darapl… · web viewtotal word count: 4689. abstract. background: insulin resistance in type 2...

Teuku Heriansyah, MD

OX-LDL, LYSOPHOSPATIDYL CHOLINE,PLATELET ACTIVATING FACTOR, IL-6

EXPRESSION LEVEL AND FOAM CELL NUMBER WITH DARAPLADIB

TREATMENT IN EARLY DEVELOPMENT OF ATHEROSCHELROSIS: IN VIVO

STUDY FOR TYPE 2 DIABETES MELLITUS MODEL

Teuku Heriansyah, MD*, Bambang Budi Siswanto, MD**, Anwar Santoso, MD**, Djanggan Sargowo***, Imam Subekti, MD****, Aulani’Am, DEA*****, Renan Sukmawan, PhD**, Nurjati Hairani Siregar, MD******, Saptawati Bordosono, MD*******, Titin Andri Wihastuti, PhD********* Department of Cardiology and Vascular Medicine, Faculty of Medicine,University of Syiah Kuala, Banda Aceh, Indonesia** Department of Cardiology and Vascular Medicine, Faculty of Medicine,University of Indonesia, Jakarta, Indonesia*** Department of Cardiology and Vascular Medicine, Faculty of Medicine,University of Brawijaya, Malang, Indonesia**** Department of Internal Medicine, Division of Metabolic and Endocrinology,Faculty of Medicine, University of Indonesia, Jakarta, Indonesia***** Department of Biochemistry, Faculty of Veterinary Medicine, University ofBrawijaya, Malang, Indonesia****** Department of Anatomy Pathology, Faculty of Medicine, IndonesiaUniversity, Jakarta, Indonesia******* Department of Nutrition, Faculty of Medicine, Indonesia University,Jakarta, Indonesia******** Department of Biomedicine, Faculty of Medicine, Brawijaya University,Malang, Indonesia

Short Tittle: Effect of Darapladib in Atherogenesis Development

Name and adress of the author responsible for correspondence:Titin Andri Wihastuti, PhDJaksa Agung Suprapto I/47, Malang, East Java, Indonesia

Total word count: 4689


ABSTRACT

Background: Insulin resistance in type 2 Diabetes Melitus (T2DM) is a condition of extensive

tissue damage due to vascular inflammation and oxidative stress. Lp-PLA2 has antiinflammatory

role as it hydrolyze atherogenesis mediators such as oxLDL and platelet activating factor (PAF)

and produces lysophosphatidylcholine (lysoPC) and oxidized fatty acid (oxFA) that have pro-

inflammatory, proliferative and pro-atherogenic effect.

Methods and results: This study aimed to discover the expression of ox-LDL level, LysoPC,

PAF, IL-6 and foam cells numbers in aorta of T2DM in vivo model with darapladib treatment.

True experimental laboratory and only post test with control group design using 30 spraque

dowley rats that is divided into 3 main groups: normal, T2DM, and T2DM with darapladib

administration group. Each group consisted of 2 serials treatment time: 8-weeks and 16-weeks

treatment group. Parameter measured was IL-6, oxidized LDL and PAF, LysoPC, IL-6, foam

cells and also blood glucose, lipid profile, and insulin plasma level. ANOVA test result showed

that darapladib were significantly (p: 0.000) lowering ox-LDL level, LysoPC, PAF, IL-6

expression and number of foam cells in aorta on T2DM in vivo model.

Conclusions: This study conclude that dalapladib proved to have a role as anti atherogenesis on

T2DM model.

Keywords: Type 2 DM, atherosclerosis, darapladib

1. Introduction

To this day, CVD remains a serious global health problem and has become the leading cause of

death in both developed and developing countries. The incidence of CVD in Indonesia turned out

to be much higher than the developed countries with the number of cases is 150.8 compared to

only 80.5 in the United States and 31.2 per 100,000 population in Japan. The death rate from


CVD in Indonesia reaches 243,048 or 17.05% of total deaths. This condition puts CVD as the

first cause of death from 20 leading causes of death in Indonesia[1]. Atherosclerosis is the most

common cause of CVD, including coronary heart disease (coronary artery disease/CAD),

cerebrovascular disease (stroke) and peripheral blood vessel diseases (peripheral artery

disease /PAD)[2].

