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Indian Journal of Pharmacology, Vol. 38, No. 3, May-June, 2006, pp. 205-206 Research Letter Antioxidant activity of ezetimibe in hypercholesterolemic rats Pandya N, Santani D, Jain S Department of Pharmacology, L.M. College of Pharmacy, Navrangpura, Ahmedabad Code Number: ph06051 Hypercholesterolemia is a significant risk factor for the development of coronary heart disease, which is a leading cause of mortality and morbidity in many countries. Results from several landmark clinical trials have shown that, 3-hydroxy-3methylglutaryl-coenzyme A reductase inhibitors (i.e., statins) effectively and significantly reduce elevated serum low-density lipoprotein cholesterol (LDL-C) concentrations, improve cardiovascular outcome and reduce the risk for coronary events. Despite the availability of statins and other lipid lowering drugs, a significant number of patients with hypercholesterolemia do not achieve adequate cholesterol reduction, having blood cholesterol concentrations higher than desired. Thus, treatment options with drugs possessing different mechanisms of action and improved safety profile, are needed. Ezetimibe, 1-(4-fluorophenyl)-3(R)-(3-(4-fluorophenyl)-3(S)-hydroxypropyl)-4(S)-(4-hydroxyphenyl)-2-azetidinone, is a selective cholesterol absorption inhibitor that effectively blocks intestinal absorption of cholesterol. Such drugs prevent the absorption of cholesterol, by inhibiting the passage of dietary and biliary cholesterol across the intestinal wall. These drugs represent a new class of pharmaceutical agents that can be used to treat patients with hypercholesterolemia. Ezetimibe alone or in combination with statins, effectively reduce LDL-C concentrations. It also increases high-density lipoprotein cholesterol (HDL-C) and may reduce elevated triglyceride (TG) concentrations.[1] The role of free radicals, is almost clear in many disorders. In atherosclerosis, oxidative stress generates free radicals, which has harmful effects on every organ. This free radical oxidizes LDL-C to convert the latter into oxidized LDL (oxLDL), which leads to the atherosclerosis. Ezetimibe has not been evaluated so far, for its effects on free radical scavenging activity. Therefore, the present investigation was undertaken to evaluate the effects of Ezetimibe on hypercholesterolemic rats, with special reference to antioxidant activity. Effect of Ezetimibe was observed on lipid profile, as well as lipid peroxidation, in rats receiving a high cholesterol diet. Vitamin E was used as standard for antioxidant activity. Sprague Dawley (SD) rats of either sex, maintained at a 12 h light/dark cycle, were used for the study. Animals were housed under standard laboratory conditions, with free access to food (commercial rat cubes from Pranav Agro Industries Ltd, Baroda, India, consisting of 23% crude protein, 4.3% crude oil, 3.1% crude fiber, 7.1% ash, 1.22% sand silica) and water, ad libitum . Hyperlipidemia was induced by feeding a high cholesterol diet (Regular diet mixed with 2 % w/w cholesterol and 1% w/w sodium cholate and 2.5 % w/w coconut oil) to healthy rats for five days. Rats were divided into four groups containing six animals each; Group1 received normal diet (normal); group 2 received high cholesterol diet (control); group 3 received ezetimibe 3 mg/kg, p.o. and group 4 received Vitamin E 60 mg/kg, p.o. for 6 days. At the end of the 5th day, food was withdrawn and on the 7th day, fasting blood samples were collected by retro-orbital puncture technique in a coagulant free vessel and were kept at room temperature for 1 h. Samples were centrifuged at 4000-5000 rpm to separate serum, that was subjected for the estimation of lipid profile [2] viz., total cholesterol (TC), TG, LDL-C, HDL-C, atherogenic index (AI).
