak at m/z 619 in the positive spectrum, which indicated the molecular
+
+
+
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748 [ M + H
I+
461
444
[M+Na I +
L 800
300
400
600
500
700
154
7 4 6 [ M-H 1-
I
[M-2H+Na 1 -
.. .., .. ... .. 200
300
400
1
. ,. ... ... .. , . . .. . . .. 500
600
' I '
700
800
Figure 1. Mass spectra of pravastatin metabolite (CM-I), the glutathione conjugate. The positive secondary ion mass spectrum (upper) and the negative secondary ion mass spectrum (lower). The M-80 double-focusing mass spectrometer (Hitachi) with an M-300 data manipulator was used. Measurement used a first acceleration voltage for positive of -8kV, and negative of - 9 kV; and a second acceleration voltage of 3 kV; the first filament electric current was 35A, the first collision gas was X'(2 x lo-* torr), and the matrix was glycerol and thioglycerol.
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weight of the product was 618. T h e decreased number of 129 mass unit agreed with the molecular weight of glutamic acid. Similarly, the hydrolysate with DHP-I had an m/z of 561 in the positive mode, and the decreased number of 58 mass unit corresponded to the liberation of the glycine moiety. This hydrolysate gave a product of molecular weight of 602 after acetylation with acetic anhydride. T h e enzymically cleaved products of CM-1 noted above were also anaiysed by t.1.c. As shown in figure 2, on treatment with y-GTP and DHP-I (dipeptidase), CM1 gave new products with different R, values, and after acetylation a further product was formed, which provide evidence for CM-1 being a glutathione conjugate and the absolute structure was assigned by n.m.r. spectroscopy. Concerning CM-2, since mass spectrometric analysis indicated a dihydrodiol form of the parent compound, structural determination was carried out using n.m.r. spectrometry, as for CM-1. T h e details on structural assignments are reported elsewhere (Nakamura et al. 1991), and the absolute structures of CM-1 and CM-2 are shown in figure 3.
Formation of the glutathione conjugate (CM-1)and the dihydrodiol (CM-2) Since the formation of the glutathione conjugate CM-1 was thought to be derived from an epoxide, chemical synthesis of the epoxides on the decalin ring was undertaken. After making the lactone at the carboxy moiety, selective oxidation of the double bonds on the decalin ring was effected with m-chloroperbenzoic acid, with or without modification at the hydroxy group considering steric hindrance. As a result, two epoxides were synthesized. Figure 4 shows the structure of these two expoxides, the 4'aP, S'P-epoxide and the 3'a, 4'a-epoxide. Each epoxide thus obtained was incubated at 37°C for 15 min in rat liver cytosol, since hopefully one of these epoxides would be the intermediate substrate to form the
Figure
2.
Enzyme hydrolysis and acetylation of the isolated glutathione conjugate (CM-1).
CM-1 was treated with y-GTP and dipeptidase successively, and the resulting product was acetylated. Each reaction product A, B, C, D was separated by t.1.c.on silica gel F 254 (E. Merck) in n-butanol-acetic acid-water (4 : 1 : 1 by vol.) a6 a solvent. Spots were visualized by contacting with X-ray film.
Metabolism of pravastatin sodium in rat hepatocytes
HO HO I G
Figure 3.
"OH
OH
SG: Glutathione
Glutathione conjugate of pravastatin (CM-1 )
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\ .
HO
493
Dihydrodiol metabolite of pravastatin ( CM-2 )
Structures of the pravastatin metabolites CM-1 and CM-2.
4ap, Sp-epoxide
3a,@a-epoxide
NaOOCyoH
Figure 4.
H.p.1.c. profiles of the products obtained on incubation of pravastatin epoxides with rat hepatic cytosol.
H.p.1.c. was conducted in a Hitachi model 655 liquid chromatograph with a U.V.detector and a D-2000 integrator system. The column used was YMC-Pack ODS A-312 ( 6 m m x 1 5 0 m m , Yamamura Kagaku Co.) and was monitored by U.V. detector at 210 n m with water-acetonitrile-Pic A (ion-pairing reagent, low u.v., Waters) (83 : 17 : 0 3 , by vol.) as mobile phase with a flow rate of 1ml/min.
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z
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E
'-'T
0.0
0.5
1.0
1.5
Concn. of 4ap, S'pepoxide (epoxide(1)) ( rnM ) Figure 5. Formation of CM-1 and CM-2 from the epoxide (1) in rat hepatic cytosol. The epoxide (1) was incubated in liver cytosol at 37°C for 15min and the CM-1 and CM-2 produced were determined by h.p.1.c. whose conditions are shown in figure 4: A and A represent CM-1 formed in cytosol and dialysed cytosol, respectively, and e, 0 and 0 represent those of CM-2 in cytosol, dialysed cytosol and in medium buffer (without cytosol), respectively. Each point represents the mean of triplicate determinations; SD at each point was less than 10%.
glutathione conjugate (CM-1) with the possible help of glutathione-S-transferase. T h e reaction were followed by h.p.1.c and the resulting typical chromatograms (see figure 4) show that the 4'ap, 5'p-epoxide produced CM-1. T h e diol CM-2 was also produced from the same 4'aB, S'B-epoxide. These findings strongly indicate that pravastatin sodium was metabolized to form CM-1 and CM-2 exclusively via the 4'a/?, S'B-epoxide in rat. T h e rate of formation of CM-1 and CM-2 from the 4'ab, 5'p-epoxide in liver cytosol was investigated by incubation at 37°C for 15 min under various conditions, and the results are shown in figure 5. T h e rate of formation of CM-1 was nearly saturated with increase of substrate concentration, while that of CM-2, the dihydrodiol, was increased linearly with increase of substrate concentration. For confirmation of this, pravastatin sodium was incubated in liver cytosol after dialysis to remove glutathione. T h e dialysed cytosol no longer produced the conjugate although the dihydrodiol was still produced; the glutathione conjugating ability of the dialysed cytosol was restored by addition of glutathione to the reaction medium. T h e 4'ap, S'p-epoxide was relatively unstable and its rate of decomposition to the dihydrodiol in the buffer used in the incubation was almost the same as in the cytosol. This leads to the conclusion that glutathione conjugation (CM-1) was an enzymic process and that the dihydrodiol is formed by non-enzymic hydrolysis of the +a@,S'b-epoxide, accompanied by the intramolecular migration of the double bond in the decalin moiety. The reaction product (CM-1) obtained in the cytosol system was shown to be of identical structure and configuration with the conjugate obtained from isolated hepatocytes.