Type 2 Diabetes mellitus (T2DM) is one of major classical risk factors for atherosclerosis[3].The

clinical condition with insulin resistance and T2DM is a condition of an existance ofextensive

tissue damage due to vascular inflammation and Oxidative stress[4]. Hyperglycemic

environment occurred in DM type 2 increases Reactive Oxygen Species (ROS) production[5].

Enzymatic activity and expression of lipoprotein associated phospholipase A2 (Lp-PLA2) have

begun to be studied as a biomarker of CVD risk in people with CAD and healthy individual[6].

The understanding about role of Lp-PLA2 in the pathophysiology of cardiovascular disease

remains controversial and far from well established. Lp-PLA2 antiinflammatory role

demonstrated by the ability of this enzyme to hydrolyze mediators that could potentially have a

role in atherogenesis such as oxLDL and platelet activating factor(PAF). At the same time, the

products of hydrolysis of these molecules that are mediated byLp-PLA2 such as

lysophosphatidylcholine (lysoPC) and oxidized fatty acid (oxFA) have the effect of pro-

inflammatory, proliferative and pro-atherogenic role.

In the last decade, a new pharmacological therapy for atherosclerosis which actively, selectively

and reversibly inhibiting Lp-PLA2 is being developed and the result is darapladib. Darapladib is

a substance that works to hydrolyze Lp-PLA2 substrate. Darapladib forms non-covalent bonds

and reversibly with human recombinant Lp-PLA2. Darapladib is lipophilic and has good


membrane permeability. Darapladib reduce the activity of caspase-3 and caspase-8 inhibits

macrophage apoptosis induced by ox-LDL.

Several studies stated that darapladib show significant results in inhibiting the process of

atherosclerosis[7]. Both with in vitro and in vivo experiments[8,9]. Positive results are also

shown in a trial study in patients undergoing carotid endarterectomy[10]. However, there are

several opinions regarding the usefulness of darapladib as a treatment for atherosclerosis because

some clinical trials show negative results of darapladib usage in reducing mortality from

cardiovascular disease, myocardial infarction, or stroke[11]. Based on that facts, this study aims

to determine the expression ox-LDL levels, LysoPC, PAF, IL-6 and the number of foam cell in

aorta of in vivo model T2DM with darapladib treatment.

2. Materials and Methods

Study group

This study used male Sprague Dawley Rats with 4 weeks old and weight around 150-200 grams

as animal model. The samples were obtained from Bogor Agricultural University, Bogor,

Indonesia. These rats were divided into three groups; normal group (N); Type 2 DM Model

groups (DM) which fed with High Fat Diet (HFD) and Streptozotocin STZ injection

intraperitoneal low dose 35 mg/KgBW, and type 2 DM model with Darapladib administration

group (DMDP). Each group was divided into two serial time which was 8 weeks (early phase)

and 16 weeks (late phase). Darapladib was obtained from Glaxo Smith Kline. Samples were

given Darapladib orally 20mg/Kg body weight once daily in duration according the time serial

groups given. In short, there were 6 groups in total. N8 and N16 for normal groups with 8 and 16

weeks serial time. DM8 and DM16 for T2DM model groups with 8 and 16 weeks serial time,


and DMDP8 and DMDP16 for T2DM model with Darapladib administration groups with 8 and

16 serial time.

The normal rats food contained total energy Calories (kcal/g) of 3.43 (67% carbohydrate,21%

protein and 12% fat), while the HFD contained total calorie energy of 5.29 (58% fat,17%

carbohydrate and 25 % protein). Each kilogram of a mixture of vitamins and minerals contained

2000000 IU of Vitamin A, 400000 IU of Vitamin D3, 0.8 grams of Vitamin B2,300 units of

Vitamin E, 0.4 gram Vitamin K, 1 gram of calcium pantothenate, 4 grams of Nicotinamide, 2.4

gram of Vitamin B12, 60 grams of Choline Chloride, 300 grams of Calcium, 2.75 grams of

Manganese, 0.1 grams of Iodine, 3 grams of Iron, 6 grams of Zinc,0.8 grams of Copper and 0.18

grams of Cobalt[12]. Everyday 30 grams of food were given foreach rat. Measurements of

parameters were done at the Central Laboratory of BiologicalSciences, Brawijaya University.,

Brawijaya University, Malang, Indonesia. Slicing and staining samples were done at

Pathological Anatomy Laboratory, Faculty of Medicine University of Brawijaya, Malang,

Indonesia

Blood glucose measurements

A dose of 20 mg/kg body weight STZ was administered prior to the first measurement of blood

glucose to induce type 2 diabetes mellitus. Type 2 diabetes mellitus was diagnosed after

measurement of the blood glucose level with a GlucoDR blood glucose test meter (AllMedicus

Co. Ltd, Dongan-gu, Anyang-si, Korea). Type 2 diabetes mellitus was diagnosed after obtaining

blood glucose levels >200 mg/dL in the rats.


