Evaluation of Combination of Low Dose Atorvastatin plus Ezetimibe and Aspirin in the Treatment of Atherosclerotic Vascular Disease

Narasimha Kumar, Jyothi, Pullaiah, Sastha Ram, Dhanunjaya, and Dayanand Reddy: Evaluation of Combination of Low Dose Atorvastatin Plus Ezetimibe and Aspirin in the Treatment of Atherosclerotic Vascular Disease

Authors

INTRODUCTION

Atherosclerotic vascular disease (AVD) is the major leading cause of the cardio vascular diseases in India and worldwide.1,2 As AVD development involves several steps, usage of combinational drugs ameliorates its progression by acting in different ways. Usage of conventional drugs has significant effect in decreasing the lipid levels.3 Statins play a major role in treatment of AVD and cardiovascular diseases.4 Co-administration of ezetimibe, a cholesterol absorption inhibitor along with statins resulted in decrease in elevated lipid levels.5 As AVD involves platelet aggregation in the last stage which results in severe outcomes like heart attack or stroke, anti-platelet drugs like aspirin at low doses can be used for the attenuation of consequences. Anti-inflammatory drugs may also account in reduction of generation of free radicals. The drugs which increase levels of HDL-C like niacin may improve the condition as HDL-C was involved in the reverse cholesterol transport.6-8

At present in the market several combinational drugs are available for the treatment of AVD like Ezetimibe + Atorvastatin, Atorvastatin + clopidogrel, Ezetimibe + Simvastatin etc. In most of the combinations the common drug used is Hydroxy methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, which inhibits biosynthesis of cholesterol in the liver. A moderate reduction of Cholesterol results in cell membrane domain aggregation and activation of T cell signalling events.9 As a result cell membrane integrity is lost and leakage of cell contents to the outside of cell occurs i.e., muscle damage occurs commonly known as rhabdomyolysis. Rhabdomyolysis is the most common side effect of (HMG-CoA) reductase inhibitors.10 Atorvastatin is the most commonly used drug of this category which is administered in the range of 10-80 mg once daily. Studies with atorvastatin even at low dose of 10mg have revealed its ability to decrease the increased lipid levels but incidence of rhabdomyolysis was also observed. Thus, in spite of its hypolipidemic potential, due to muscle pain caused by rhabdomyolysis, abrupt discontinuation of atorvastatin among patients is high. As atorvastatin is commonly employed in the treatment of cardiovascular disorders along with many other categories of drugs, the problem of rhabdomyolysis needs to be solved to improve the quality of life of patients.11 Thus the hypothesis of the study was to find out the incidence of rhabdomyolysis at selected low doses of atorvastatin without losing the hypolipidemic activity in the management of AVD.

Ezetimibe is a synthetic 2-azetidinone that selectively inhibits the intestinal reabsorption of hepatic ally excreted biliary cholesterol and dietary cholesterol and its mechanism of action is complementary to that of statins.12 Due to the potential risk of myopathy with statins alone, or when used in combination with fibrates or niacin, ezetimibe becomes an attractive choice to use alone or with a statin to achieve lipid targets.13-16 Further ezetimibe was only rarely reported to induce myopathy alone and also is not usually known to exacerbate statin induced myopathy.17 Aspirin is an anti-platelet agent frequently employed to manage cardiovascular disorders involving platelet aggregation. Thus, in the present study ezetimibe and aspirin were combined with atorvastatin.

The aim of the present study was to evaluate the effect of combination of low dose of atorvastatin 1.027 mg/kg, 0.770 mg/kg, 0.513 mg/kg (Human dose 10mg, 7.5 mg & 5 mg respectively) plus Ezetimibe 0.986 mg/kg (Human dose 10 mg) and aspirin, 7.708 mg/kg (Human dose 75 mg) in the treatment of AVD in albino rats. The objective of the study was to determine the levels of triglycerides, total cholesterol, high density lipo proteins (HDL), low density lipoproteins (LDL) in the serum. Very low-density lipoproteins (VLDL) and atherogenic index were also determined based on lipid values. Grip strength was also determined at the end of the study period to know the incidence of rhabdomyolysis. Anatomical changes in the aorta were evaluated by histopathological examination.

MATERIALS AND METHODS

Animals

Adult male Wistar rats weighing 150-200 g were selected for the study and were purchased from Raghavendra enterprises, Bangalore. The animals were housed in standard environmental conditions (22-28°C, 60-70% relative humidity, 12 hr light and dark cycle) and fed with rat chow and supplied with water. The experimental protocol was approved by the institutional animal ethical committee (SPSP/CPCSEA/IAEC-1016/a /2014/ 001) of Sri Padmavathi School of Pharmacy, Tirupathi, India.

