In vivo Evaluation for the Anticoagulant Activity of Dipyridamole Matrix  Tablets

 

Ashwini Gawade1*, Sanjay Boldhane2, Anil Pawar1, Rohini Pujari1, Ashwin Kuchekar1

1School of Health Sciences and Technology, Dr. Vishwanath Karad,

MIT World Peace University, Kothrud, Pune - 411038, Maharashtra, India.

2Development Micro Labs Limited, Micro Advanced Research Centre (Marc).2, No. 58/3, Kudlu Village,

Anekal Taluk, Singasandra Post Bangalore - 560068, Karnataka India.

*Corresponding Author E-mail: ashwinigawade890@gmail.com

 

ABSTRACT:

Dipyridamole (DYP) is potent drug that prevents the thromboembolic risk. It has been clinically used for chronic treatment of angina pectoris treatment and during the valve replacement. heart valve replacement and long-term angina pectoris treatment and is well absorbed in the stomach with BCS class II drug and low oral bioavailability. The present research investigation was focused on the formulation of matrix tablets of Dipyridamole cocrystals and the evaluation of In vivo anticoagulant activity. The results of the study showed that the formulated matrix tablets of dipyridamole cocrystals showed improved efficacy in comparison with the plain drug by enhancing the pre-compression parameters such as bulk density, tap density, Carr's index, angle of repose and Hausner's ratio and post-compression parameters like thickness and weight variation, hardness and friability, In vitro dissolution parameters. The improved efficacy was confirmed by improvement in the pharmacodynamic parameters such as cutaneous bleeding time and clotting time indicative of enhanced bioavailability of dipyridamole. Thus, it can be concluded that the dipyridamole matrix tablets prove to be more effective in producing the anticoagulant effect in clinical practice as compared to the plain drug resulting in more patient compliance.

 

KEYWORDS: Dipyridamole, Cocrystals, Anticoagulant, Matrix, Cutaneous bleeding, Clotting time.

 

 


INTRODUCTION: 

Dipyridamole (DYP) is a thromboembolic risk preventive drug that has been used clinically for platelet- mediated thrombotic and antiplatelet therapy disease for heart valve replacement and long-term angina pectoris treatment1,2.

 

DYP produces anticoagulant activity through various pathways such as platelet cAMP-phosphodiesterase inhibition, enhancing of inhibition of platelet function by adenosine by blocking its reuptake by blood and vascular cells; degradation of adenosine and enhancement of biosynthesis and antiaggregatory activity of PGI2. Unlike other anticoagulants, it doesn’t produce severe adverse effects, which is an added advantage of it.

 

DYP is a BCS class II drug i.e., low solubility and high permeability class. It is insoluble in water. It has good solubility in acidic pH but at alkaline pH, it is practically insoluble. DYP shows a narrow range of absorption and is absorbed through the stomach primarily. Its oral bioavailability ranges between 38-65% and has a short half-life of 45mins3.

 

The main aim of any drug delivery system is to make a therapeutic amount of the drug available at the appropriate site in the body to promptly achieve and then maintain the desired concentration of the drug4,5. Drug delivery through oral route is the most commonly used route of drug administration. It is used for systemic delivery through several pharmaceutical products in various dosage forms. In chronic therapy for the treatment of chronic diseases, conventional formulations are needed to be frequently administered in multiple dosage regimens and hence exhibits several untoward effects6-8. Therefore, to overcome the shortcomings associated with multiple dosage forms, the controlled or sustained release tablets have been developed in past9-11.

The attempt is made to formulate the matrix tablet dosage form of dipyridamole cocrystals to improve the rate of dissolution, absorption, bioavailability and efficacy of the DEM and to carry out its In vivo anticoagulant activity to confirm the same.

 

MATERIALS AND METHODS:

Materials:

Dipyridamole was obtained as a gift sample from Microlab research lab Bangalore India. Dipyridamole cocrystals are prepared in-house. Microcrystalline cellulose was obtained as a gift sample from Colorcorn Asia Pvt. Ltd., Goa, India. Crosspovidone was received as a gift sample from Concept Pharmaceutical Pvt. Ltd., Aurangabad, India. All other chemicals and reagents are of analytical grades.