Measurement of lipid profile levels

Lipid profiles were measured using rat blood serum and calculating total cholesterol, HDL (High

Density Lipoprotein), and LDL (Low Density Lipoprotein) using kit EnzyChrom™ which is a

Bioassay system.

Measurement of insulin resistance

Insulin in rat’s blood plasma were measured using sandwich ELISA by using Rat INS (insulin)

ELISA kit (Cat. No. E-EL-R2466). The result obtained are still in ng / mL units. Forthe

conversion of plasma insulin levels of ng / mL to IU / L, WHO formula were used by dividing

the result by 0.0347, as 1 IU is equivalent to 0.0347 mg / L[13]. Measurements of insulin

resistance can be performed with HOMA-IR (homeostatic model assessment-insulin resistance)

formula especially in rat, which require data from fasting glucose and plasma insulin levels by

the following formula[14]:

Explanation:

HOMA-IR : Homeostatic Model Assessment-Insulin Resistance

FBG : Fasting Blood Glucose (mmol/L)

FINS : Fasting Insulin Plasma (μU/mL)

The interpretation from HOMA-IR calculation in rats is if the result > 1,716 then it can be

categorized as insulin resistance with a sensitivity of 83.87% and a specificity of 80.56%

(95%confidence interval) [15].

HOMA – IR = FBS x FINS

14,1


Biochemical tests

Expression IL6 measurement

IL-6 were measured by immunofluorescence of aortic tissues that were previously fixed with

PHEMO buffer (68 mM PIPES, 25 mM, HEPES, pH 6.9, 15 mMEGTA, 3 mM MgCl2, 10%

[v/v] dimethyl sulfoxide containing 3.7% formaldehyde and0.05% glutaraldehyde) and were

processed by imumunofluoresence double labeling with anti-rat antibody Lp-PLA2 using

rhodamin secondary antibody and anti-rat antibody IL-6using fluorescein isothiocyanate (FITC)

secondary antibody (BIOS Inc., Boston, MA, USA). These three parameters were observed with

confocal laser scanning microscopy (OlympusCorporation, Tokyo, Japan) and were

quantitatively analyzed using Olympus FluoViewsoftware (version 1.7A; Olympus Corporation).

OxLDL measurement

Ox-LDL was measured using rats aorta tissue as samples. Rats had been fasted a day before the

sample obtained. Ox-LDL Level measured by Sandwich ELISA method using Rat ox-LDL

ELISA kit (Cat. No. E-EL-R0710). The first step was antigen coating, 100 μL standard and

sample was put into well that are coated with antibody before then incubated in 37 0C for 90

minutes. The residual antigen that was not bind with antibody were disposed. After that, 100 μL

Biotin-antibody was added into each well. Incubated each well in 370C for 1 hour. The fluid in

well was aspirated and washed by wash buffer for 3 times. Next, 100 μL HRPavidin was added

into each well and incubated in 370C for 30 minutes. Repeated the aspiraiton and washing

process for 5 times. 90 μL TMB substrate was added to each well then was incubated in 370 for

15 minutes in light-free area. 50 μL stop reaction was added for stopped the reaction. Each well

was read 5 minutes later with ELISA reader with 450 nm wavelength.


LysoPC Measurement

The samples used were serum and rat’s aorta homogenates. The tool used was 6460

triplequadrupole LCMS / MS System, Agilent Technologies, USA. Standard LysoPC material

(16:0) used was 1-palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphocholine (16: 0), cat 85567P, lot

16LPC-79, Avanti Polar Lipids, Inc., 700 Industrial Park drive, Alabaster, Alabama 35007-9105,

USA. Standard LysoPC internal material (16: 0) used was 1-palmitoyl (D31) -2-Hydroxy-sn-

Glycero-3-Phosphocholine (16: 0), paint 85567P, lot 16 LPC-79, Avanti PolarLipids, Inc. , 700

Industrial Park Drive, Alabaster, Alabama 35007-9105, USA. StandardLysoPC material (18:0)

used was 1-Stearoyl-2-Hydroxy-sn-Glycero-3-Phosphocholine(16:0), cat 85567P, lot 16LPC-79,

Avanti Polar Lipids, Inc, 700 Industrial Park Drive,Alabaster, Alabama 35007-9105, USA.

