Design and Development of Novel Synergistic Formulation of Pravastatin and Aspirin for the Treatment of Atherosclerosis and its Evaluation

 

Pramod S. Salve*, Nikhil Bali, Navleen Saini

University Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University Campus, Mahatma Jyotiba Fuley Shaikshanik Parisar, Amravati Road, Nagpur-440033, (M.S), India.

*Corresponding Author E-mail: pramodsalve77@yahoo.com

 

 

 

Received on 30.04.2015       Accepted on 17.05.2015     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech. 2016; 6(2): 61-69.

DOI: 10.5958/2231-5713.2016.00009.X

 

 

ABSTRACT:

Coronary artery diseases (CAD) represent a condition in which the blood supply to the heart muscle is partially or completely blocked. In atherosclerosis, the hardening of arteries occurs with decreasing elasticity of the arteries. For management of disease, cholesterol lowering agents like Pravastatin and anti-platelet agent like aspirin are commonly used. Various studies have shown that these agents when given in combination results in synergistic pharmacological activity, however they are not chemically compatible. In present work, formulation approaches utilizes principles of pelletization and coating technologies to form a stable drug delivery system. The enteric coating of the core aspirin pellets was done using Eudragit L 100 (6%) with polyethylene glycol 6000 (0.6%) till 2.5, 5 and 7.5 % weight gain was obtained. A barrier coating on enteric coated pellets was applied using hydroxyl propyl methyl cellulose (HPMC) 15 cps solution. For the layering of Pravastatin over the barrier coated pellets, Pravastatin was dispersed in 2.5% HPMC solution.  The coating solution was sprayed over the pellets till a dose strength equivalent to 10 mg of Pravastatin per 250 mg of pellets was obtained. The optimized pellets were subjected to evaluation of drug content, bulk density, crushing strength, friability, size distribution, packing ability and in-vitro drug release, stability studies and in-vivo performance using trition X 100 induce hyperlipidaemia rat model. The developed formulation showed an effective cholesterol lowering effect as compared to standard marked formulation of Pravastatin. It indicates the development of stable combination formulation in which aspirin forms the enteric coated sustained release core and that of Pravastatin fast releasing outer layers of the pellets.

 

KEY WORDS: Atherosclerosis, Pravastatin, aspirin, pelletization, trition X 100 induce hyperlipidaemia rat model.

 

 


 

INTRODUCTION:

In the United States, cardiovascular disease is leading cause of death among both sexes, and coronary artery disease is the most common type of cardiovascular disease, occurring in about 5 to 9 % of people aged 20 and older. On average, men develop it about 10 years earlier than women, because until menopause, women are protected from disease by high levels of estrogen. However, after menopause, it becomes more common among women [1, 2]. Arteriosclerosis, hardening of arteries, is a general term for several diseases in which wall of an artery becomes thicker and less elastic. Atherosclerosis develops when levels of cholesterol in the blood injure artery's lining, causing an inflammatory reaction and enabling cholesterol and other fatty materials to accumulate there [2].

 

Pravastatin, is a competitive inhibitor of 3-hydroxy-3-methyl CoA  reductase an enzyme that catalyses the rate limiting step in cholesterol biosynthesis resulting in up regulation of low density lipoproteins (LDL) receptors in response to the decrease in intracellular cholesterol. It is administered in its active form as a sodium salt and 34% of oral dose is absorbed. Peak plasma concentration of drug is reached 1-1.5 hours after dosing and elimination half-life is approximately 77 hours [11, 12]. Aspirin, a non-steroidal anti-inflammatory agent also inhibits platelet activation and aggregation for the lifetime of the platelet by irreversibly inhibiting prostaglandin cyclooxygenase. Peak plasma levels of aspirin occur within 1-2 hours of dosing and the plasma half-life is approximately 6 hours [12].

 

Today, the scenario of pharmaceutical drug delivery is changing from conventional dosage form to new drug delivery system with main objective of patient compliance. Combination formulation of two or more active ingredients, are being designed to combat many clinical conditions like cardiovascular diseases [9, 10]. Since patient with coronary artery diseases have to take multiple medications for long period of time. Thus today pharmaceutical industries are diverting more towards developing the combination formulation for diseases with multiple manifestations [6].