Antioxidant enzymes namely malondialdehyde (MDA),[3] superoxide dismutase (SOD),[3] catalase,[3] and reduced glutathione (GSH),[3] were studied in liver homogenate. One g of liver tissue was homogenized with 10 ml Tris-hydrochloride buffer, the homogenate was centrifuged and the supernatant was collected and used for the estimation of antioxidant enzymes. All data are presented as mean±SEM. To investigate the relationship among the groups, one-way ANOVA followed by Tukey′s multiple range test, was performed using Sigmastat 2.03 software. Correlations were considered significant only when P was less than 0.05. High cholesterol diet caused a significant increase in serum TC, LDL-C, VLDL-C, TG and AI, with a significant decrease in the HDL-C and HDL/LDL ratio. [Table - 1] Ezetimibe pretreatment showed significant decrease in TC, LDL-C and AI, with insignificant increase in the HDL-C and HDL/LDL ratio, as compared to the control group, whereas the Vitamin E treated group showed a significant decrease in TG, VLDL-C and the atherogenic index, with no significant alteration in LDL-C, TC, HDL-C and the HDL/LDL ratio, as compared to control group. [Table - 1] Further, the antioxidant parameters measured in the liver homogenates were in terms of lipid peroxidation (MDA), superoxide dismutase (SOD) and catalase (CAT) and reduced glutathione (GSH). A high cholesterol diet produced insignificant increase in MDA, significant rise in SOD and insignificant decrease in CAT levels. [Table - 2] Ezetimibe treatment produced a significant decrease in SOD and increase in CAT activity, along with insignificant decrease in MDA and GSH levels. Vitamin E treatment showed significant decrease in MDA and SOD, but did not show any marked change in GSH and CAT activity. [Table - 2] Hypercholesterolemia is a significant risk factor for the development of coronary heart disease, which is a leading cause of mortality and morbidity in many countries. In the present study, ezetimibe at the dose of 3 mg/kg orally in rats, prevented hypercholesterolemia (70%), when compared with serum cholesterol levels in rats receiving high cholesterol diet. It has further shown reduction in VLDL-C as well as TG levels to the tune of 8%, albeit statistically not being significant. Van heek, et al (1997) have also reported attenuation of hypercholesterolemia in atherogenic rats. They attributed attenuation of hypercholesterolemia to LDL-C reduction. In addition, an indirect increase in cholesterol synthesis after multiple dosing of this class of cholesterol absorption inhibitors is quite likely, since an increase in hepatic HMG Co-A reductase activity has been reported after chronic treatment of ezetimibe.[4] Vitamin E at 60 mg/kg dose orally, showed reduction in serum cholesterol content in rats, that was mainly due to decrease in VLDL and LDL levels. It thus reduced the atherogenic index, which was 70%, as compared to respective control group. These findings are quite similar with the reports of Paul et al, .[5] Measurement of MDA gives an indirect evidence of LDL oxidation. The increase in SOD activity in the control group observed by us, may be due to adaptive mechanism to oxidative stress. It has been reported that oxidative stress increases SOD production.[6] Further, increase in MDA content might be due to increase in lipid peroxidation. The decrease in catalase can be due to overproduction of superoxide anion, which inactivates catalase by converting the resting ferric enzyme to the poorly active ferro-oxy form. In the present study, Vitamin E treatment showed significant decrease in both MDA and SOD levels. Ezetimibe treatment showed an insignificant decrease in MDA and a significant decrease in SOD level. This suggests that ezetimibe reduces oxidative stress, thereby prevents the generation of free radical and finally inhibits development of atherosclerosis. It can thus be concluded from this study, that ezetimibe reduces the cholesterol level with a significant increase in antioxidant activity, as evident from significant reduction in LDL-C parameter. Acknowledgment We are thankful to Alembic Pharma. Ltd. Vadodara, India, for providing gift sample of ezetimibe[7].References
Copyright 2006 - Indian Journal of Pharmacology The following images related to this document are available:Photo images[ph06051t1.jpg] [ph06051t2.jpg] |
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