Inhibitory effects of fluvastain and its metabolites on the formation of several reactive oxygen species

Life Sciences 69 (2001) 1381–1389

Inhibitory effects of fluvastain and its metabolites on the formation of several reactive oxygen species Akinori Nakashimaa,*, Masakatsu Ohtawaa, Kazuhide Iwasakia, Mitsuhiro Wadab, Naotaka Kurodab, Kenichiro Nakashimac a

b

Tsukuba Research Institute, Novartis Pharma K.K., Ohkubo 8, Tsukuba-shi, Ibaraki 300-2611, Japan School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki, 852-8521, Japan c Graduate School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki, 852-8521, Japan Received 12 September 2000; accepted 14 February 2001

Abstract We investigated the inhibitory effects of fluvastain (FV) and its metabolites (M-2, M-3, M-4, M-5, and M-7) on the formation of several reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide anion (O22), hydroxy radical (.OH), hypochlorite ion (OCL2, and linoleic acid peroxide (LOO.). Inhibitory effects of pravastatin (PV), simvastatin (SV), probucol (PR) and a-tocopherol (TOC) were also tested. The inhibitory effects of 5-hydroxy FV (M-2) and 6-hydroxy FV (M-3) on the formation of 1O2, O22, .OH, and OCL2 were strongest. Scavenging of 1O2 by M-4, M-5, (1)-FV, and (2)-FV was also noted. The inhibitory effects of (1)-FV on the formation of 1O2 were comparable to those of (2)-FV. PV, SV, PR and M-7 had little or no inhibitory effect on the formation of several ROS. In conclusion, FV and its metabolites, particulary M-2 and M-3, have the potential to protect against oxidative stress mediated by several ROS. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Oxidative stress; Antioxidant; HMG-CoA reductase inhibitor; Fluvastatin; Metabolite; Reactive oxygen species

Introduction Oxidation of low-density lipoprotein (LDL) is considered an important step in the development of atherosclerosis (1). Antioxidant drugs such as probucol have been reported to inhibit the progression of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits (2). There are relatively few reports directly demonstrating the effects of antioxidants on progression of atherosclerosis in human, because of the difficulty of evaluation of antioxidant * Corresponding author. Tel.: 181-298-65-2383; fax: 1-298-65-2383 E-mail address: [email protected] (A. Nakashima) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 2 3 -1

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effect of drug (3). However, oxidative modification of LDL is thought to play a very important role in the development of atherosclerosis in humans, and lowering plasma LDL level is important in preventing progression of atherosclerosis (4). In this respect, oxidative stress is considered a major risk factor for atherosclerosis. Hypercholesterolemia is also major risk factor for atherosclerosis; in hypercholesterolemic patients, reduced LDL receptor activity in the liver contributes to increased plasma LDL concentration. The plasma LDL in these patients is more “aged” and thus more susceptible to oxidative modifications than LDL derived from healthy individuals (5). Fluvastatin (FV) is the first totally synthesized HMG-CoA reductase inhibitor and has clinical anti-hypercholesterolemic effects (6). Further, the antiatherogenic properties of FV may not be limited to its hypocholesterolemic effect, but may also be related to its ability to reduce LDL oxidizability (7, 8). Antioxidant effects of FV in vivo have also been reported in humans (7–9) and animals (10,11). After oral 3H-FV administration to healthy volunteers, FV and its metabolites, 5-hydroxy FV (M-2), 6-hydroxy FV (M-3), and des-isopropylpropionic acid derivative of FV (M-4), were detected in human plasma (12). In vitro data indicated that FV was metabolized by human liver microsomes to M-2, M-3, and des-isopropyl-FV (M-5) (13). In order to estimate antioxidant capacity after oral administration of FV, it is important to characterize the antioxidant effects of FV and its metabolites. We previously reported antioxidant effects of FV and its metabolites on NADPH-induced lipid peroxidation using rat liver microsomes (14). The antioxidant effects of FV and its metabolites varied depending on microsomal concentration (0.025–0.2 mg/ml, final concentration). These results suggest that each compound has different inhibitory effects on the formation of several reactive oxygen species (ROS). We therefore investigated the inhibitory effects of FV ((1)-FV, (2)-FV) and its metabolites (M-2, M-3, M-4, M-5, and M-7) on the formation of several ROS, such as singlet oxygen (1O2), superoxide anion (O22), hydroxy radical (.OH), hypochlorite ion (OCL2), and linoleic acid peroxide (LOO.). Materials and methods Materials (1)-FV, (2)-FV, and M-5 were supplied by Novartis Pharmaceuticals Corporation (East Hanover, NJ). M-2 and M-3 were donated by Tanabe Seiyaku Corporation (Japan). M-4 and M-7 were synthesized at Daiichi Pure Chemicals (Japan). The chemical structures of FV and its metabolites are shown in Fig.1. Pravastatin (PV) was obtained from Sankyo Co., LTD (Japan). Simvastatin (SV) was obtained from Banyu Pharmaceutical Corporation., LTD (Japan). Probucol (PR), a-tocopherol (TOC), luminol, xanthine oxidase (XOD), lactoperoxidase (LPO), hypoxanthine (HX), N-2-hydroxyethylpiperazine-N9-2ethanesulphonic acid (HEPES), diethylentriaminepentaacetic acid (DETAPAC) and linolenic acid were purchased from Sigma Chemical Corporation (St Louis, MO). 2-Methyl6- (p-methoxyphenyl)-3,7-dihydroimidazo[1, 2-a]pyrazin-3-one (MCLA) was purchased from Tokyo Kasei (Japan). All other chemicals were of the highest grade commercially available.