Standards calibration range used were 250-16000 ng/ mL. Dilution factors of aorta homogenates

samples 16-7 for LysoPC Stearoyl was 2X.Dilution factors of serum sample was 20X, except for

palmitoyl (16: 0) Code 8-1 and 8-2 itwas 40X, and Stearoyl (18: 0) Code 8-1 was 80X. Standard

material used specifically forresearch (Research Use Only). LysoPC analysis procedure refers to

journals: Takatera A,et.al. Blood Lysophosphatidylcholine (LPC) Levels and Characteristic

Molecular Species inNeonates: Prolonged Low Blood LPC Levels in Very Low Birth Weight

Infants.Pediatr Res:477-482, 2007.

PAF measurement

Measurement of PAF concentrations in blood plasma samples of rat conducted by competitive

ELISA method using Rat Platelet Activating Factor (PAF) ELISA kit (Cat. No.MBS722041).

Competitive ELISA begins with Coating antigen. Standard and a sample of100 μL inserted into

the well (to form using PBS). Then added 50 μL Conjugate to each well(except blank well) was

incubated at a temperature of 370C for 1 hour. Aspirated and washed with wash buffer 5 times.


Then added 50 μL Substrate A and 50 μL Substrate B to each well and incubated for 10-15

minutes at a temperature of 370C (avoid from light). To stop reaction, add 50 μL stop solution to

each well. After 5 minutes read by ELISA reader at awavelength of 450 nm.

Tissue sampling

Tissues from rats were observed at the end of this study after surgically removing aortic tissues

and blood samples via cardiac puncture. The aortic tissues were prepared by a frozen section

technique and hematoxylin eeosin staining prior to being examined microscopically in the

Laboratory of Pathology and Anatomy, Brawijaya University.

Measurements of foam cells

The aortic tissues were prepared by a frozen section technique and stained with

hematoxylineeosin. After preparation of the aortic tissues, the foam cells was measured usinga

microscope with a magnification of 400_and dotSlide Olyvia version 2.4 software(Olympus

America inc., Center Valley, PA, USA). Examination and calculation of the number of foam

cells were performed for each 10 microscope fields after taking images of the aortic tissues.

Ethics

We obtained ethical approval for the animal treatment and experimental processes in this study

from the Animal Care and Use Committee Brawijaya University Number 229-KEP-UB

Statistical analysis

The one-way analysis of variance (ANOVA) test was used to determine the effects of time series

factor, treatment of darapladib and the interaction of time and treatment Darapladib onmRNA

Lp-PLA2 expresion, OxLDL, PAF, LysoPC, PVAT and foam cells in Spraque Dowley rats with

type 2 diabetes mellitus. This analysis was continued by the post hoc testusing the Duncan


method to detect differences in parameters in each treatment group. SPSS software version 20

(IBM Corporation, New York, NY, USA) was used for data analysis.

3. Results

Total cholesterol levels observed ranged from 56.560 to 123.002 (mg/dL). The highest total

cholesterol levels can be observed in the DM8 group with an average of 123.002

mg/dL.Meanwhile, the lowest total cholesterol level found in N16 group with an average of

56.560mg/dL. HDL levels observed ranged from 4.958 to 35.767 (mg/dL). The highest HDL

levels can be observed in N16 with an average of 35.767 mg/dL. Meanwhile, lowest HDL levels

found in the DM8 group with an average of 4.958 mg/dL. Levels of VLDL/LDL observed

ranged from 19.241 to 95.531 (mg/dL). The highest Levels of VLDL/LDL can beobserved in

DM8 group with an average of 95.531 mg/dL. Meanwhile, the lowest levels of VLDL/LDL

obtained at N16 group with an average of 19.241 mg/dL. Fasting glucose levels observed ranged

from 79.6 to 147.8 (mg/mL). The highest fasting glucose levels canbe observed in DM16 group

with an average of 147.8 mg/mL. Meanwhile, the lowest fasting glucose levels found in N16

group with an average of 79.6 mg/mL. Plasma insulin levels observed ranged from 4.664 to