Drugs and Chemicals

Atorvastatin, Ezetimibe, Aspirin, Ketamine, Xylazine, diagnostic kit for triglycerides, cholesterol-HDL kit was purchased from Span diagnostics Pvt. Ltd. India. Sudan III (Sigma Aldrich, India) and all other chemicals used were of analytical grade (Bross Scientifics, Tirupathi).

Instruments

Auto analyzer (Mispa Excel), UV-visible spectrophotometer (Analytical systems, model no: AUV 2060), Electronic balance (Shimadzu, model no: DS- 852 J), Colorimeter (Inco, model no: CL- 157), Homogenizer (Ever shine, model no: 607), Cooling centrifuge (Remi, model no: KKLO- 9013).

High fat diet model

Hypercholestremia was the major risk factor for the development of cardiovascular diseases and it involves the deposition of lipid in the intima of arteries. In the present study rats were fed with high fat diet composed of casein, cholesterol, choline, corn starch, soya bean oil, cellulose, and vitamin mixture, salt mixture was given to the rats.18

Experimental design

The total study period was 10 weeks and effect of low doses of atorvastatin plus ezetimibe and aspirin on high fat diet induced hyperlipidemia in rats was evaluated by monitoring body weights and by estimation of lipid profiles of rats biweekly. Except normal group animals remaining all animals were supplied with high fat diet. After completion of 4th week, serum lipid profiles were monitored in animals and induction of hyperlipidemia was confirmed by the elevated lipid levels, except a decrease in HDL-C levels in experimental rats. Thus, after completion of 4th week treatment with drugs was started in test groups and lipid profiles were estimated biweekly throughout the study period. So, duration of treatment was six weeks.

Male Wistar rats were divided into five groups six in each as follows:

Normal Control - Received normal diet, Disease Control - Received high fat diet, Test group I - Received high fat diet and atorvastatin (1.027 mg/kg) + ezetimibe (0.986 mg/kg) + aspirin (7.708 mg/kg). Test group II - Received high fat diet and atorvastatin (0.770 mg/kg) + ezetimibe (0.986 mg/kg) + aspirin (7.708 mg/kg). Test group III – Received high fat diet and atorvastatin (0.513 mg/kg) + ezetimibe (0.986 mg/kg) + aspirin (7.708 mg/kg). Drugs were administered to rats in test groups p.o. dissolved in Carboxy methyl cellulose (CMC).

For biochemical estimations blood samples of rats were collected from retro-orbital plexus. Rhabdomyolysis was evaluated by determining grip strength by using Rota rod apparatus at the end of study period. At the end of study period, all the animals were anaesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg)19 and were dissected and aortas of rats was isolated and sent to histopathological examination.

Biochemical Estimations: Estimation of Lipid profile

Triglycerides (TG) were measured by GPO/PAP method,20 total cholesterol by CHOD/POP method,21 high density lipo protein cholesterol (HDL-C) by PEG-CHOD-PAP method by using auto-analyser.22 Very low density lipo proteins (VLDL) and atherogenic index were calculated by Freidewald’s formula.23

VLDL = TG/5

Atherogenic index = TG/ HDL

Grip strength of animals by Rota-rod apparatus

Grip strength was calculated at the end of the study period. With the Rota rod apparatus muscle coordination, balance, strength and condition were tested. Rats were placed on the rod that accelerated from 5 to 45 rotations per minute within 15s. When a rat ran for 500s without falling from the rod, the test session was ended. Rat that fell off within 500s were given a maximum of two more tries. The longest running time was used for analysis.24

Statistical Analysis

All the data was expressed as mean ± SEM (standard mean error). The statistical significance between groups was analyzed using two-way analysis of variance (ANOVA). The significance was expressed by ‘p’ values, and a ‘p’ value of <0.05 was taken as significant.

RESULTS

Body weight

A gradual increase in body weights was observed biweekly in disease control group animals when compared with the normal control group animals. In test group animals there was a minor decrease in body weights when compared with disease control group animals (Table 1).

Lipid profile

The levels of triglycerides, cholesterol, LDL, and VLDL were markedly increased from zero weeks to tenth week in disease control group animals when compared with the normal control group animals. In test group animals, there was a slight decrease in all these lipid levels when compared with control animals. The same trend was observed with atherogenic index also where as in case of HDL-C levels it was vice-versa. (Table 2,3).