 

It was observed that when these two agents were given in combination, synergistic pharmacological activity results [6, 7]. The main complication encountered with these two drugs was incompatibility of aspirin with statin especially Pravastatin, a hydrophilic statin with limited side effects compared to other statins. Aspirin is acidic compound and Pravastatin a very acid labile basic compound when both are formulated together aspirin get hydrolyzed with degradation of Pravastatin [6, 12]. For developing a stable combination formulation of Pravastatin with aspirin, pelletization and coating technologies are explored.

 

Pellets offer better statistical assurance of complete drug release as the risk of dose dumping is minimized. High local concentration of drug, which may inherently be irritating, can be avoided. As pellets uniformly distributed throughout the gastrointestinal tract, they invariably maximize drug absorption, reduce peak plasma fluctuations and minimize potential side effects without appreciably lowering bioavailability [3, 4]. Control release pellets enable a smoother absorption sorption profile. Combined delivery of two or more bioactive agent, which may or may not be chemically compatible at the same site or at different site within gastrointestinal tract is possible [5, 6].

 

The aim of present study is to develop stable combination formulation containing Pravastatin and aspirin for the management of CAD (atherosclerosis). The sustaining of aspirin release which needs multiple dosing and hence reducing dosing intervals is envisaged. To achieve the above objectives, drug is incorporated in core pellets and another as coated layer with a barrier coating with hydrophilic polymer hydroxyl propyl methyl cellulose.

 

MATERIALS:

Pravastatin sodium was obtained as gratis sample from Biocon Pvt. Ltd (India). Aspirin was obtained from Zim Laboratories (India). Microcrystalline cellulose was obtained from Chemfields Ltd. (India). Hydroxy propyl methyl cellulose was obtained from Colorcon (India). Eudragit L100 was procured from Rohm Pharma (India). Polyethylene glycol 6000 was purchased from S. D. Fine Chemicals (India).   

 

EXPERIMENTAL METHODS:

A] Preparation of enteric coated Aspirin pellets

Three formulation batches A1, A2 and A3 of core aspirin pellets containing aspirin and microcrystalline cellulose (MCC) PH 101 in ratio of 75:25, 50:50, and 25:75 respectively were prepared by extrusion spheronization technique at the speed of 1500 rpm for 10 minutes using water as binder (Table 1). No significant changes were observed in the physical properties of core pellets prepared with different ratios of drug: MCC PH 101. Hence 75:25 drug:MCC  PH 101  ratio was optimized to reduce dose size.  The pellets of 10-14 # were selected for coating as small pellets create agglomeration problem while large pellets are not suitable for animal ingestion during in-vivo study. The 6% w/v solution of Eudragit L100 with 10 %w/v PEG 6000 in isopropyl alcohol having 44 cps viscosity was used for coating core aspirin pellets.

 

Table.1. Composition of core aspirin pellets

Sr. No

Formulation batch

Microcrystalline

Cellulose PH 101 (% w/w)

Aspirin

(% w/w)

Binder

1

A1

25

75

Water

2

A2

50

50

Water

3

A3

75

25

Water

 

B] Preparation of Eudragit L100 coating solution

Weighed quantity of Eudragit L100 was dissolved in IPA and transferred PEG 6000 solution under stirring condition. Talc was dispersed in solution and stirring continued until uniform solution was obtained (Table 2). Coating process was done in conventional coating pan on batch size of 25 g. Coating solution at pressure of 20 psi was sprayed over cascading aspirin pellets rotating at speed of 30 rpm. The process continued till 2.5, 5 and 7.5 % coating weight gain was achieved.

 

Table. 2. Preparation of Eudragit L100 coating solution

Ingredients

Quantity (%)

Eudragit L100

6.0

Polyethylene glycol 6000 (PEG 6000)

0.6

Talc

1.0

Isopropyl alcohol (IPA)

85

Water

5.0

 

 

Barrier coating of enteric coated aspirin pellets

The coating procedure for aspirin pellets was followed for barrier coating using hydroxyl propyl methyl cellulose (HPMC) 15 cps as coating material. The composition of barrier coating solution is shown in table 3. As aspirin and Pravastatin are chemically incompatible (prone to chemical degradation), HPMC coating is used to prevent their direct physical contact in a single unit dosage form.

 

Table 3. Composition used for preparation of barrier coating solution

Ingredients

Quantity (%)

Function

HPMC 15 cps

2.5

Polymer for coating

Diethyl phthalate

0.5

Plasticizer

Titanium dioxide

0.5

Opacifier

Cetyl alcohol

0.5

Stabilizer

Talc

0.5

Lubricant

Dichloromethane

66

Solvent

 

Layering of pravastatin over precoated aspirin pellets

The pre-weighed quantity of pravastatin sodium was dispersed in 2.5 %w/v HPMC solution. The dispersed drug solution was coated over precoated aspirin pellets with barrier coating to prevent contact between pravastatin and aspirin. The coating solution was sprayed over pellets until the pellets gain the desirable weight equivalent to 10 mg of pravastatin dose in 250 mg of pellets.