40.220 (ng/mL). The highest plasma insulin levels canbe observed in DM16 group with an

average of 40.220 ng/mL. Meanwhile, the lowest plasma insulin levels obtained in N8 group

with an average of 4.664 ng/mL. HOMA-IR values observed ranged from 0.462 to 3.789. The

highest HOMA-IR values can be observedin DM16 group with an average of 3.789. Meanwhile,

the lowest value of HOMA-IR wasfound in N16 group with an average of 0.462.

Ox-LDL levels observed ranged from 1.347 to 34.049 (ng/mL). The highest Ox-LDL levels can

be observed in DM16 group with an average of 34.049 ng/mL. Meanwhile, the lowest Ox-LDL

levels obtained at N8 group with an average of 1.347 ng/mL. Anova test showed that oxLDL


level were significantly (p<0.050) influenced by time series factor, darapladib treatment and

interaction of time series factor with treatment darabladib factor.

LysoPC levels observed ranged from 28.427 to 266.677 (ng/g aorta). The highest LysoPC levels

can be observed in N8 group with an average of 266.677 ng/g aorta. Meanwhile, the lowest

LysoPC levels obtained in DMDP16 group with an average of 28.427 (ng/g aorta). Anova test

showed that LysoPC level were significantly (p<0.050) influenced by time series factor,

darapladib treatment and interaction of time series with treatment darabladib factor.

PAF levels observed ranged from 0.726 to 1.963 (ng/mL). The highest PAF levels can be

observed in DM16 group with an average of 1.963 ng/mL. Meanwhile, the lowest PAF levels

found in N8 group with an average of 0.726 ng/mL. Anova test showed that PAF level were

significantly (p<0.050) influenced by time series factor, darapladib treatment and interaction of

time series with darabladib treatment factor.

IL-6 expression observed ranged from 634.60552 to 792.131 (AU). The highest IL-6 expression

can be observed in DM16 group with an average of 792.131 AU. Meanwhile, the lowest

expression of IL-6 was found in the DMDP8 group with an average of 634.60552 AU. ANOVA

test showed that the expression of IL-6 were significantly (p <0.050) affected by darapladib

treatment factor. Meanwhile, time series factor and interaction of time series with darapladib

factor didn’t significantly (p>0.050) influenced IL-6 expression. Number of foam cells observed

ranged from 80 to 138.8 (cell). The highest number of foam cells can be observed in DM16

group with an average of 138.8 cells. Meanwhile, the lowest number of foam cells found in N8

group with an average of 80 cells. ANOVA test showed that the number of foam cells was

significantly (p <0.050) affected by darapladib administration factor. Meanwhile, time series


factor and interaction of time series factor anddarapladib treatment didn’t significantly (p>0.050)

influenced the amount of foam cell.

4. Discussion

High-Fat diet (HFD) can increase the incident of dyslipidaemia and insulin resistance that

canlead to the increase of blood sugar level[16]. In the DM model, the increasing levels of Total

Cholesterol (TC) and Low-Density Lipoprotein (LDL) and decreasing levels of high-

densitylipoprotein(HDL) were observed. Dyslipidaemia that occur in this study lead to insulin

resistance. Insulin receptor on all cells can be decrease in the event of system downregulation

due to the high content of glucose in the blood. If these events occur continuously and

chronically, insulin receptor desensitization will be happening. It leads to a higher need of insulin

in blood plasma to incorporate glucose into cell tissue. In this research, an increase in plasma

insulin levels in the DM8 and DM16 groups occurred. That conditions may increase the

incidence of insulin resistance. By using HOMA-IR Rat formula it’s found that insulin resistance

occurred in DM16 and DM8 groups (CI 95%; sensitivity 83.87%; specificity 80.56%). DM8

group has shown an increase in HOMA-IR values compared to other groups, but has not been

able to reach the cut-off value. This shows that DM8 group is almost in insulin resistance state or

pre-diabetic condition. Abnormalities in lipoprotein metabolism are also associated with type 2

diabetes and trigger the development of atherosclerosis[17]. Glucose and lipid metabolism

disorders are synergistic in the developmentof atherosclerosis[18]. In atherosclerosis, vascular

damage occurs as a result of inflammation and oxidative stress [4,5]. Metabolic disorders

associated with type 2 diabetes such as hyperglycaemia, the formation of AGEs, increased FFA

and lipoprotein abnormalities can induce vascular inflammation and then promote atherosclerosis

progressivity[19].


