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INTERNATIONAL JOURNAL FOR RESEARCH IN EMERGING SCIENCE AND TECHNOLOGY, VOLUME-2, ISSUE-4, APRIL-2015 E-ISSN: 2349-7610 Investigation into Beneficial Effect of Ketoconazole in Management of Complications Associated with Upasana Bhalani1 and Pravin Tirgar 2 1School of Pharmacy, RK University, Kasturbadham, Rajkot, Gujarat, India.
[email protected] ABSTRACT
Cardiovascular diseases (CVDs) have been reckoned amongst the top reasons for early deaths in the country. One of the major risk factors for developing CVDs is hyperlipidemia, an elevated condition of lipid levels in the body. Hyperlipidemia has been known to speed up a process of hardening of the arteries called atherosclerosis that may prove fatal in the development of various CVDs. Many drugs are available in the market for treatment of hyperlipidemia. This study was done with an objective to have a better alternative for treating complications associate with hyperlipidemia than the existing treatment. An anti-fungal drug ketoconazole was taken under studies for treating major complications associated with hyperlipidemia in High Fat Diet induced hyperlipidemic rats. With ketoconazole treatment there was a significant improvement in complications of hyperlipidemia similar to that of the standard drug atorvastatin. Thus the results helped to conclude that ketoconazole can be helpful in improving and managing the complications associated with hyperlipidemia along with lowering og cholesterol level hyperlipidemic conditions. Keywords: Hyperlipidemia, High Fat Diet, Ketoconazole, Atorvastatin
1. INTRODUCTION
the extra cholesterol circulating in the bloodstream forms the Hyperlipidemia is a medical condition characterized by an basis for plaque lining the arteries. Plaque slows the flow of elevation of any or all lipid profile or lipoproteins in the blood. blood through the arteries, which is especially dangerous when It is the major cause of coronary artery disease, ischemic it occurs in the heart. Coronary artery disease can result in cerebrovascular disease, peripheral vascular disease, etc. angina or a heart attack. During a heart attack, a section of the Lipids are water-insoluble organic compounds, which are heart muscle receives no oxygen because blood circulation in essential for many normal functions of living organisms: they the heart arteries is blocked by plaque. Plaque can also break are important components of cell membranes, they are used to off from an artery wall and circulate in the body, causing a store energy, and they play a significant role as enzyme co- stroke or peripheral arterial disease. Coronary artery disease factors, hormones, and intracellular messengers.[1] Of the (CAD) is the most common cause of congestive heart failure many groups of lipids, three are most important from a clinical (CHF) in the developed world, accounting for 50% of cases. [3] perspective: fatty acids, sterols (mainly cholesterol), and acyl- In itself, high cholesterol does not cause symptoms. Many glycerols (mainly triglycerides).[1][2] Cholesterol is the main people do not discover that they have high cholesterol until sterol in animal tissues. Dietary intake is the major source of after plaque has formed. Unless a person has regular checkups cholesterol, but it can also be synthesized endogenously by the that include laboratory testing, high cholesterol may silently liver and other tissues. It plays a fundamental role in central cause plaque build up in the arteries until symptoms of heart metabolic pathways, such as bile acid metabolism and steroid disease appear. Angina, heart attack, and stroke are all hormone and vitamin D synthesis.[1][2] It is dangerous because possible results of untreated high cholesterol. VOLUME-2, ISSUE-4, APRIL-2015 COPYRIGHT 2015 IJREST, ALL RIGHT RESERVED 24

INTERNATIONAL JOURNAL FOR RESEARCH IN EMERGING SCIENCE AND TECHNOLOGY, VOLUME-2, ISSUE-4, APRIL-2015 E-ISSN: 2349-7610 This medical condition or problem divided into two subtypes Present study was focused on an anti-fungal drug ketoconazole to give beneficial effect in management of complications associated with hyperlipidemia. Ketoconazole Primary or familial hyperlipidemia which is usually occurs as is an imidazole derivative antifungal agent developed for a result of genetic problems i.e., mutation within receptor treatment of human mycotic infections and plays an essential protein. It is classified according to the Fredrickson role in antifungal chemotherapy. Ketoconazole was first classification, which is based on the pattern of lipoproteins on discovered at Janssen Pharmaceuticals. The IUPAC name