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0
10 15
30
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Time of incubation ( min.) Figure 6. Elimination of pravastatin sodium (upper) and formation of its metabolites (lower) in isolated rat hepatocytes. Data are means of three animals, L- SEM indicated by bars. Pravastatin sodium; 0 ,CM-1; 0, R-416 (see Arai et 01. 1988); A , Non-polar metabolites; W , CM-2; 0,Polar metabolites; A , CM-3.
+,
Metabolic profile of pravastatin sodium in the isolated rat hepatocytes T h e metabolic profile of pravastatin sodium in isolated rat hepatocytes is depicted in figure 6, which shows the time-course of formation and elimination of the metabolites and the parent compound, quantified by t.1.c. using an imaging analyser. T h e two t.1.c. methods were designed for separation of relatively polar and non-polar metabolites as described in 'T.1.c. of the metabolites'. The amount of pravastatin remaining in the incubation medium was 79% at 30 min, 75% at 60 min and 66% at 90min, respectively, when 0.1 mM substrate was incubated. With the decrease of pravastatin sodium, four major metabolites were produced in the course of incubation. Among these metabolites, the isomer of pravastatin sodium, was identified based on its R, value in t.1.c. and the retention time in h.p.1.c. T h e other three metabolites, CM-1, CM-2 and CM-3, were isolated by t.1.c. followed by h.p.1.c. Metabolite CM-3, as well as other minor ones, noted as polar and nonpolar metabolites in figure 6, will be reported in a subsequent publication. Metabolism of pravastatin sodium in P-450-induced and -inhibited hepatocytes Since the structures of the main metabolites of pravastatin sodium in the isolated rat hepatocytes system were shown to be the glutathione conjugate (CM-I) and the dihydrodiol (CM-2), the effects of induction and inhibition of cytochrome P-450 on the formation of these two metabolites was examined in the cell system. Induction of P-450 was carried out by pretreatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) prior to the preparation of the cells, and P-450 activity was inhibited by the addition of SKF-525A to the incubation medium. As shown in figure 7, PB caused a marked increase in metabolite formation while M C
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0
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30
m
90
Tlrne of incubation ( rnln. )
.,
Figure 7. Effect of phenobarbital, 3-methylchoranthrene and SKF-525A on (upper) elimination of pravastatin sodium, and (middle) formation of CM-1, and (lower) CM-2 in isolated rat hepatocytes. induced with MC; Substrate: 0 1 mM: 0 , Normal hepatocytes; A , induced with PB; 0, inhibited with SKF-525A.
showed a i.!oderate increase. T h e PB-treated cell system produced more than three times the glutathione conjugate and twice the dihydrodiol compared to that formed by normal hepatocytes, whereas MC-induced cells produced about 1.5 times the amount of glutathione conjugate and twice the amount of dihydrodiol produced by non-induced hepatocytes after incubation for 60 min. In contrast, addition of SKF525A completely inhibited the formation of the glutathione conjugate and the dihydrodiol, as well as the elimination of the substrate pravastatin. These findings strongly indicate an association of the hepatic monooxygenase system in the metabolism of pravastatin sodium.
Discussion T h e in vitto metabolic studies of pravastatin sodium in isolated rat hepatocytes were carried out to elucidate the structures of the two major polar metabolites and their mechanism of formation. The major metabolite (CM-1) produced in hepatocytes was isolated and shown to be a glutathione conjugate, both by enzymic hydrolysis and mass spectrometric and n.m.r. spectroscopic analysis. This glutathione conjugate could also be prepared by incubating the 4'aB, 5'B-epoxide, obtained synthetically, in rat liver cytosol. The structure of the other major metabolite, the dihydrodiol of the parent drug, was established by n.m.r. spectroscopy. One of the possible routes for glutathione conjugate formation is via an epoxide (Hammarstrom et al. 1979). From the structural consideration of the metabolites, as
- -
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Metabolism of pravastatin sodium in rat hepatocytes /
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-
HO
Epoxide (1)
Pravastatin sodium
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HO
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i
Epoxide (3)
-
OH
Dihydrodiol( CM-2 )
:
Epoxide (2)
GS
SG
dH
SG: Glutathione
Figure 8. Possible formation of CM-1 and CM-2 from pravastatin epoxides.
well as the results of the enzyme induction and inhibition studies, it seemed probable that the biotransformation of pravastatin proceeds through an epoxide. Figure 8 illustrates the four kinds of possible epoxides on the decalin moiety and the presumed formation routes for the glutathione conjugate (CM-1) and the dihydrodiol (CM-2). In this scheme the epoxides which are likely to produce CM-2 are probably epoxide (1) and epoxide (2), as it seemed impossible to form a dihydrodiol from the other two epoxides on the basis of positional and the stereochemical consideration of the hydroxy groups on the decalin ring. According to this hypothesis, four possible epoxides were synthesized, and two of them, the
Glutathione-
H
O
W
no
H
-
'y
COOH
CONH -COOH
Glutathione conjugate (W-1)
HO
cap, Sp-epoxide
\\ Non-enzymic
-
Pravastatin sodium OH HO
I
'
""OH
OH
Dihydrodiol( CM-2)
Figure 9.
Probable mechanisms of formation of CM-1 and CM-2.
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epoxide (1) and epoxide (2) obtained were incubated in the rat liver cytosol which contained glutathione-S-transferase. Epoxide (4)was isolated as a minor by-product in the fermentation product of pravastatin sodium and the structure wiH be reported elsewhere (Hamano, K., Kinoshita, M. and Hosobuchi, M., Sankyo in-house report, unpublished); epoxide (4) did not produce CM-1 and CM-2 by the incubation with rat liver cytosol. As a result, it was only 4'aP, 5'P-epoxide (epoxide (1)) that could produce the glutathione conjugate identified as CM-I. Concerning the dihydrodiol, the hydroxy group was not introduced into the same position as the glutathione moiety in the conjugate when the epoxide was opened. T h e reason for this might be postulated as a nucleophilic attack of the hydroxy ion at the 3'-C position instead of the 4'a-C, due to protonation on the oxygen of the epoxide followed by concerted migration of the double bond. The mechanism of formation of the glutathione conjugate and the dihydrodiol is summarized in figure 9. In general, glutathione conjugates are excreted as mercapturates in the urine after being acetylated in the kidney, and the further metabolism of this glutathione conjugate both in vitro and in vivo needs to be studied.