Table 1

Effect of low dose of Atorvastatin plus Ezetimibe and Aspirin on body weight of experimental rats

GroupZero weekSecond weekFourth weekSixth weekEighth weekTenth week
Normal Control120 ±0.8135±0.5165±0.4190±0.8200±0.8210±0.7
Disease Control125±0.9150±0.6*190±0.7*251±0.5*274±0.3*298±0.7*
Test group-I122±0.8152±0.8192±0.5205±0.5**221±0.7**215±0.5**
Test group-II125±0.6151±0.6189±0.6214±0.5**225±0.5**218±0.7**
Test group-III123±0.7155±0.7191±0.7235±0.7**230±0.5**223±0.5**

[i] All values are shown as Mean ± SEM and n=6, * indicate p<0.05, when compared to Normal control, ** indicate p<0.05, when compared to disease control.

Table 2

Effect of low dose of Atorvastatin plus Ezetimibe and Aspirin on Triglyceride, Total Cholesterol and LDL-C levels in experimental rats

GroupPara-meterZero weekSecond weekFourth weekSixth weekEighth weekTenth week
Normal ControlTG47.8±0.659.4±0.563.6±0.465.3±0.469.7±0.475.6±0.4
TC130.8±0.5141.4±0.5144.3±0.3151±0.7158.4±0.4152.3±0.5
LDL79.4±0.379.4±0.482.7±0.388.4±0.381.2±0.384.1±0.3
Disease ControlTG49.7±0.374.8±0.3*121.5±0.7*183.9±0.5*208.9±0.4*241.7±0.6*
TC127.4±0.4149.3±0.4*192.5±0.4*229.9±0.4*251.4±0.5*283.4±0.4*
LDL77.3±0.492.4±0.4*132.3±0.3*160.3±0.3*179.4±0.2*207.4±0.3*
Test group-ITG49.3±0.677.7±0.4119.2±0.3117.4±0.9**119.1±0.6**127.5±0.4**
TC129.6±0.3147.4±0.4191.3±0.4183.1±0.4**190.5±0.3**180.5±0.3**
LDL76.2±.390.4±0.3130.3±0.4110.4±0.2**102.3±0.3**98.5±0.2**
Test group-IITG47.5±0.579.4±0.4116.3±0.4118±0.4**124.4±0.6**132.4±0.2**
TC126.6±0.5144.7±0.4195.7±0.4192.5±0.4**209.7±0.3**191.2±0.3**
LDL76.2±0.489.5±0.4129.2±0.2119.3±0.2**108.4±0.4**110.4±0.3**
Test group-IIITG46.9±0.678.4±0.4116.9±0.2128.6±0.4**132.2±0.8**149.8±0.3**
TC124.4±0.4149.52±0.9197.6±0.4205.5±0.4**224.6±0.3**217.3±0.5**
LDL74.5±0.288.3±0.4131.3±0.3148.6±0.2**144.4±0.3**159.6±0.2**

[i] All values are shown as Mean ± SEM and n=6, * indicate p<0.05, when compared to Normal control, ** indicate p<0.05, when compared to disease control. TG=Triglycerides, TC=Total Cholesterol, LDL-C= Low Density Lipoprotein- Cholesterol

Table 3

Effect of low dose of Atorvastatin plus Ezetimibe and Aspirin on HDL-C, VLDL-C and Atherogenic Index in experimental rats

GroupPara-meterZero weekSecond weekFourth weekSixth weekEighth weekTenth week
Normal ControlHDL41.2±0.342.4±0.441.5±0.545.8±0.447.7±0.442.8±0.8
VLDL9.5±0.311.9±0.312.6±0.313.4±0.414.1±0.415.0±0.5
AI1.21±0.31.4±0.31.5±0.31.1±0.31.4±0.11.7±0.06
Disease ControlHDL41.8±0.841.7±0.436.2±0.3*31.8±0.3*29.4±0.4*27.4±0.4*
VLDL9.9±0.414.6±0.3*24.6±0.4*36.2±0.4*41.0±0.4*48.2±0.4*
AI1.25±0.31.7±0.2*3.3±0.2*5.8±0.2*7.1±0.2*8.8±0.2*
Test group-IHDL40.6±0.341.9±0.338.2±0.245.5±0.4**51.4±0.4**56.2±0.3**
VLDL9.8±0.315.2±0.223.5±0.423.4±0.4**23.7±0.4**25.2±0.4**
AI1.24±0.31.8±0.33.2±0.1**2.5±0.3**2.3±0.3**2.2±0.3**
Test group-IIHDL40.7±0.440.7±0.236.2±0.243.2±0.2**46.9±0.3**49.78±0.3**
VLDL9.4±0.315.4±0.325.6±0.423.5±0.3**24.4±0.8**26.5±0.3**
AI1.1±0.21.92±0.23.2±0.2**2.7±0.2**2.4±0.2**2.6±0.3**
Test group-IIIHDL41.4±0.340.6±0.437.2±0.338.2±3**40.2±0.3**39.0±0.7**
VLDL9.3±0.415.2±0.423.5±0.525.7±0.4**26.3±0.4**30.4±0.3**
AI1.17±0.11.90±0.13.1±0.4**3.3±0.1**3.3±0.1**3.8±0.04**