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Fig. 1. Chemical structures of fluvastatin and its five metabolites.

Reactive oxygen species assay The inhibitory effect of each compound on the formation of several ROS was investigated in the following reaction system (15,16). Each compound tested was dissolved in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) at concentrations of 1.0, 2.0, and 4.0 mM (the final concentration of each compound was set at 5, 10, and 20 mM, respectively). For assay of several ROS, 100 mM acetate buffer, pH4.5 (buffer A), 100 mM HEPES buffer, pH7.4 (buffer B), and 50 mM borate buffer,pH9.5 (buffer C) were prepared. Assays for singlet oxygen A mixture composed of 6mL test sample solution and 300 mL of 0.4% (w/v) H2O -buffer A was added to 300 mL of 80 mM NaBr-buffer A and 300 mL of 0.8 mM-buffer A suspension

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to start the reaction. After preincubation for 10 min at 37 °C, 300mL of 10mg/mL LPO-buffer A was added. Assays for superoxide anion A mixture composed of 6mL test sample solution and 600 mL of 0.8 mM HX-buffer B was added to 300 mL of 1.6 mM Luminol-buffer B to start the reaction. After preincubation for 10 min at 37 °C, 300 mL of 1.0 unit/mL XOD-buffer B was added. Assays for hydroxy radical (.OH) A mixture composed of 6 mL of test sample solution and 600 mL of 0.8 mM Luminolbuffer B was added to 8 mM DETAPAC- buffer B and 150 mL of 1.6 % H2O2 (w/v)-buffer B to start the reaction. After preincubation for 10 min at 37 °C, 300 mL of 200 mM FeCl2-buffer B was added. Assays for Hypochlorite ion A mixture composed of 6 mL test sample solution was added to 900 mL of 0.53 mM Luminol-buffer C to start the reaction. After preincubation for 10 min at 37 °C, 300 mL of 40 mM of sodium hypochloride-buffer C solution was added. Assays for linoleic acid peroxide A mixture composed of 6 mL test sample solution was added to 900 mL of 5.0 mM linoleic acid-buthanol solution (this solution was incubated for 2 hr at 37 °C before assay) to start the reaction. After preincubation for 3 min at 37 °C, 300mL of 4mM MCLA-buthanol solution was added. Chemiluminescence measurements Chemiluminescence measurements were performed for 1 min at room temperature, approximately 25 °C (LUMATAG ANALYZER AUTO-250, Berthold, UK). Inhibitory effects on the formation of several ROS are indicated as percentages in relative luminescence units (% RLU) against the value for vehicle control (i.e., DMSO or DMF). Statistical analysis The data are expressed as the mean 6 S.E.M.Statistical analysis of data was performed using Tukey’s multiple comparison test. Statistical significance was accepted at P,0.05. Results Inhibitory effects of each compound on the formation of singlet oxygen (Table1) M-2 had the strongest 1O2 scavenging activity (IC50: about 5 mM). Inhibitory effects of M-2 were significantly different compared with those for (1)-FV(P,0.001, at 5, 10 and 20 mM). The scavenging activities of M-3 and M-4 for 1O2 were comparable to that of M-2. The inhibitory effect of M-5 was less potent than those of M-2, M-3 and M-4. The % RLU values

TOC

97.3 6 0.1 88.3 6 0.9 99.0 6 0.2 83.8 6 1.2 97.7 6 1.0 76.6 6 1.1

91.2 6 0.9 68.9 6 1.9 87.2 6 2.4 57.9 6 2.9 86.7 6 2.2 48.8 6 0.8

98.1 6 0.4 97.9 6 0.5 97.8 6 0.3 98.3 6 0.4 97.2 6 0.4 98.3 6 0.5

Note. All data presented mean 6 SEM (n53). Each compound was dissolved in DMSO or DMF. The final concentration of each compound was set at 5, 10, and 20 m M, respectively. Inhibitory effects on the formation of singlet oxygen, superoxide anion, hydroxy radical, hydroclorite ion, and linoleic acid peroxide were indicated percentages of relative luminescence units (% RLU) against the value for vehicle control.