References ARAI,M., SERIZAWA, N., TERAHARA, A., TSUJITA, Y.,TANAKA, M., MASUDA, H. and ISHIKAWA, S., 1988, Pravastatin sodium (CS-514), a novel cholesterol-lowering agent which inhibits HMG-CoA reductase. Annual Reports of Sankyo Research Laboratories, 40,1-38. S., MURPHY, R.C., SAMUELSSON, B., CLARK, D. A,, MIOSKOWSKI, C., and COREY, E. J., HAMMARSTROM, 1979, Structure of leucotriene C, identification of the amino acid part, Biochemical and Biophysical Research Communications, 91, 1266-1272. HARUYAMA, H., KUWANO, H., KINOSHITA, T., TERAHARA, A., NISHIGAKI, T., and TAMURA, C., 1986, Structure elucidation of the bioactive metabolites of ML-236B (mevastatin) isolated from dog urine. Chemical and Pharmaceutical Bulletin, 34, 1459-1467. HIROTA, T., NISHIKAWA, Y., TAKAHAGI, H., IGARASHI, T., and KITAGAWA, H., 1985, Simultaneous purification and properties of dehydropeptidase-I and aminopeptidase-M from rat kidney. Research Communications in Chemical Pathology and Pharmacology, 49, 435-445. KOMAI, T., h A W A I , K., SHIGEHAR, A. E., KUROIWA, C., KAWASAKI, Y., KITAZAWA, E., and TANAKA, M., 1988, Metabolic study of pravastatin sodium(I), Metabolism and disposition of 14C-pravastatin in experimental animals, Second Znternational ZSSX Meeting, 16-20 May, Kobe, Japan. I., and ORRENIUS, S., 1978, Isolation and use of liver cells. Methods in MOLDBUS,P. HOEGBERG, Enzymology, 52, 6&71. H., MIYAGUCHI, K., MURAMATSU, S., TAKAHAGI, H., and NAKAMURA, T., YODA, K., KUWANO, KINOSHITA, T., 1991, Metabolism of pravastatin sodium in isolated rat hepatocytes. 11. Structure elucidation of the metabolites by n.m.r. spectroscopy. Xenobiotica, 21, 277-293. TSUJITA, Y., KURODA, M., SHIMADA, Y., TANZAWA, K., ARAI,M., KANEKO, I., TANAKA, M., MASUDA, H., TARUMI, C., WATANABE, Y.and FUJII,S., 1986, CS-514, a competitive inhibitor of 3-hydroxy-3methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species. Biochimica et Biophysica Acta, 877, 5 M O .
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Metabolism of pravastatin sodium in isolated rat hepatocytes. I. Glutathione conjugate formation reaction S. Muramatsu, K. Miyaguchi, H. Iwabuchi, Y. Matsushita, T. Nakamura, T. Kinoshita, M. Tanaka & H. Takahagi To cite this article: S. Muramatsu, K. Miyaguchi, H. Iwabuchi, Y. Matsushita, T. Nakamura, T. Kinoshita, M. Tanaka & H. Takahagi (1992) Metabolism of pravastatin sodium in isolated rat hepatocytes. I. Glutathione conjugate formation reaction, Xenobiotica, 22:5, 487-498 To link to this article: http://dx.doi.org/10.3109/00498259209053111
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Date: 26 January 2016, At: 21:56
XENOBIOTICA,
1992, VOL. 22,
NO.
5, 487-498
Metabolism of pravastatin sodium in isolated rat hepatocytes. I. Glutathione conjugate formation reaction S. MURAMATSU, K. MIYAGUCHI, H. IWABUCHI, Y. MATSUSHITA, T. NAKAMURA, T. KINOSHITA, M. TANAKA and H. TAKAHAGI
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Analytical and Metabolic Research Laboratories, Sankyo Co. Ltd., 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan
Received 16 January 1990; accepted 22 January 1992
1. T h e metabolic fate of pravastatin sodium (sodium ( +)-(3R, 5R)-3,5-dihydroxy-7-
{ (1’S,2’S,6‘S,8’S,8’aR)-6‘-hydroxy-2’methyl-8’-[(S)-~-methylbutyryloxy]-l’,~,6‘,7’,8‘, 8’a-hexahydro-l‘-naphthyl}heptanoate) was studied in isolated rat hepatocytes. 2. Two polar metabolites were isolated and identified as a glutathione conjugate and a dihydrodiol. 3. Both metabolites were formed via an epoxide which has been identified as the 4‘ap,S’P-epoxide on the decalin moiety. 4. Formation of the glutathione conjugate was enzymic, while the dihydrodiol was formed by non-enzymic hydrolysis of the epoxide accompanied by the intramolecular migration of the double bond.
Introduction Pravastatin sodium (sodium (+)-(3R,5R)-3,5-dihydroxy-7-{(l’S,2’S,6’S,8’S, 8’aR)-6‘-hydroxy-2’-methyl-8’- [( S)-2”-methylbutyryloxy]- 1’,2’,6’,7’,8’,8’a-hexahydro-1’-naphthy1)heptanoate) is a tissue-selective inhibitor of 3-hydroxy-3methylglutaryl coenzyme A reductase, a key enzyme in cholesterol biosynthesis (Haruyama et al. 1986, Tsujita et al. 1986). When l4C-labe1led pravastatin sodium was administered to animals, several metabolites were detected in plasma and bile (Arai et al. 1988, Komai et al. 1988). For elucidation of the structure of these metabolites, as well as of their mechanism of formation, a metabolism study was undertaken in isolated rat hepatocytes and in a cell-free system of rat liver. This paper describes the isolation and structure of the glutathione conjugate and the dihydrodiol, the major metabolites in the cell system, their mechanism of formation, and the metabolic profile of pravastatin sodium in the isolated hepatocytes. Experimental Radiolabelled pravastatin sodium 14C-labelled mevastatin, the precursor of pravastation was prepared by fermentation with P. citrinum using sodium I4C-acetate as the starting material. This compound was converted biologically by S. carbophylus (Arai et al. 1988) into 14C-pravastatin lactone (23.4pCi/mg, 98.0% pure) which was hydrolysed with equimolar NaOH in ethanol (T. Komai, unpublished).
Animals Male Sprague-Dawley (SD) strain rats weighing 200-250 g were pre-treated daily with an i.p. dose of 3-methylcholanthrene (MC, Wako Chemical Industries, Osaka, Japan, 80 mg/kg per day, in rape oil) and 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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phenobarbital (PB, Wako Chemical Industries, 80mg/kg per day, in aqueous solution) for 4 days to induce the cytochromes P-450 in the liver. Preparation of isolated rat hepatocytes Isolated rat hepatocytes were prepared according to the perfusion method of Moldbus et al. (1978). Collagenase was purchased from Boehringer-Mannheim (Clostridium hzstolyticum, 0 1 5 U/mg).