[i] All values are shown as Mean ± SEM and n=6, * indicate p<0.05, when compared to Normal control, ** indicate p<0.05, when compared to disease control. HDL-C= High density lipoprotein Cholesterol, VLDL-C= Very low density lipoprotein Cholesterol, AI= Atherogenic Index.

Table 4

Effect of low dose of Atorvastatin plus Ezetimibe and Aspirin on grip strength in experimental rats.

GroupTime (sec)
Normal Control402.5±8.732
Disease Control286.2±3.859*
Test group-I188.7±4.080*
Test group-II303.5±4.041*
Test group-III385.3±2.011*

[i] All values are shown as Mean ± SEM and n=6, * indicate p<0.05, when compared to Normal control

Grip Strength

Grip strength of animals was estimated at the end of study. There was greater grip strength in animals of test group I and II when compared with the animals of test group III (Table 4).

Histopathology of Aorta

Histopathological examination (magnification-10X) of abdominal aorta in normal control group animals had shown no abnormality (Figure 1.a). Disease control group animals had shown intimal thickening, and accumulation of lipids in the aorta (Figure 1.b). There was a slight decrease in lipid accumulation in test group animals when compared to disease control group animals (Figure 2 ab & Figure 3 a).

DISCUSSION

Currently AVD is effectively managed with a combination therapy which may include a statin, ezetimibe, clopidogrel or aspirin and niacin. As statins helps in reduction of hepatic cholesterol synthesis, they play a major role in the treatment of AVD and other lipid disorders.25 Among statins atorvastatin is the most prescribed drug to the patients and the dosing may be continued up to six months or more based on serum cholesterol levels. The most important drawback of statins is rhabdomyolysis causing abrupt discontinuation by patients.26 Ezetimibe given at a dose of 10 mg once daily is a cholesterol absorption inhibitor through duodenal cells by inhibiting Niemann Pick C1 like protein which is a transporter for cholesterol. Ezetimibe usage in AVD condition can improve the patient condition by decreasing the plasma concentration of cholesterol.27 Aspirin, at a dose of 75-100 mg selectively inhibits cyclooxygenase-1 in platelets by acetylating a single serine residue at position 529 in cyclooxygenase-1 leading to inhibition of Thromboxane A2 production, thus resulting in anti-platelet activity.28

Studies with atorvastatin at its lowest dose i.e. 10mg in the management of cardiovascular disorders have shown that there is a prominent decrease in lipid levels with incidence of rhabdomyolysis.11 Patients will be benefited if atorvastatin can retain its hypolipidemic potential at doses less than 10mg without causing rhabdomyolysis. Thus, in the present study, we contemplated to test whether atorvastatin at selected doses i.e., 10mg, 7.5mg and 5mg, was able to produce rhabdomyolysis, along with ezetimibe (10mg) and aspirin (75mg) in the management of AVD, without losing its hypolipidemic potential in albino rats. 10mg dose was included in the study as it is the lowest therapeutic dose of atorvastatin. Rat doses of the above mentioned human doses were calculated as per Jang-Woo Shin et al., 2010.29

In the present study high fat diet model was used to induce hypercholestremia as it has same pathophysiology in the development of AVD as that of humans. A high fat diet containing cellulose, choline, cholesterol, soyabean oil, casein, salt mixture, vitamin mixture was supplied to the male wistar rats throughout the study period of 10 weeks except for the normal control group animals. Casein is a milk protein which can increase the levels of lipids in the triglyceridemic condition. Cholesterol can directly increase the plasma levels of cholesterol after absorbing through duodenal cells into the systemic circulation. Soyabean oil when taken in normal quantities it has protective effect against free radical generation but in higher amounts triglyceridemic condition will result. Choline intake directly increases the levels of lipids when taken.12