84.7 6 5.1 96.4 6 5.9 98.2 6 4.7 122.7 6 5.4 108.0 6 4.2 114.2 6 3.7 81.3 6 5.7 90.2 6 3.4 103.2 6 2.3 98.7 6 3.7 120.4 6 5.4 103.7 6 5.2 116.5 6 3.2 64.9 6 4.8 98.3 6 1.4 104.9 6 5.4 122.6 6 13.9 121.4 6 5.9 123.3 6 8.1 115.3 6 4.6 40.0 6 2.0

110.6 6 0.6 108.4 6 1.3 111.9 6 1.6 110.6 6 1.9 110.9 6 1.4 109.0 6 1.6

99.7 6 0.5 98.4 6 0.6 99.3 6 0.0

Hydroclorite ion 5 mM 106.3 6 1.1 104.4 6 0.8 41.0 6 1.5 34.1 6 2.0 101.5 6 0.7 103.3 6 1.5 102.7 6 1.4 10 mM 104.8 6 1.1 103.7 6 0.6 26.6 6 0.9 13.1 6 0.7 99.9 6 0.9 103.4 6 1.2 94.7 6 0.6 20 mM 105.1 6 0.7 103.9 6 0.8 21.6 6 0.7 12.4 6 0.3 98.5 6 1.6 102.2 6 1.3 84.7 6 0.8

Linoleic acid peroxide 5 mM 119.4 6 3.9 112.7 6 7.7 98.5 6 1.7 80.3 6 3.3 10 mM 131.2 6 2.1 101.7 6 3.9 86.3 6 2.6 87.3 6 0.3 20 mM 116.8 6 3.3 102.9 6 2.8 78.9 6 1.5 58.5 6 2.5

PR

94.7 6 1.4 102.2 6 0.2 98.4 6 0.7 87.5 6 0.7 102.3 6 0.7 94.3 6 0.7 67.7 6 0.3 101.9 6 0.8 88.8 6 1.5

SV

96.7 6 2.6 102.5 6 1.3 101.6 6 0.3 99.4 6 1.9 101.8 6 1.2 95.9 6 2.0

92.3 6 1.1 92.1 6 1.4 90.6 6 0.8

99.3 6 0.3 100.0 6 0.2 100.4 6 0.2

95.0 6 1.1 90.8 6 1.3 88.3 6 1.1

PV

88.3 6 1.4 86.4 6 0.7 87.5 6 1.3

72.7 6 3.2 62.0 6 3.8 46.7 6 3.9

99.1 6 1.0 98.6 6 1.3 93.5 6 0.6

M-7

95.6 6 1.2 63.6 6 1.7 38.8 6 4.3 95.1 6 1.0 46.7 6 2.6 25.6 6 3.6 91.0 6 1.0 30.1 6 2.5 16.5 6 2.9

75.3 6 0.7 46.9 6 0.2 18.5 6 0.3

M-5

Hydroxy radical 5 mM 105.9 6 1.8 10 mM 94.4 6 2.2 20 mM 94.0 6 1.6

56.5 6 1.1 22.3 6 0.8 5.4 6 0.1

M-4

98.1 6 0.2 46.2 6 0.0 35.0 6 0.2 101.5 6 0.2 100.7 6 0.4 99.6 6 0.3 98.8 6 0.3 32.0 6 0.1 21.6 6 0.2 100.2 6 0.4 101.6 6 0.2 100.0 6 0.2 98.4 6 0.4 19.9 6 0.1 12.0 6 0.2 101.9 6 0.2 100.7 6 0.4 99.1 6 0.4

M-3

Superoxide anion 5 mM 98.2 6 0.3 10 mM 97.4 6 0.6 20 mM 98.8 6 0.6

M-2

82.2 6 0.3 46.3 6 1.3 63.7 6 1.9 59.1 6 0.4 16.7 6 0.5 24.1 6 0.9 26.9 6 0.7 3.4 6 0.1 5.2 6 0.1

(2)-FV

Singlet Oxygen 5 mM 80.5 6 0.3 10 mM 61.2 6 0.2 20 mM 26.8 6 0.6

(1)-FV

Table 1 Inhibitory effects of each compound on the formation of several reactive oxygen species