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Preparation of rat liver cytosol Rat liver was obtained after carotid bleeding under ether anaesthesia. The liver was homogenized in a Polytron (Kinematica GmbH, Switzerland) in 3 vol. of 1.15%KC1/10mM potassium phosphate buffer, pH 7.4. T h e homogenate was centrifuged at 105 OOOg for 60min at 4°C and the supernatant was stored at - 70°C (cytosol fraction). Incubation of prawastatin sodium with isolated rat hepatocytes Hepatocytes were diluted to 2 x lo6 cells/ml in Krebs-Henseleit buffer p H 7.4 supplemented with 1OmM HEPES, after preincubation at 37°C for 5min. ‘‘C-pravastatin sodium was added ( 0 1 mM final conc. 0.5 pCi/mg) and incubated in a round-bottomed flask which was rotated (30 rpm) in an incubation bath at 37°C. During incubation, carbogen gas (95% 0,-5%CO,) was applied continuously to the surface of the incubation medium. Aliquots of the incubation media were taken at regular intervals, and substrate and metabolite concentrations were determined by h.p.1.c. T h e cells were kept at 4°C before incubation. Cell viability was 88-96%, on the basis of trypan blue staining method, throughout the experiment. Incubation of pravastatin sodium was also carried out in the P-450-induced rat hepatocytes prepared from PB- and MC-treated rats and inhibition of P-450 was carried out in the presence of SKF-525A (a gift of Smith Kline & Franch Labs. Ltd, 0.2 mM final conc.) in hepatocytes before adding substrate. Isolation of the major metabolites from the isolated hepatocytes T h e two major polar metabolites (CM-1 and CM-2) obtained from the isolated rat hepatocytes were isolated and purified for structure analysis. All the supernatant solution (250 ml) of the medium incubated for 2 h with hepatocytes was adjusted to p H 3 with formic acid and charged onto 30 Bond-Elut C,, cartridges (Analytichem International, CA, USA) for clean-up procedure. After washing with water (twice), one metabolite, CM-1, was eluted with 300ml of water-methanol (80: 20v/v), and the other metabolite, CM-2, was eluted with 100mlof water-methanol (50 : 50 v/v). The CM-1 fraction was further purified by two procedures of h.p.l.c., as shown; 1. Column: YMC-ODs, S-343-15 (20mm x 250mm, Yamamura Kagaku, Osaka, Japan); mobile phase: 0.05% aq. trifluoroacetic acid-acetonitrile (80 : 20 v/v); flow rate 4 ml/min; detection U.V. (210 nm). 2. Column: YMC-ODS, A-312 (6mm x 150mm, Yamamura Kagaku, Osaka, Japan); mobile phase same as (1); flow rate 1ml/min; detection same as (1). The eluates at around 8min for CM-1 and at 12.5 min for CM-2 with h.p.1.c. condition (2) were collected and evaporated to dryness under reduced pressure. Purified CM-1 (0.9mg) and CM-2 (1.4mg) were obtained from 60mg (3 pCi) of pravastatin sodium. Incubation of the various epoxides in rat liver cytosol Rat liver cytosol(4OOpl of 25% w/v) was incubated at 37°C with 50pl of 1-16 mM epoxide and 50mM glutathione (Sigma Chem. Co., USA) in 1OmM phosphate buffer (pH 7.4) containing 1.15% KCI. These reactions were also carried out in cytosol after dialysis using cellulose tubing (UC 20-32-100, Viscase Corp., USA) for 4 h, and in medium without cytosol. After 15 min incubation the reaction was stopped by adding 4vol. of methanol, and the substrate epoxides and products were determined by h.p.1.c. as described in figure 4. Preparation of metabolites and their intermediates 1. Synthesis of 4‘ag, S’/l-epoxypravastatin sodium (epoxide (1)) (1) 4’a/l, 5‘B-epoxypravastation lactone. T o an ice-cold solution of pravastatin lactone in lOml of ethyl acetate were added 2ml of 5%aq. NaHCO, and 431 mg of m-chloroperbenzoic acid, successively. After stirring for 1 h under cooling by ice, lOml of ethyl acetate was added to extract the product, the organic layer was separated, washed with water, 2% aq. Na,CO, and 2% aq. Na,SO, and water, successively and dried over anhydr. Na,SO,. After removal of the solvent the crude product was purified by column chromatography on alumina (Xlumina B, activity grade IV, ICN Biomedicals, dichloromethane-methanol(95 : 5, v/v) as eluent) to afford 260mg of the 4’ag,S’fi-epoxy derivative. ‘H-n.m.r. (CDCI,, 6ppm): 0.90 (3H, t, J=7.7Hz, 4”-CH,), 0.99 (3H, d, J=7.3 Hz, 2’-CH,), 1.14 (3H, d, J=7.0Hz, 2”-CH3),2.61 ( l H , ddd, J=1.5, 3.7, 17.6Hz, 2-CHH), 2.74 ( l H , dd, J=4.8, 17.6Hz, 2-CHH), 3.36 ( l H , br, 5’-CH),4,14(1H,m,6‘-CH), 4.38(1H,m, 3-CH), 4.60(1H, m, 5-CH), 5.08 ( l H , d, J=9.5Hz, 4‘-CH=), 5.22 ( l H , m, 8’-CH), 6.28 ( l H , dd, J=6.2, 9.9Hz, 3’-CH=).