Lp-PLA2 is an enzyme that is able to catalyze many important biological reactions and allegedly

played an important role in the pathogenesis of atherosclerosis. Expression of Lp-PLA2 in this

study tended to increase in type 2 diabetes condition and tends to decrease overtime increment.

This scheme shows the role of Lp-PLA2 in the early stages ofatherosclerosis. Darapladib

administration in this study had no significant effect on the expression of Lp-PLA2. The

increasing of Lp-PLA2 expression is associated with oxLDLlevels. A study shows that oxLDL

plays an important role in the pathogenesis of atherosclerosis and it is easy to form and increase

in T2DM state[19]. OxLDL level in this study is significantly influenced by the time series

factor, darapladib treatment factor and also the interaction of time series and treatment

darapladib factor. An increasing of oxLDL levels occurred in the development of adipocytes,

insulin resistance, and type 2 diabetes[20].

LysoPC, the main products of Lp-PLA2 activity, is the proatherogenic property of Ox-LDL

which can induce the synthesis of proteoglycans and play an important role in the thickening of

the intimal layer of the endothelium [21]. LysoPC in this study is significantly influenced by the

time series factor, treatment darapladib factor and interaction of time series with treatment

darapladib factor. The increased of Lp-PLA2 activity are also increase LysoPClevels that are

cytotoxic on blood vessels and smooth muscle cells [22]. Based on preclinical studies, darapladib

is proven to reduce atheroma which has LysoPC and some expression of multiple genes related

to macrophage and T lymphocyte function in large enough quantities to the plaque and necrotic

core area [23]. Research conducted by Melissa et al in 2012 stated that the increased levels of

LysoPC occur at the onset of the Type 2 diabetes mellitus and obesity condition [24].

Lp-PLA2 has a role as an anti-inflammatory through its ability to hydrolyze oxPC and PAF

(Platelet Activating Factor) that are pro-inflammatory and pro-thrombotic. PAF is a


potentinflammatory agent that has a very important role in the atherosclerosis formation[25]. In

thisstudy, PAF levels were significantly (p <0.050) influenced by time series factor, darapladib

treatment factor and interaction of time series with darapladib treatment factor. Previous study

explained, darapladib and rilapladib can inhibit PAF receptor, therefor reducing the biological

activity of PAF, as well as interfere with the biosynthesis of PAF [26].

IL-6 is one of pro-inflammatory cytokine that is involved in atherogenesis. IL-6 in type 2

diabetes mellitus condition can increases the basal glucose uptake, alter insulin sensitivity,

improve adhesion molecule expression by endothelial cells, have pro-coagulant effects on

platelets and increase fibrinogen release by the liver. In addition, IL-6 is also capable of blocking

the action of lipoprotein lipase and stimulate the process of lipolysis[27]. In this study,

expression of IL-6 were significantly (p <0.050) affected by darapladib treatment.While the time

series factor and the interaction of time series and treatment darapladib factor does not

significantly affect the expression of IL-6 (p> 0.050). Increased expression of Lp-PLA2 followed

by increased expression of cytokines IL-6 in type 2 diabetes. OxLDL stimulate macrophages to

phagocyte lipid which would then form a foam cell that signed the ongoing process of

atherogenesis [28]. This study shows that the number of foam cells was significantly (p <0.050)

affected by darapladib treatment. While the time series factor as well as the interaction of time

series and treatment darapladib factor does not significantly affect the amount of foam cell (p>

0.050) The formation of foam cells induced glucose indicates that glucose uptake is required by

cells, namely D-glucose (which is taken up by the cells) but not L-glucose (which can’t be

attached to the cell membrane) which then could induce macrophage oxidative stress[29].


5. Conclusion

Darapladip is Lp-PLA2 inhibitor, it is lipophilic and has a stupendous membrane permeability.

Recent studies explained that darapladip showed significant result in inhibiting atherosclerosis

process, whether by in vitro or in vivo test[7-9]. Darapladib were significantly affected tissue ox-

LDL level, plasma PAF, IL-6 expression and number of foamcells.

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