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Fig. 2. Inhibitory effects of FV and its metabolites on the formation of singlet oxigen. All data were meaned from the results of three different experiments.

of (1)-FV at 5, 10 and 20 mM were 80 %, 61% and 27%, respectively and no statistical differece were detected compared with those for (2)-FV (Fig.2). The other compounds had little or no inhibitory effect on the formation of 1O2. Inhibitory effect of each compound on the formation of superoxide anion (Table1) M-3 had the highest O22 scavenging activity (IC50: under 5 mM). Inhibitory effects of M-3 were significantly different compared with those for (1)-FV(P,0.001, at 5, 10 and 20 mM). Scavenging activity on the formation O2- of M-3 was comparable to that of M-2. All compounds except M-2 and M-3 had little or no inhibitory effect on the formation of O22. Inhibitory effects of each compound on the formation of hydroxy radical (Table1) M-3 had the highest .OH scavenging activity (IC50: under 5 mM). The scavenging capacity of M-2 was the second strongest (IC50: about 10mM). Inhibitory effects of M-2 and M-3 were significantly different compared with those for (1)-FV(P,0.001, at 5, 10 and 20 mM). The inhibitory effect of M-4 on the formation of .OH was comparable to that of TOC (IC50: about 20mM). The other compounds had little or no inhibitory effect on the formation of .OH. Inhibitory effects of each compound on the formation of Hypochlorite ion (Table1) M-3 had the highest OCL2 scavenging activity (IC50: under 5 mM). Inhibitory effects of M-3 were significantly different compared with those for (1)-FV(P,0.001, at 5, 10 and 20 mM). The scavenging potency of M-2 was comparable to that of M-3. All compounds except M-2 and M-3 had little or no inhibitory effect on the formation of OCL2.

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Inhibitory effect of each compound on the formation of Linoleic acid peroxide (Table1) The scavenging activity of TOC for LOO. was highest ( IC50:10–20 mM). Inhibitory effects of TOC were significantly different compared with those for (1)-FV(P,0.001, at 10 and 20 mM).All compounds except TOC had little or no inhibitory effect on the formation of LOO.. Discussion We investigated the inhibitory effects of FV and its metabolites on the formation of several ROS. Our results clearly showed that FV and its metabolites, especially M2 and M-3, had potent scavenging activities. FV has two chiral carbons in its chemical structure and it is clinically used as a racemic mixture((1)-FV,(2)-FV). The inhibitory effects of (1)-FV on the formation of 1O2 were comparable to those of (2)-FV (and were not enantioselective), although the HMG-CoA reductase inhibitor activity of (1)-FV was 30-fold that of (2)-FV (17). The present results clearly demonstrated that two enantiomers had equal 1O2 scavenging ability. Similarly, a nonenantioselective antioxidant effect of FV on copper ion-induced LDL oxidation has also been reported (18). The antioxidant effect of FV on NADPH-induced lipid peroxidation in rat liver microsomes was due to .OH scavenging activity (19). Effects of scavenging by FV and its metabolites on the formation of .OH have also been reported, and the effect of FV on the formation of .OH was comparable to that of M-4 (20). In this study, no inhibitory effects of (1)-FV and (2)-FV on the formation of .OH were found, although an inhibitory effect of M4 was observed. These results suggested that the antioxidant property of FV is independent of its HMG-CoA inhibitory activity, and that (2)-FV may have an antiatherosclerotic effect in vivo medicated through its antioxidant effect. In in vitro studies using normal human aortic endothelial cells, saturable uptake of FV was not observed, and FV may be taken up by normal human aortic endothelial cells via nonspecific simple diffusion (21). These results suggested that FV prevented oxidative modification of LDL in the space between endothelial cells and matrix. The scavenging activities of M-2 and M-3 against the formation of 1O2, O22, .OH, and OCL2 were strongest among all compounds tested in this study. M-2 and M-3 each have a phenolic group in their indole moiety. Many indole groups including phenolic hydroxy groups have potent antioxidant effects, e.g., melatonin (22–24), indole-2carboxamide and cycloalkeno [1,2-b] indole derivatives (25),and indole alkaloids (26). Inhibitory effects of M-4 and M-5 on the formation of 1O2 were also found. M-4 had a .OH scavenging effect. Inhibition by TOC on the formation of LOO. was also found. On the other hand, M-7 (4, 5-pentenoic acid derivative of FV), PV, SV and PR had little or no inhibitory effect on the formation of several ROS. After oral 3H-FV administration to healthy volunteers, M-2 (24% dose), M-3 (24% dose), and M-5 (11% dose) were predominant in feces (12). FV was metabolized to M-2, M-3, and M-5 by human liver microsomes. In addition, previous animal studies showed that the highest level of radioactivity was found in the liver 2hr after administration of 14C-FV to rats, about 50 times that in whole blood (27). These results suggest that antioxidant effects of FV and its metabolites in liver have the potential to alter the characteristics of LDL. LDL oxidiz-