electrophoresis or ultracentrifugation. It was later adopted by of this molecule is 1-[4-[4-[[2-(2, 4-dichloropheny)-2-
the World Health Organization (WHO). It does not directly account for HDL, and it does not distinguish among the yl]methoxy]phenyl]piperazin-1-yl]ethan-1-one. Its chemical different genes that may be partially responsible for some of formula is C26H28Cl2N4O4 with a molecular weight of these conditions.[4] 531.43092 gm/mol. [15][16] Acquired or secondary hyperlipidemia may mimic primary Ketoconazole is contains heterocyclic ring imidazole and forms of hyperlipidemia and can have similar consequences. interferes with fungal synthesis of ergosterol, a constituent of They may result in increased risk of premature atherosclerosis fungal cell-membrane as well as certain enzymes. It inhibits or, when associated with marked hypertriglyceridemia, may the enzyme cytochrome p-450 14 alpha demethylase lead to pancreatitis and other complications of the (p45014DM). This enzyme participates in sterol biosynthesis chylomicronemia Syndrome, as a result of other underlining pathway that forms ergosterol from lanosterol. Similarly diseases like diabetes, renal failure, hypothyroidism and drug induced like corticosteroids, oral contraceptive, beta blockers biosynthesis. From the same pathway of lanosterol dihydrosterols are synthesised and from dihydrosterols Lipoproteins are spherical structures that consist of a cholesterol is formed. So from this assumption was made as hydrophobic core containing lipids (i.e. triglycerides and/or for the inhibition of 14- alpha demethylase in synthesis of cholesterol esters), and an amphophilic (i.e. both hydrophobic ergosterol in fungus will also inhibit the cholesterol formation and hydrophilic) outer layer of phospholipids, free cholesterol, via same pathway thus lowering the level of cholesterol in and proteins that forms a protective envelope surrounding the serum and thus will also help in manageing various lipid core.[1][2][5][6][7][8] It can be divided based on their hydrated complications caused by hyperlipidemia. [17] density into the following major classes - chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins 2. MATERIALS AND METHODS
(LDL), and high-density lipoproteins (HDL).[9][10][11] Drugs used in treatment of hyperlipidemia currently include 2.1 Selection of Animals
statins (simvastatin, pravastatin), resins (cholecystyramine) Male Wistar albino rats of weight 180- 200 gm were used for and fibrates (gemfibrozil). Less commonly used drugs include the present study. The animals were procured from animal nicotinic acid, probucol, clofibrate and colestipol. These house, Department of Pharmacology, School of Pharmacy existing drugs have one or the other side effects along with its R.K.University, Rajkot. Animals were house at a temperature beneficial effect on improving lipid profile in hyperlipidemic of 24±2˚C and relative humidity of 30 – 70 %. A light and condition. Some of them are Myopathy including Myalgia, dark cycle was followed. All the experimental procedures and protocols used in the study were reviewed and approved by abdominal cramps, constipation, diarrhea, heartburn, Hepatitis the Institutional Animal Ethical Committee (IAEC) of School by elevating hepatic enzyme Alanine Amino Transferase of Pharmacy, RK University and care of laboratory animals (ALT) level in serum are found. This gave an influence in this were taken as per the guidelines of Committee for the Purpose research for getting a better alternative of it with similar of Control and Supervision of Experiments on Animals efficacy and lesser side effects. [12][13][14]. (CPCSEA).The animals were used after approval of IAEC VOLUME-2, ISSUE-4, APRIL-2015 COPYRIGHT 2015 IJREST, ALL RIGHT RESERVED 25

INTERNATIONAL JOURNAL FOR RESEARCH IN EMERGING SCIENCE AND TECHNOLOGY, VOLUME-2, ISSUE-4, APRIL-2015 E-ISSN: 2349-7610 protocol by Ministry of Social Justice and Empowerment, difference between mean body weights in each group was Government of India (Protocol No. RKCP/COL/RP/15/59).