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(2) 4‘a8, 5’8-epoxy pravastatin sodium T o a solution of 260 mg of the above epoxy derivative in 3 ml of acetonitrile was added 6.2 ml of 0-1 MNaOH under cooling by ice. After stirring for 30min, 300ml of acetonitrile was added and the solution was evaporated in vucuo, to afford 285 mg o f the title compound as white powder. Retention time by h.p.1.c.: 13.15 min (condition; column: Cosmosil 5C18 6 m m x 150mm, eluent: 50mM potassium phosphate buffer (pH 7,4)-acetonitrile (8 : 2 v/v), flow rate l.Oml/rnin, detection u.v., 210nm). ‘H-n.m.r. (D,O, 6ppm): 0 8 8 (3H, t, J = 7 . 0 H z , 4”-CH,), 0.95 (3H, d, J = 7 . 0 H z , 2’-CH,), 1.12 (3H, d , J=6.8Hz, 2”-CH,), 1.27-1.63 (9H, m), 1.86 ( l H , m, 1’-CH), 2.14 ( l H , m, 7’-CHH), 2.20 ( l H , dd, 5=4.0,12.2Hz,8’a-CH), 2.31 (lH,dd,J=8.0,14.8Hz, 2-CHH),2.37(1H,dd,J=5.4,14.8Hz, 2-CHH), 2.45 (2H, m, 2‘7-CH), 3.50 ( l H , br, 5’-CH), 3.70 ( l H , ddt, J=3.3, 5.0, 7,7Hz, 5-CH), 4.08 ( l H , tt, J = 5-5,8.0 Hz, 3-CH), 4.25 (ZH, ddd, J=2.0,5.5,11.5 Hz, 6‘-CH), 5.1 1 ( l H , d, J=9.7 Hz, 4’-CH=), 5.23 ( l H , tbr, J = 3 , 9 H z , 8’-CH), 6.45 ( l H , dd, J=6.4, 7,8Hz, 3’-CH=). 2. Synthesis of 3’a,4a-epoxypravastatin sodium (epoxide (2)) (1) 3,6’-bis-t-Butyldimethylsilylpravastatinlactone T o a solution of 2.03 g of the pravastatin lactone in 10 ml of dimethylformamide were added 0.82 g of imidazole and 1.81 g of t-butyldimethylsilylchloride,successively. The whole mixture was stirred for 17 h at room temp. Ethyl acetate was added and washed with water and dried over anhydr. Na,SO,. After removal of the solvent the crude products were purified by silica gel column chromatography (ethyl acetate-hexane (2 : 8 v/v) as eluent) to afford 2.67 g of the bis-silyl derivative. ‘H-n.m.r. (CDCI,, Spprn): 0.8M.94 (24H, t-butyl x 2, CH, x 2), 1.12 (3H, d , J=7.0Hz, 2”-CH,), 4.28 ( l H , m, 3-CH), 4.43 ( l H , m, 6’-CH), 4.58 ( l H , m, 5-CH), 5.37 ( l H , m, 8’-CH), 5.48 ( l H , br, 5’-CH=), 5.85 ( l H , dd, 5 ~ 5 . 89.5H2, , 3’-CH=), 5.99 ( l H , d, 5=9.5Hz, 4’-CH=) (2) 3’,6‘-bis-t-Butyldimethylsilyl-3‘a,4‘a-epoxypravastatin lactone T o a solution of 2.1 3 g of the above bis-silyl derivative in 25 ml of ethyl acetate were added 10 ml of 5% aq. NaHCO, and 1.16g of m-chloroperbenzoic acid, successively. After stirring at room temp. for 20min, 30ml of ethyl acetate was added and the organic layer was separated and washed with 2%aq. Na,CO,, 2% aq. Na,SO, and water, successively, and dried over anhyd. Na,SO,. After removal of the solvent, the crude products were separated by silica gel column chromatography (hexane-ethyl acetate (75 : 25 v/v) as eluent) to give 1.Og of the major product of and 0.59 g of the minor one with R, values of 0 3 7 and 045, respectively, by t.1.c. on silica gel (hexane-ethyl acetate (75 : 25 v/v) as developing solvent). T h e major product was confirmed to be the 3’a,4’a-epoxide isomer by n.m.r. ‘H-n.m.r. (CDCl,, 6 ppm) 085-095 (24H, t-butyl x 2, CH, x 2), 1.12 (3H, d , J = 7.0 Hz, 2”-CH,), 3.29 ( l H , m, 3’-CH), 3.42 ( l H , d ,J=3.7Hz,4’-CH),4.28(1H,m,3-CH),4.42(1H,m, 6‘-CH),4,55(1H,rn, 5-CH), 5.26(1H, br, 8’CH), 5.94 ( l H , br, 5’-CH=). T h e minor product with R, value of 0.45 was confirmed to be the 4’a/?,S’j-epoxide isomer by n.m.r. ‘H-n.m.r. (CDCI,, Gppm): 0.85-0.95 (21H, t-butyl x 2, CH,), 0.97 (3H, d, J = 7 . 0 Hz, 2’-CH3), 1.41 (3H, d, J=7.0Hz, 2”-CH3), 2.5C2.67 (2H, m, 2-CH2), 3.21 ( l H , br, 5’-CH), 4 1 8 4 . 3 3 (2H, m, 6’,3-CH), 4.58 ( l H , m, 5-CH), 5.07 ( l H , d, 5=9.9Hz, 4‘-CH=), 5.18 ( l H , m, 8’-CH), 6.24 ( l H , dd, J=6.6, 9.9Hz, 3‘-CH=). (3) 3’a, 4’a-Epoxypravastatin lactone T o a solution of 326 mg of bis-silyl-3’a, 4’a-epoxide in 5 ml of tetrahydrofuran was added 5 ml of 1 Mtetrabutyl-ammonium fluoride in tetrahydrofuran and 0 6 3 ml of acetic acid at 0°C. After stirring at room temperature for 17 h, 10 ml of ethyl acetate was added, the organic layer was separated, washed with water and dried over anhydr. Na,SO,. After removal of the solvent, the crude products were separated by column chromatography with silica gel (dichloromethane-methanol(95.5v/v) as eluent) to give 139mg of the 3’a, 4’a-epoxypravastatin lactone. T.1.c.: R, = 0.21 (silica gel, ethyl acetate as developing solvent). ‘H-n.m.r. (CDCl,, Sppm) 0.88 (3H, t, J = 7 . 7 H z , 4”-CH3), 0.94 (3H, d, J = 7 . 3 H z , 2’-CH,), 1.11 17.6Hz, 2-CHH),273(1H,dd,J=5.1, 17.6Hz2(3H, d,J=7~0Hz,2“-Me),2.62(1H,ddd,J=1.1,4.0, CHH), 3.39 ( l H , m, 4’-CH), 3.46 ( l H , d , J=3.7 Hz, 4‘-CH), 4.35 ( l H , m, 3-CH), 4.41 ( l H , m, 6‘-CH), 4.58 ( l H , m, 5-CH), 5.31 ( l H , br, 8’-CH), 6.04 ( l H , br, 5’-CH=). (4) 3’a,4’t~-Epoxypravastatinsodium To a solution of 42.2 mg of 3’a,4’a-epoxypravastatin lactone in 0.5 ml of acetonitrile was added 0.1 M NaOH solution under cooling by ice. After stirring at room temperature for 1 h, the whole mixture was purified by column chromatography (CHP-20P (MCI Gel, 75-1 50pm, cu. 20m1, Mitsubishi Chemical Industries, Tokyo, Japan), stepwisegradient with water(100 ml), water-acetone (95 : 5 v/v, SOml), wateracetone (90: 1Ov/v, 50 ml), water-acetone (85 : 15 v/v, 50ml), and water-acetone (80: 20v/v, SOml), successively) to afford 32 mg of the desired product. Retention time by h.p.1.c.: 6-23min (column: Cosmosil 5C18, 6 m m x 150mm. eluent: 50mM potassium phosphate buffer (pH 7,4)-acetonitrile (8 : 2 v/v), flow rate l.Oml/rnin, detection U.V. 210nm).
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'H-n.m.r. (D,O, 6ppm): 086 (3H, t, J=7.5Hz, 4"-CH3), 0.94 (3H, d, J = 6 6 H z , 2'-CH,), 1.10 (3H, d, J=7.0Hz, 2"-CH), 1.27-1.69(10H,m), 2.3&2.50(6H,m), 3.50(1H, tbr,J=3-4Hz, 3'-CH), 3.65(1H,d, J=4.0Hz, 4'-CH), 3.71 (lH, m, 5-CH), 4.08 (lH, tt, J=60, 7.3 Hz, 3-CH), 440 (lH, ddt, J = 105, 6.6, 3.3 Hz, 6'-CH), 5.31 (lH, m, 8'-CH), 6.08 ( l H , br, 5'-CH).