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ability is related to its particle size, and small LDL is more susceptible than large LDL to oxidative stress (5,7). FV and its metabolites appeared to prevent oxidative modification of VLDL, since small LDL is derived from damaged VLDL by ROS in liver. In conclusion, FV and its metabolites, particularly M2 and M-3, have the potential to protect against oxidative stress mediated by aqueous ROS such as 1O2, O22, .OH, and OCL2 and primarily inhibit the initiation of lipid peroxidation. On the other hand, the inhibitory effect of TOC on the formation of non-aqueous ROS such as LOO. was mainly due to inhibition of progression process of lipid peroxidation. Acknowledgements We are thankful to Mr.Naoki Masuda and Mr.Hiroshi Morikawa for their great support. References 1. Steinberg D, Parthasarathy S, Carew TE, Khoo JC and Wiztum JL. Beyond Cholesterol modification of lowdensity lipoprotein that increase its atherogenicity. The New England Journal of Medicine 1989;320:915– 924. 2. Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, Yoshida H. Probucol prevents the progression of atherosclerosis in Watanabe heritatable hyperlipidemic rabbit, an animal model for familial hyperchcholesterolemia. Proceeding of the National Academy of Sciences of the USA 1987;84:5928–5931. 3. Rice-Evance CA, Diplock AT. Current status of antioxidant therapy. Free Radical Biology & Medicine 1993;15:77–96. 4. Lavy A, Brook JG, Dankner G, Ben AA, Aviram M. Enhanced in vitro oxidation of plasma lipoproteins derived from hypercholesterolemic patients. Metabolism 1991;40:794–799. 5. Aviram M, Hussein O, Rosenblat M, Schleziger S, Hayek T, Keidar S. Interactions of platelets, Macrophages, and lipoproteins in hypercholesterolemia:antiatherogenic effects of HMG-CoA reductase inhibitor therapy. Journal of Cardiovascular Pharmacology 1998; 31:39–45. 6. Kathawala FG. HMG-CoA reductase inhibitors:an exciting development in treatment of hyperlipoproteinemia. Medicinal Research Reviews 1991;11:121–146. 7. Hussein O, Schleziger S, Rosenblat M, Keidar S, Aviram M. Reduced susceptibility of low density lipoprotein (LDL) to lipid peroxidation after fluvastatin therapy is associated with the hypocholesterolemic effect of the drug and its binding to the LDL. Atherosclerosis 1997;128:11–18. 8. Bellosta S, Bernini F, Ferri N, Quarato P, Canavesi M, Arnaboldi L, Fumagalli R, Paoletti R, Corsini A. Direct vascular effects of HMG-CoA reductase inhibitor. Atherosclerosis 1998;137:S101–S109. 9. Leonhardt W, Kurktschiev T, Meissner D, Lattke P, Abletshauser C, Weidinger G, Jaross W, Hanefeld M. Effects of fluvastatin therapy on lipids, antioxidants, oxidation of low density lipoproteins and trace metals. European Journal of Clinical Pharmacology 1997;53:65–69. 10. Bandoh T, Mitani H, Niihashi M, Kusumi Y, Ishikawa J, Kimura M, Totsuka T, Sakurai I, Hayashi S. Inhibitory effect of fluvastatin at doses insufficient to lower serum lipids on the catheter-induced thickening of intima in rabbit femoral artery. European Journal of Pharmacology 1996;315:37–42. 11. Mitani H, Bandoh T, Ishikawa J, Kimura M, Totsuka T, Hayashi S. Inhibitory effects of fluvastatin: a new HMG-CoA reductase inhibitor, on the increase in vascular ACE activivty in cholesterol-fed rabbits. British Journal of Pharmacology 1996;119:1269–1275. 12. Dain JG, Fu E, Gorski J, Nicoletti J, Scallen TJ. Biotransformation of fluvastatin sodium in humans. Drug Metabolism and Disposition 1993;21:567–572. 13. Fischer V, Johanson J, Heitz F, Tullman R, Graham E, Baldeck JP, Robinson WT. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor fluvastatin:effect on human cytochrome P-450 and implications for metabolic drug interaction. Drug Metabolism and Disposition 1999;27:410–421.

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