 

Evaluation of Aspirin pellets

1] In-vitro drug release study of combination formulation                                                                           

The in-vitro release of drug from pellets was performed in triplicate using by USP dissolution test apparatus type I (basket method) using 900 mL of pH 1.2 phosphate buffer for 2 hrs and pH 6.8 phosphate buffer for 6-8 hrs. The sample size was taken is equivalent to 10 mg of pravastatin and 150 mg of aspirin. The filtered samples of pravastatin and aspirin were analyzed spectrophotometrically at 237 nm and 265 nm respectively.

 

2] Surface Topography study

Surface topography of coated pellets from optimized formulation was studied using scanning electron microscopy (SEM).  The samples were sputtered with gold for 15 min. (JEOL JFC- 1100E ion sputtering device) before characterization with SEM.  The electric current used for sputtering was 10 mA and the sputtering gas was argon.

 

3] Assessment of Antihyperlipidemic activity of combination formulation 

Experimental Animals

Sprague Dawley rats of either sex (200-250 g) were used. Animals were housed in well ventilated standard condition of temperature (25 ± 1 °C) and kept them for 18 hrs fasting with free access to water before study.

 

Trition X 100 induced hyperlipidemic rat model  

Hyperlipidemia in rats was induced by surfactant triton X 100. The systemic administration of surfactant triton to rats or mice results in a biphasic elevation of plasma cholesterol and triglycerides. It increases the serum cholesterol level sharply 2-3 times after 24 hrs (Phase-I). The hypercholesterolemia decreases nearly to control levels within next 24 hrs (Phase-II). The test drugs employed or the solvent for control are administered simultaneously with triton injection for 22 hrs thereafter. Serum cholesterol analysis were made 6, 24, 48 hrs after triton injection [8, 9].

 

Table. 4. Treatment groups

Groups

Treatment and route of administration

Number of animals (SD rats)

Dose

Non hyperlipidemic control

Vehicle (Glycerin) (oral)

05

0.5-1 mL

Hyperlipidemic control

Triton (s.c)

05

200 mg/kg

Hyperlipidemic animals treated with market formulation

Triton (S.C) + Market formulation (Oral)

05

Pravastatin 10 mg/kg

Hyperlipidemic animals treated with combination formulation

Triton (S.C) + Combination formulation (Oral)

05

245 mg pellets equivalent to 10 mg pravastatin

                                                      

Results and Discussion

A] Preparation of core pellets

Effect of moisture level on physical properties of pellets

Table. 6. Effect of moisture level on physical properties of pellets

Sr. No

Material

Moisture (%)

Size distribution (#)

Shape

Yield (%)

1

Drug-MCC

25

14- 18

Spherical

> 60

2

Drug-MCC

50

14- 18

Spherical

< 90

3

Drug-MCC

75

10- 18 (wide)

Dumble + Spherical

< 90

 

 


Effect of spheronization speed and time on physical properties pellets

Table. 7. Effect of spheronization speed and time on physical properties of pellets

Sr. No.

Material

Spheronization speed (rpm)

Spheronization Time (min)

Shape of pellets

Size of pellets (mm)

1

Drug-MCC

500

10

Dumble

Above 2.5

2

Drug-MCC

500

15

Dumble

Above 2.5

3

Drug-MCC

750

10

Dumble

Above 2

4

Drug-MCC

750

15

Dumble + Spherical

Above 2

5

Drug-MCC

1000

10

Dumble + Spherical

Above 2

6

Drug-MCC

1000

15

Spherical

1- 2

7

Drug-MCC

1500

10

Spherical

1-2

8

Drug-MCC

1500

15

Spherical

1-2

 


B] Enteric coating of core aspirin pellets

Effect of different coating levels on drug release.

The comparative release profile of 5% and 7.5% of aspirin in pH 1.2 and 7.5 phosphate buffer is shown in figure 1.

 

 

Fig.1. Comparative drug release profile of aspirin at different coating levels

 

 

In-vitro drug release profile of optimized enteric coated pellets

 

Figure. 2. In-vitro drug release curve of aspirin

 

In-vitro  dissolution profile of optimized combined formulation

In-vitro drug release curve of pravastatin at pH 1.2 and aspirin at pH 6.8 in optimized formulation is shown in figure 3.