calculated to determine the change in the body weight between the first day and 28th day.[19] The daily food intake for each 2.2 Grouping of Animals
group of rats was measured at an interval of 7 days and Male Wistar rat with a weight ranging between 180-200gm expressed as mean daily food intake for each group of 6 rats. were divided into five groups each consisting of 6 animals. The blood samples were collected on 28th day, using Retro- Group I: Normal control - Regular Normal low fat diet Orbital Plexus method and the serum was separated by centrifugation at 5000 rpm for 30 minutes at stabilization.[14] Group II: Disease control - High Fat diet Group III: Standard treatment - High Fat Diet+ Atorvastatin The parameters like body weight, total Cholesterol, CK MB, (dose 10mg/kg p.o) SGOT, SGPT, serum Creatinine and Atherogenic Index were Group IV: Treatment 1 - High Fat Diet + Ketoconazole (dose evaluated using the kits of Span Diagnosis.[20][21][22]. 2.3 Statistical Analysis
High fat diet is a hyper caloric diet and was prepared by To check the significance of the data obtained of the mixing the below given constituents in fixed percentage. The parameters evaluated following statistical tests were mentioned quantity is for 1000 gm diet. The feed was prepared and given to animals with 25% fructose water. Diet was given  ANOVA - to see the variability within all the groups for 21 days and the initial weight of the animals was noted.  TUCKEY'S TEST – to get the honest significance The weight gain was observed in rats by noting the weight on difference between all the groups 7th, 11th, 21st and 27th day, therefore confirming the  INSTAT SOFTWARE – to derive all the statistical development of obesity in rats. On 21st day the High Fat Diet terms like Standard Error of Mean (SEM), P-value, was stopped and the treatment protocol of the drug was given Standard Deviation, ANOVA, Degree of Freedom, etc. for 7 days. Study was continued for 28 days. Table 1: High Fat Diet (HFD) composition [18]
3. RESULT AND DISCUSSION
3.1 Beneficial Effect of Ketoconazole on Body Weight
in High Fat Diet Induced Hyperlipidemic Rats
High Fat Diet when introduced to rats showed a significant difference from normal control group to that of disease control group. There was a significant increase in body weight from 271.66 ± 5.27 gm to 498.33 ± 4.77 gm at the end of 21 days. This increase in body weight proves that there was an induction of hyperlipidemia in rats after consuming High Fat Diet. After the treatment of 7 days with drugs there was a lowering in the body weight in standard group and treatment- 1. This decrease in the body weight indirectly gives an indication that there will be lowering of serum lipid level in the rats. (Table 2) A body weight for each group of rat was recorded on day 7, 14, 21 and 28 day during the period of experiment. The VOLUME-2, ISSUE-4, APRIL-2015 COPYRIGHT 2015 IJREST, ALL RIGHT RESERVED 26

INTERNATIONAL JOURNAL FOR RESEARCH IN EMERGING SCIENCE AND TECHNOLOGY, VOLUME-2, ISSUE-4, APRIL-2015 E-ISSN: 2349-7610 Table 2: Effect of Ketoconazole on body weight on Hyperlipidemic rats
Physical
3.2 Beneficial Effect of Ketoconazole on Serum Total
treatment with Ketoconazole there was a significant decrease Cholesterol
in the SGOT level (27.87 ± 3.01 U/L) in T-1 group. By this After consuming High Fat Diet there was a significant observation we can say that ketoconazole gives beneficial increase in serum total cholesterol level in the rats. The serum effect in decreasing serum SGOT and thus can be said a lesser cholesterol in disease control was found to be 248.08 ± 5.33 damage to liver and heart. (Table 3) mg/dl compared to normal control (118.08 ± 2.83 mg/dl). 3.5 Beneficial Effect of Ketoconazole on Serum SGPT
After 7 days of treatment, the outcome was with standard atorvastatin (172.15 ± 5.20 mg/dl) and ketoconazole (175.2 ± High Fat Diet significantly increased the serum SGPT level in 5.20 mg/dl). Thus we can say that there was a significant the disease control group (48.90 ± 2.75 IU/L) compared to difference in the decrease in total cholesterol level in both the normal control group (14.03 ± 0.86 IU/L). After the treatment groups compared to disease control. So it helps in improving of 7 days with Atorvastatin the serum SGPT was found to be the lipid profile. (Table 3) decreased (20.16 ± 1.85 IU/L). Similarly with 7 days of treatment with Ketoconazole there was a significant decrease 3.3 Beneficial Effect of Ketoconazole on Serum