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3. Preparation of the glutathione conjugate (CM-1) with rat liver cytosol To 45 ml of rat liver cytosol, 20mg of the synthetic epoxide (1) was added and incubated at 37°C for 30min as described previously. The reaction was stopped by addition of 2201-1-11 of methanol, and the reaction mixture filtered through celite (Hyflo Super-Cel, John-Manville/Iwai Kagaku Co., Tokyo, Japan). The filtrate was concentrated to about 20ml under reduced pressure, adjusted to p H 4 with 2 M-HCI and purified by column chromatography on porous polymer CHP-2OP (ca. 20 ml) by gradient elution with water (250ml), water-acetone (95 : 5 v/v, 200ml), water acetone (90: lOv/v, 200ml), and water-acetone (80:20v/v, 200 ml), successively. The glutathione conjugate was obtained from the 5% and 10% aqueous acetone fractions after concentration under reduced pressure; 7.7 mg of purified conjugate (CM-1) was obtained from 20mg of epoxide (1). Structure of the conjugate was shown to be pravastatin 4'aa glutathione conjugate by n.m.r. and mass spectrometry, as described elsewhere (Nakamura et al. 1991). Identification of glutathione conjugate by specific enzyme reaction The isolated cell metabolite, the glutathione conjugate, CM-1, was incubated with y-glutamyl transpeptidase (y-GTP, 20 units, Sigma, USA) in 2ml of 5 0 m Tris ~ buffer (pH 7-4) containing 5 0 m ~ glycylglycine and 1OmM MgCI, at 37°C for 1h. A part of the reaction mixture was further incubated after addition of 10 units of dehydropeptidase-I (DHP-I, Hirota et al. 1985) at 37°C for 30min. The enzymically cleaved products with p G T P alone and p G T P followed with DHP-I were charged onto Bond-Elut C,, cartridges and each product was eluted with water-methanol (90: lOv/v) and (85 : 15 v/v), respectively, after washing with water. A part of the hydrolysis product with DHP-I was acetylated by addition of a drop of acetic anhydrideacetone (30 : 70v/v) in an ice bath. The enzymic cleavage process and acetylated product thus obtained were separated by t.1.c. (conditions, see analytical procedures, t.1.c. (2)). After development, the t.1.c. plate was contacted with X-ray film SB (Kodak Co., USA) in the dark. Identification of the glutathione conjugate by mass spectrometry The structure of the glutathione conjugate, CM-1, was determined by secondary ion mass spectrometry (M-80A, Hitachi, Tokyo, Japan). Glycerol was used as a matrix.
T.1.c. of the metabolites T h e hepatocyte incubation mixture was centrifuged, and the supernatant was freeze-dried, dissolved in 0.41~11of water-methanol (20 : 80 v/v) and 10 jd aliquots of the sample solution were analysed by t.1.c. as follows: (1) silica gel 60 F254 (250p-1, E. Merck, Germany) in chloroform-methanol-acetic acid (9 : 1 : 1 by vol.), (2) silica Gel 60 F254 in n-butanol-acetic acid-water (4 : 1 : 1 by vol.). After development, the concentration of the individual metabolites were determined by Bio-Image Analyzer BA-100 (Fuji Photo Film Co. Ltd, Tokyo, Japan).
Results Identification of major polar metabolites by specific enzyme reaction and mass spectrometry The major polar metabolite CM-1 was analysed by secondary ion mass spectrometry after separation and purification by two procedures of h.p.1.c. From the mass spectra of CM-1 shown in figure 1, CM-1 had intense ions at m/z748 (M H)+ and m / z 770 (M Na)+ in the positive spectrum and at m / z 746 (M-H)and m / z 768 (M-2H Na)- in the negative spectrum, indicating that the molecular weight of CM-1 was 747. T h e fact that the molecular weight of the metabolite was 323 higher than that of the parent compound (mol. wt, 424) indicated the formation of a glutathione conjugate. T h e metabolite CM-1 was then subjected to enzyme cleavage reaction with y-GTP followed by DHP-I, and the reaction product was analysed by secondary ion mass spectrometry after monitoring the reaction products by t.1.c. and h.p.1.c. and peak fractionation. y-GTP is an enzyme which hydrolyses glutathione, liberating glutamic acid. DHP-I hydrolyses the cysteinylglycine moiety acting on the hydrolysate of y-GTP. T h e hydrolysis product with y-GTP showed an intense peak at m/z 619 in the positive spectrum, which indicated the molecular
+
+
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7 4 6 [ M-H 1-
I
[M-2H+Na 1 -
.. .., .. ... .. 200
300
400
1
. ,. ... ... .. , . . .. . . .. 500
600
' I '
700
800
Figure 1. Mass spectra of pravastatin metabolite (CM-I), the glutathione conjugate. The positive secondary ion mass spectrum (upper) and the negative secondary ion mass spectrum (lower). The M-80 double-focusing mass spectrometer (Hitachi) with an M-300 data manipulator was used. Measurement used a first acceleration voltage for positive of -8kV, and negative of - 9 kV; and a second acceleration voltage of 3 kV; the first filament electric current was 35A, the first collision gas was X'(2 x lo-* torr), and the matrix was glycerol and thioglycerol.
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weight of the product was 618. T h e decreased number of 129 mass unit agreed with the molecular weight of glutamic acid. Similarly, the hydrolysate with DHP-I had an m/z of 561 in the positive mode, and the decreased number of 58 mass unit corresponded to the liberation of the glycine moiety. This hydrolysate gave a product of molecular weight of 602 after acetylation with acetic anhydride. T h e enzymically cleaved products of CM-1 noted above were also anaiysed by t.1.c. As shown in figure 2, on treatment with y-GTP and DHP-I (dipeptidase), CM1 gave new products with different R, values, and after acetylation a further product was formed, which provide evidence for CM-1 being a glutathione conjugate and the absolute structure was assigned by n.m.r. spectroscopy. Concerning CM-2, since mass spectrometric analysis indicated a dihydrodiol form of the parent compound, structural determination was carried out using n.m.r. spectrometry, as for CM-1. T h e details on structural assignments are reported elsewhere (Nakamura et al. 1991), and the absolute structures of CM-1 and CM-2 are shown in figure 3.