 

Figure. 3. In-vitro drug release curve of combination pellet formulation

 

Table 8 enlists correlation coefficient values calculated from kinetic models for combination formulation. The mechanism of drug release from coated pellets was determined by fitting the in-vitro release profiles with zero order, first order, Higuchi, Hixson-Crowell, Korsmeyer-Peppas kinetics models [17]. The highest correlations were observed with Korsmeyer-Peppas model. Korsmeyer-peppas equation gave higher value for the correlation coefficient as compared to other release kinetic models. The curve fitting of drug release data to peppas equation indicates that the drug release was due to diffusion and erosion through coated pellets.

 

 

Table. 8. Correlation coefficient values for combination formulation

Sr. No.

Release Kinetics model

Correlation coefficient

1

Zero order

0.997

2

First order

0.9958

3

Higuchi

0.9983

4

Hixson-crowell

0.9989

5

Korsmeyer-peppas

1.000

 

Fourier’s Transform Infra-Red (FT-IR) study

The FT-IR spectra of aspirin, pravastatin, aspirin + pravastatin, HPMC, HPMC + pravastatin are shown in figures 4, 5, 6, 7 and 8 respectively. The FT-IR peaks (in cm-1) and its functional groups are shown in table 9.

 

Table. 9. FT-IR peaks and functional groups

Sr. No.

Material

Peak cm-1

Characteristic functional group

1

Aspirin

800-650

1700

1600-1400

Ar-H

C=O

Aromatic ring

2

Pravastatin Sodium

4600-4000

3400-3000

3000-2500

1700

1300-1200

Aliphatic C-H

O-H

C-H

C=O

CH3CO-

3

HPMC

3400-3000

1600-1400

4600-4000

O-H

CH3CO-

Aliphatic CH-

 

 

Figure. 4. FTIR spectra of aspirin

 

Figure. 5. FTIR spectra of pravastatin sodium

 

 

Figure. 6. FTIR spectra of hydroxypropyl methyl cellulose

 

Figure. 7. FTIR spectra of mixture (aspirin: pravastatin) in 1:1 ratio

 

 

Figure. 8. FTIR spectra of Pravastatin: HPMC in 1:1 ratio

 

 

Differential Scanning Calorimetry (DSC) study

Differential scanning calorimetry thermo gram of aspirin, pravastatin sodium and 1:1 mixture of drugs are shown in figures 9, 10 and 11 respectively.

 

Figure. 9. DSC thermogram of aspirin

 

 

Figure. 10. DSC thermogram of pravastatin sodium

 


Figure. 11. DSC thermogram of pravastatin: aspirin (1:1) mixture

 

Surface Topography of Pellets

Morphological details of the coated pellets were observed under scanning electron microscopy (SEM). Photograph of coated intact pellets was shown in figure 12

 

Figure. 12. Surface Topography of pellets

 

 

 

 

 

 


Pharmacological screening of combination formulation for antihyperlipidemic activity

                       

Figure 13. Total Serum Cholesterol in rats

 

                       

Figure 14. Total Triglycerides level in rats

 

 

 

                      Figure 15. HDL cholesterol levels in rats

          

                                                            Figure 16. VLDL cholesterol levels in rats

 

                         

                                                                    Figure 17. LDL cholesterol levels in rats


The reduced cholesterol and triglycerides levels are shown in figures 13, 14, 16 and 17 respectively while raised HDL cholesterol levels are shown in figure 15.

 

DISCUSSION:

A] Effect of moisture level on physical properties of pellets

From table 10, it is observed that, the size and shape of pellets was found to depend on the amount of water added to form the damp mass before extrusion. The increase in amount of water increased the pellets diameter where as low amount of moisture resulted in reduction of the yield of pellets [18, 22, 25]. Thus, the amount of moisture was kept at 50 % to get desired size pellets with maximum yield.

 

B] Effect of spheronization speed and time on physical properties pellets

At the speed of 1500 rpm for 10 minute desired size pellets (1-2 mm) were obtained (15, 16, 20) hence 1500 rpm and 10 minute was selected as optimized speed and time for the formulation of the core pellets and the time period.