in the SGPT level (21.81 ± 3.01 IU/L) in T-1 group. By this Creatinine
observation we can say that ketoconazole gives beneficial High Fat Diet significantly increased the serum creatinine effect in decreasing serum SGPT and thus can be said a lesser level in the disease control group (3.2 ± 0.36 mg/dl) compared damage to liver. (Table 3) to normal control group (63.59 ± 2.147 mg/dl). After the 3.6 Beneficial Effect of Ketoconazole on Serum CK
treatment of 7 days with Atorvastatin the serum creatinine was found to be decreased (1.01 ± 0.015 mg/dl). Similarly with 7 days of treatment with Ketoconazole there was a significant High Fat Diet significantly increased the serum CK MB decrease in the creatinine level (1.061 ± 0.03 mg/dl) in T-1 level in the disease control group (283.24 ± 5.07 U/L) group. By this observation we can say that ketoconazole gives compared to normal control group (89.22 ± 9.51 U/L). beneficial effect in decreasing serum creatinine and thus can After the treatment of 7 days with Atorvastatin the be said a lesser damage on kidney is found. (Table 3) serum CK MB was found to be decreased (103.88 ± 6.16 U/L). Similarly with 7 days of treatment with 3.4 Beneficial Effect of Ketoconazole on Serum
Ketoconazole there was a significant decrease in the CK MB level (104.82 ± 5.09 U/L) in T-1 group. By this High Fat Diet significantly increased the serum SGOT level in the disease control group (64.92 ± 3.19 U/L) compared to observation we can say that ketoconazole gives normal control group (19.135 ± 0.99 U/L). After the treatment beneficial effect in decreasing serum CK MB and thus of 7 days with Atorvastatin the serum SGOT was found to be can be said a lesser damage to heart. (Table 3) decreased (25.92 ± 3.51 U/L). Similarly with 7 days of VOLUME-2, ISSUE-4, APRIL-2015 COPYRIGHT 2015 IJREST, ALL RIGHT RESERVED 27

INTERNATIONAL JOURNAL FOR RESEARCH IN EMERGING SCIENCE AND TECHNOLOGY, VOLUME-2, ISSUE-4, APRIL-2015 E-ISSN: 2349-7610 3.7 Beneficial Effect of Ketoconazole on Atherogenic
hyperlipidemic rats by decrease in cholesterol level and body The probable mechanism for anti-hyperlipidemic activity of High Fat Diet significantly increased the serum atherogenic ketoconazole seems to be improving the complications index in the disease control group (4.507 ± 1.105) compared associated with hyperlipidemia by decreasing 29.55% of total to normal control group (1.63 ± 0.108). After the treatment of cholesterol, 66.8% of creatinine, 57.07 % of SGOT, 55.39 % 7 days with Atorvastatin the atherogenic index was found to of SGPT, 62.99 % of CK MB and 57.84 % of Atherogenic be decreased (1.63 ± 0.14). Similarly with 7 days of treatment with Ketoconazole there was a significant decrease in the The assumed mechanism for anti-hyperlipidemic activity of atherogenic index (1.90 ± 0.04) in T-1 group. By this ketoconazole was found to be inhibiting 14-alpha demethylase observation we can say that ketoconazole gives beneficial enzyme, which is one of the enzyme responsible for effect in decreasing the atherogenic index and thus can be said biosynthesis of cholesterol from dihydrosterols as well as as cardio protective action. (Table 3) responsible for inhibiting formation of ergosterol from lanosterol in fungi. Thus dihydrosterols are not formed and Table 3: Effect of Ketoconazole on Biochemical
ultimately reduces biosynthesis of cholesterol and thus helps parameters
Biochemical
Parameter
Ketoconazole can also be helpful in decreasing body weight. 118.08 248.51 172.15 175.02 Cholesterol
5. FUTURE ASPECTS
Toxicity studies for ketoconazole can be done to check the extent of damage to body organs on prolong use. Creatinine
Clinical trials can be done to check the anti-hyperlipidemic activity of ketoconazole and to note the improvement in the lipid profile when exposed to humans. SGOT(U/L)
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