Formation of the glutathione conjugate (CM-1)and the dihydrodiol (CM-2) Since the formation of the glutathione conjugate CM-1 was thought to be derived from an epoxide, chemical synthesis of the epoxides on the decalin ring was undertaken. After making the lactone at the carboxy moiety, selective oxidation of the double bonds on the decalin ring was effected with m-chloroperbenzoic acid, with or without modification at the hydroxy group considering steric hindrance. As a result, two epoxides were synthesized. Figure 4 shows the structure of these two expoxides, the 4'aP, S'P-epoxide and the 3'a, 4'a-epoxide. Each epoxide thus obtained was incubated at 37°C for 15 min in rat liver cytosol, since hopefully one of these epoxides would be the intermediate substrate to form the
Figure
2.
Enzyme hydrolysis and acetylation of the isolated glutathione conjugate (CM-1).
CM-1 was treated with y-GTP and dipeptidase successively, and the resulting product was acetylated. Each reaction product A, B, C, D was separated by t.1.c.on silica gel F 254 (E. Merck) in n-butanol-acetic acid-water (4 : 1 : 1 by vol.) a6 a solvent. Spots were visualized by contacting with X-ray film.
Metabolism of pravastatin sodium in rat hepatocytes
HO HO I G
Figure 3.
"OH
OH
SG: Glutathione
Glutathione conjugate of pravastatin (CM-1 )
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\ .
HO
493
Dihydrodiol metabolite of pravastatin ( CM-2 )
Structures of the pravastatin metabolites CM-1 and CM-2.
4ap, Sp-epoxide
3a,@a-epoxide
NaOOCyoH
Figure 4.
H.p.1.c. profiles of the products obtained on incubation of pravastatin epoxides with rat hepatic cytosol.
H.p.1.c. was conducted in a Hitachi model 655 liquid chromatograph with a U.V.detector and a D-2000 integrator system. The column used was YMC-Pack ODS A-312 ( 6 m m x 1 5 0 m m , Yamamura Kagaku Co.) and was monitored by U.V. detector at 210 n m with water-acetonitrile-Pic A (ion-pairing reagent, low u.v., Waters) (83 : 17 : 0 3 , by vol.) as mobile phase with a flow rate of 1ml/min.
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z
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E
'-'T
0.0
0.5
1.0
1.5
Concn. of 4ap, S'pepoxide (epoxide(1)) ( rnM ) Figure 5. Formation of CM-1 and CM-2 from the epoxide (1) in rat hepatic cytosol. The epoxide (1) was incubated in liver cytosol at 37°C for 15min and the CM-1 and CM-2 produced were determined by h.p.1.c. whose conditions are shown in figure 4: A and A represent CM-1 formed in cytosol and dialysed cytosol, respectively, and e, 0 and 0 represent those of CM-2 in cytosol, dialysed cytosol and in medium buffer (without cytosol), respectively. Each point represents the mean of triplicate determinations; SD at each point was less than 10%.
glutathione conjugate (CM-1) with the possible help of glutathione-S-transferase. T h e reaction were followed by h.p.1.c and the resulting typical chromatograms (see figure 4) show that the 4'ap, 5'p-epoxide produced CM-1. T h e diol CM-2 was also produced from the same 4'aB, S'B-epoxide. These findings strongly indicate that pravastatin sodium was metabolized to form CM-1 and CM-2 exclusively via the 4'a/?, S'B-epoxide in rat. T h e rate of formation of CM-1 and CM-2 from the 4'ab, 5'p-epoxide in liver cytosol was investigated by incubation at 37°C for 15 min under various conditions, and the results are shown in figure 5. T h e rate of formation of CM-1 was nearly saturated with increase of substrate concentration, while that of CM-2, the dihydrodiol, was increased linearly with increase of substrate concentration. For confirmation of this, pravastatin sodium was incubated in liver cytosol after dialysis to remove glutathione. T h e dialysed cytosol no longer produced the conjugate although the dihydrodiol was still produced; the glutathione conjugating ability of the dialysed cytosol was restored by addition of glutathione to the reaction medium. T h e 4'ap, S'p-epoxide was relatively unstable and its rate of decomposition to the dihydrodiol in the buffer used in the incubation was almost the same as in the cytosol. This leads to the conclusion that glutathione conjugation (CM-1) was an enzymic process and that the dihydrodiol is formed by non-enzymic hydrolysis of the +a@,S'b-epoxide, accompanied by the intramolecular migration of the double bond in the decalin moiety. The reaction product (CM-1) obtained in the cytosol system was shown to be of identical structure and configuration with the conjugate obtained from isolated hepatocytes.
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0
10 15
30
60
90
Time of incubation ( min.) Figure 6. Elimination of pravastatin sodium (upper) and formation of its metabolites (lower) in isolated rat hepatocytes. Data are means of three animals, L- SEM indicated by bars. Pravastatin sodium; 0 ,CM-1; 0, R-416 (see Arai et 01. 1988); A , Non-polar metabolites; W , CM-2; 0,Polar metabolites; A , CM-3.
+,
Metabolic profile of pravastatin sodium in the isolated rat hepatocytes T h e metabolic profile of pravastatin sodium in isolated rat hepatocytes is depicted in figure 6, which shows the time-course of formation and elimination of the metabolites and the parent compound, quantified by t.1.c. using an imaging analyser. T h e two t.1.c. methods were designed for separation of relatively polar and non-polar metabolites as described in 'T.1.c. of the metabolites'. The amount of pravastatin remaining in the incubation medium was 79% at 30 min, 75% at 60 min and 66% at 90min, respectively, when 0.1 mM substrate was incubated. With the decrease of pravastatin sodium, four major metabolites were produced in the course of incubation. Among these metabolites, the isomer of pravastatin sodium, was identified based on its R, value in t.1.c. and the retention time in h.p.1.c. T h e other three metabolites, CM-1, CM-2 and CM-3, were isolated by t.1.c. followed by h.p.1.c. Metabolite CM-3, as well as other minor ones, noted as polar and nonpolar metabolites in figure 6, will be reported in a subsequent publication. Metabolism of pravastatin sodium in P-450-induced and -inhibited hepatocytes Since the structures of the main metabolites of pravastatin sodium in the isolated rat hepatocytes system were shown to be the glutathione conjugate (CM-I) and the dihydrodiol (CM-2), the effects of induction and inhibition of cytochrome P-450 on the formation of these two metabolites was examined in the cell system. Induction of P-450 was carried out by pretreatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) prior to the preparation of the cells, and P-450 activity was inhibited by the addition of SKF-525A to the incubation medium. As shown in figure 7, PB caused a marked increase in metabolite formation while M C
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0'
0
15
30
m
90
Tlrne of incubation ( rnln. )
.,
Figure 7. Effect of phenobarbital, 3-methylchoranthrene and SKF-525A on (upper) elimination of pravastatin sodium, and (middle) formation of CM-1, and (lower) CM-2 in isolated rat hepatocytes. induced with MC; Substrate: 0 1 mM: 0 , Normal hepatocytes; A , induced with PB; 0, inhibited with SKF-525A.
showed a i.!oderate increase. T h e PB-treated cell system produced more than three times the glutathione conjugate and twice the dihydrodiol compared to that formed by normal hepatocytes, whereas MC-induced cells produced about 1.5 times the amount of glutathione conjugate and twice the amount of dihydrodiol produced by non-induced hepatocytes after incubation for 60 min. In contrast, addition of SKF525A completely inhibited the formation of the glutathione conjugate and the dihydrodiol, as well as the elimination of the substrate pravastatin. These findings strongly indicate an association of the hepatic monooxygenase system in the metabolism of pravastatin sodium.