 

C] Effect of different coating levels on drug release

From figure 1 the result revealed that, 2.5 % coating weight gain failed to give enteric effect whereas at 5 % and 7.5 % coating levels, drug release in pH 1.2 buffer was less than 5 % and 3 % respectively. The 7.5 % coating level also resulted in sustaining the release of aspirin in pH 6.8 buffer for > 6 hrs and was selected as optimum coating level. Both the coating levels 5 % and 7.5 % had released < 5 % of aspirin in pH 1.2 buffer after 2 hrs, complying with the official requirement for enteric coated dosage formulations. Pellets with 5% coating levels released > 95 % of drug in later 4 hrs in pH 6.8 buffer whereas pellets with 7.5 % coating level only release 72.60 % in 4 hrs. After 6 hrs, 99 % aspirin was released from pellets coated up to 5% coating weight gain, whereas only 88.43% aspirin was released after 6 hrs from pellets coated up to 7.5 % weight gain. Hence, 7.5 % coating weight gain was selected as optimum coating level which provide the enteric effect and sustains the release of aspirin (19, 24, 26).

 

D] In-vitro drug release profile of optimized enteric coating batch

Figure 2 shows that, release of aspirin < 3% was observed in pH 1.2 buffer for first 2 hours complying with the official standard for enteric coated dosage form. In pH 6.8 phosphate buffer, formulation shown sustained release of aspirin, 88.43% release in later 6 hrs.

 

E] Fourier’s Transform Infra-Red (FT-IR) study 

From table 13 and figures 4, 5, 6, 7 and 8, it was concluded that FT-IR spectra of drug mixture shown that there was presence of interaction, while in optimized drug mixture with excipients no such changes in peaks were observed. 

 

F] Differential Scanning Calorimetry (DSC) study

The thermo gram of aspirin is shown in figure 9 indicates the sharp endothermic peak at 150 oC corresponding to its melting. The thermo gram of pravastatin is shown in figure 10 shows the endothermic peak at 142o C corresponding to its melting point. While in thermo gram of mixture shown in figure 11 the peak of aspirin shifted to 180 oC and that of pravastatin to    95 oC with one extra exothermic peak in thermo gram reveals the presence of interaction.

 

G] Pharmacological screening of combined formulation for antihyperlipidemic activity      

i) Effect of Triton on rats cholesterol levels

The subcutaneous injection of triton X-100 (200 mg/kg) significantly increased the serum cholesterol levels in rats up to 50 % within 24 hours than normal levels. After 24 hrs, it shown the decline in raised cholesterol levels, while in case of HDL levels, the serum cholesterol levels does not show significant rise as compared to normal HDL levels after triton administration.

 

ii) Effect of treatment of formulation on cholesterol levels

In order to check the antihyperlipidemic activity of combination formulation, the rats were treated with combination formulation and compared with the marketed formulation. After formulation administration the blood samples were collected after 6 hrs, 24 hrs and 48 hrs respectively and centrifuged (150x g, 10 minutes) for collection of serum and analyzed for total triglycerides and total cholesterol. The in-vivo study revealed that the increased levels of cholesterol by triton X-100 injection were declined to normal ranges following the treatment with formulation.

 

iii) Effect of treatment of formulation on the hyperlipidemic parameters

The results obtained from in-vivo study of formulation revealed that the raised cholesterol and triglycerides level with triton return to normal levels. While the formulation shown the positive rise in the HDL cholesterol levels with no effect of triton. From figures 13, 14, 15, 16 and 17, it reveals that formulation have significant antihyperlipidemic activity which reduced the raised cholesterol levels and raised HDL levels.

 

CONCLUSION:

In the present study, the combination formulation of pravastatin along with aspirin was developed for the treatment of coronary artery diseases by exploring the pelletization and coating technology. The core aspirin pellets were coated with 6 %w/v polymer solution of Eudragit L100, followed by protective coating with 2.5 %w/v solution of HPMC.  Pravastatin was then layered over protective layer by dispersing the drug in 2.5 %w/v HPMC 15 cps solution and coating to the desired dose strength. Thus by exploring the pelletization and coating technology a stable combination formulation was developed.

 

ACKNOWLEDGEMENT:

The authors would like to thank University Grant Commission (UGC), New Delhi for the financial assistance and authors would also like to thank, Head of the Department of Pharmaceutical Sciences, R.T.M Nagpur University, Nagpur, India for constant encouragement and support for the work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

ETHICAL APPROVALS:

The animal study was conducted according to the protocol approved by Institutional Animal Ethics Committee (IAEC) (CPCSEA-IAEC/Proposal No.11/22/1/2013) of Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, India.

 

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