Figure 1

a. Histopathology of aorta of normal group rats showing normal texture (10X) B. Histopathology of aorta of disease control group rats characterised by thickness relative to normal control rats (10X). Magnification scale: 100µm

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Figure 2

a. Histopathology of aorta of test group-I rats (10X) showing absence of lipid deposits b. Histopathology of aorta of test group-II rats (10X) showing absence of lipid deposits. Magnification scale: 100µm

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Figure 3

a. Histopathology of aorta of test group-III rats (10X) showing moderate accumulation of lipids. Magnification scale: 100µm

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High fat diet was given to the rats of all groups except normal control group throughout the study period. Treatment was initiated after four weeks by which there was a prominent increase in body weight, triglycerides, cholesterol, LDL, VLDL and decrease in HDL in rats of all groups except normal control group. At the end of the treatment schedule, body weight, triglycerides, cholesterol, LDL, VLDL values and atherogenecity index were high and HDL decreased in disease control group animals when compared to normal control group animals. Test group animals showed a decrease in body weight, lipid values and atherogenecity with an elevation in HDL levels when compared to disease control. Within the test groups, test group I showed a prominent hypolipidemic effect, group-II showed a moderate effect and group-III showed low effect. From this it is evident that atorvastatin at a dose of 5mg human dose is having low hypolipidemic potential.

Evaluation of muscle strength is an essential step for researching neuromuscular disorders and multiple methods have been developed to measure muscle strength in rodents. Among these methods, grip strength tests are more convenient and give less stress to animals than other tests.30 In grip strength test, disease control animals showed moderate grip strength when compared to normal control group animals. Test group animals showed decrease in the grip strength when compared to normal group animals but grip strength was high among Group-III, moderate in Group-II and low in group-I. This gives evidence that there is a decreased incidence of rhabdomyolysis in test groups II and III. From this atorvastatin at a dose of 10mg human dose is causing rhabdomyolysis.

Histopathological examinations of aorta have shown that there was no lipid accumulation in normal group animals and there was a marked loading of lipids in disease control group animals when compared to normal control group animals. Test group animals showed a decrease in lipid accumulation but effect was pronounced in group I & II animals compared to group-III animals. Group-III animals showed moderate accumulation of lipids owing to low dose of atorvastatin employed (5 mg human dose).

Body weight changes, biochemical parameters, histopathological studies and grip strength studies, reveal that rats treated with atorvastatin at a dose of 0.770 mg/kg (clinical dose - 7.5 mg) along with ezetimibe and aspirin have shown similar lipid profile, decreased atherogenicity as in rats treated with 1.027 mg/kg atorvastatin (clinical dose - 10 mg) along with ezetimibe and aspirin with low incidence of rhabdomyolysis.

CONCLUSION

Statins are the first line therapy drugs employed in the treatment of lipid abnormalities but the most common adverse effect observed is rhabdomyolysis at therapeutic doses used, causing abrupt discontinuation by the Patient’s due to muscle pain. The present study demonstrates that 7.5mg clinical dose (0.770 mg/kg rat dose) of atorvastatin along with ezetimibe and aspirin may be used effectively to treat AVD and other lipid abnormalities with decreased incidence of rhabdomyolysis which may augment patient compliance of atorvastatin. Further clinical studies are warranted to evaluate the same in the management of AVD and other lipid disorders which can improve the quality of life of patients.

ACKNOWLEDGEMENT

Authors would like to express gratitude towards Prof. D. Ranganayakulu, Prof. C. Sridhar, Dr. B. Pushpakumari of Sri Padmavathi School of Pharmacy, Tirupathi and Dr. D. Sujatha, SPMVV, Tirupathi for their valuable inputs and support for the completion of the study.

Notes

[5] Conflicts of interest CONFLICT OF INTEREST None

ABBREVIATION USED

ANOVA

Analysis of variance

AVD

Atherosclerotic Vascular Disease

CPCSEA

Committee for the purpose of Control and Supervision of Experiments on Animals

HDL

High Density lipo Proteins

HMG-CoA

Hydroxy methylglutaryl coenzyme A

IAEC

Institutional Animal Ethics Committee

LDL

Low Density Lipoproteins

PO

Per Oral

SEM

Standard Mean Error

VLDL

Very low density lipoproteins

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