Discussion T h e in vitto metabolic studies of pravastatin sodium in isolated rat hepatocytes were carried out to elucidate the structures of the two major polar metabolites and their mechanism of formation. The major metabolite (CM-1) produced in hepatocytes was isolated and shown to be a glutathione conjugate, both by enzymic hydrolysis and mass spectrometric and n.m.r. spectroscopic analysis. This glutathione conjugate could also be prepared by incubating the 4'aB, 5'B-epoxide, obtained synthetically, in rat liver cytosol. The structure of the other major metabolite, the dihydrodiol of the parent drug, was established by n.m.r. spectroscopy. One of the possible routes for glutathione conjugate formation is via an epoxide (Hammarstrom et al. 1979). From the structural consideration of the metabolites, as
- -
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Metabolism of pravastatin sodium in rat hepatocytes /
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-
HO
Epoxide (1)
Pravastatin sodium
aOH - -
HO
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i
Epoxide (3)
-
OH
Dihydrodiol( CM-2 )
:
Epoxide (2)
GS
SG
dH
SG: Glutathione
Figure 8. Possible formation of CM-1 and CM-2 from pravastatin epoxides.
well as the results of the enzyme induction and inhibition studies, it seemed probable that the biotransformation of pravastatin proceeds through an epoxide. Figure 8 illustrates the four kinds of possible epoxides on the decalin moiety and the presumed formation routes for the glutathione conjugate (CM-1) and the dihydrodiol (CM-2). In this scheme the epoxides which are likely to produce CM-2 are probably epoxide (1) and epoxide (2), as it seemed impossible to form a dihydrodiol from the other two epoxides on the basis of positional and the stereochemical consideration of the hydroxy groups on the decalin ring. According to this hypothesis, four possible epoxides were synthesized, and two of them, the
Glutathione-
H
O
W
no
H
-
'y
COOH
CONH -COOH
Glutathione conjugate (W-1)
HO
cap, Sp-epoxide
\\ Non-enzymic
-
Pravastatin sodium OH HO
I
'
""OH
OH
Dihydrodiol( CM-2)
Figure 9.
Probable mechanisms of formation of CM-1 and CM-2.
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Metabolism of pravastatin sodium in rat hepatocytes
epoxide (1) and epoxide (2) obtained were incubated in the rat liver cytosol which contained glutathione-S-transferase. Epoxide (4)was isolated as a minor by-product in the fermentation product of pravastatin sodium and the structure wiH be reported elsewhere (Hamano, K., Kinoshita, M. and Hosobuchi, M., Sankyo in-house report, unpublished); epoxide (4) did not produce CM-1 and CM-2 by the incubation with rat liver cytosol. As a result, it was only 4'aP, 5'P-epoxide (epoxide (1)) that could produce the glutathione conjugate identified as CM-I. Concerning the dihydrodiol, the hydroxy group was not introduced into the same position as the glutathione moiety in the conjugate when the epoxide was opened. T h e reason for this might be postulated as a nucleophilic attack of the hydroxy ion at the 3'-C position instead of the 4'a-C, due to protonation on the oxygen of the epoxide followed by concerted migration of the double bond. The mechanism of formation of the glutathione conjugate and the dihydrodiol is summarized in figure 9. In general, glutathione conjugates are excreted as mercapturates in the urine after being acetylated in the kidney, and the further metabolism of this glutathione conjugate both in vitro and in vivo needs to be studied.
References ARAI,M., SERIZAWA, N., TERAHARA, A., TSUJITA, Y.,TANAKA, M., MASUDA, H. and ISHIKAWA, S., 1988, Pravastatin sodium (CS-514), a novel cholesterol-lowering agent which inhibits HMG-CoA reductase. Annual Reports of Sankyo Research Laboratories, 40,1-38. S., MURPHY, R.C., SAMUELSSON, B., CLARK, D. A,, MIOSKOWSKI, C., and COREY, E. J., HAMMARSTROM, 1979, Structure of leucotriene C, identification of the amino acid part, Biochemical and Biophysical Research Communications, 91, 1266-1272. HARUYAMA, H., KUWANO, H., KINOSHITA, T., TERAHARA, A., NISHIGAKI, T., and TAMURA, C., 1986, Structure elucidation of the bioactive metabolites of ML-236B (mevastatin) isolated from dog urine. Chemical and Pharmaceutical Bulletin, 34, 1459-1467. HIROTA, T., NISHIKAWA, Y., TAKAHAGI, H., IGARASHI, T., and KITAGAWA, H., 1985, Simultaneous purification and properties of dehydropeptidase-I and aminopeptidase-M from rat kidney. Research Communications in Chemical Pathology and Pharmacology, 49, 435-445. KOMAI, T., h A W A I , K., SHIGEHAR, A. E., KUROIWA, C., KAWASAKI, Y., KITAZAWA, E., and TANAKA, M., 1988, Metabolic study of pravastatin sodium(I), Metabolism and disposition of 14C-pravastatin in experimental animals, Second Znternational ZSSX Meeting, 16-20 May, Kobe, Japan. I., and ORRENIUS, S., 1978, Isolation and use of liver cells. Methods in MOLDBUS,P. HOEGBERG, Enzymology, 52, 6&71. H., MIYAGUCHI, K., MURAMATSU, S., TAKAHAGI, H., and NAKAMURA, T., YODA, K., KUWANO, KINOSHITA, T., 1991, Metabolism of pravastatin sodium in isolated rat hepatocytes. 11. Structure elucidation of the metabolites by n.m.r. spectroscopy. Xenobiotica, 21, 277-293. TSUJITA, Y., KURODA, M., SHIMADA, Y., TANZAWA, K., ARAI,M., KANEKO, I., TANAKA, M., MASUDA, H., TARUMI, C., WATANABE, Y.and FUJII,S., 1986, CS-514, a competitive inhibitor of 3-hydroxy-3methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species. Biochimica et Biophysica Acta, 877, 5 M O .