 

Animals:

For acute oral toxicity studies female Swiss albino mice (22-25g) were used and Wistar rats (150-200g) were used for In vivo anticoagulant activity. They were procured from the National Toxicology Centre, Pune. Animals were housed in polypropylene cages (38cm × 23cm × 10cm) under standard laboratory conditions at 25±2°C temperature, 60±5% RH and 24°C; 12:12h dark/light cycle with free access to standard pelleted diet (Pranav Agro Industries Ltd., Sangli, India) and water ad libitum. Animals were fasted overnight on the day of study. Proper care and maintenance of the animals were undertaken following the guidelines of the Committee for Prevention, Control and Supervision of Experimental Animals, Govt. of India.

 

Ethical clearance:

All the studies involving animal experiments were carried out in accordance with the experimental protocols approved by the Institutional Animal Ethics Committee of School of Pharmacy, Dr. Vishwanath Karad, MIT WPU, M.S. India (Protocol No. MIP/IAEC/2019-20/M2/01).

 

Methods:

Preparation of DYP cocrystals:

Liquid assisted grinding method involves the addition of a solvent, typically in a very small amount approximately 5ml, to the dry solids prior to the initiation of milling. Solvents such as ethanol and acetone have a catalytic role in assisting cocrystal formation and should persist for the duration of the grinding process. More efficient cocrystal formation is suggested for liquid-assisted grinding methods than with the kneading method. Dipyridamole cocrystals synthesis was performed using liquid assisted grinding technique. Screening of formation of DYP cocrystals was performed by various coformers in an optimal molar ratio (1:1, 1:2 and 1:3). A mixture of 1:1 dipyridamole and Tartaric acid was ground in mortar and pestle for 30 min. with the addition of ethanol dropwise so that damp mass was formed. The mass is then dried in a desiccator and passed through 60 mesh sieves12,13.

 

Formulation of dipyridamole cocrystal matrix tablets:

DYP cocrystals matrix tablets were prepared by direct compression method using HPMC K4M was used as a binder. Microcrystalline cellulose (MCC) and lactose monohydrate were used as diluents and Talc is used as glidant. DYP cocrystals equivalent to 75mg of DYP and all the excipients except magnesium stearate were taken in mortar. Then powder blend was mixed well for 15 to 30min. The blends were passed through the # 80 sieve. Lubrication was done using magnesium stearate. The final blend was compressed on Remake Mini Press II D Tooling 8 station compression machine equipped with concave punches to a weight of 280mg/tablet. The compressed tablets were evaluated for pre- and post-compression parameters14,15.

 

Physical evaluation of IR tablets of DEM cocrystals:

The prepared blends were evaluated for pre-compression parameters like bulk density, tap density, Carr's index, angle of repose, and Hausner's ratio and post-compression parameters like thickness and weight variation, hardness and friability, In vitro dissolution study16,17.

 

Stability studies of tablets:

Studies were carried out for 45 days for the optimized batches of DYP cocrystal matrix tablets at a temperature of 40±2°C/ RH 75±5%18.

 

Acute oral toxicity study of tablets:

The acute oral toxicity test was performed using the Acute Toxicity Class (ATC) method according to the Organization of Economic Co-operation and Development (OECD) guideline 423. Female Swiss albino mice were weighed and randomly divided into two groups (6mice/group). The first group served as the test group and was administered orally with the suspension of matrix tablets of Dipyridamole cocrystals in distilled water at a single dose of 2000mg/kg body weight. The second group served the control group and received only distilled water at a volume of 10ml/kg body weight. Observations were noted at 1, 2, 4 and 6 h after administration of test substance and recorded systematically. The visual observations like changes in the skin and fur, eyes and mucous membranes were recorded. Further, respiratory, circulatory, autonomic and central nervous systems, as well as somatomotor activity and behavioral patterns, were observed. The number of survivors was recorded initially after 48h and then a further 14days with once-a-day observation19,20.

Evaluation of In vivo anticoagulant activity of dipyridamole cocrystal matrix tablets using cutaneous bleeding time model:

Wistar rats were divided into three groups (n=6). Group I was considered as the control group and was administered with distilled water at a dose of 1ml/kg, group II was administered with the suspension of plain DYP in distilled water at a dose of 50mg/kg orally while group III was administered with the suspension of powdered tablets from an optimized batch of matrix tablets of DYP cocrystals at the dose of 50mg/kg orally. After two hours, the rats of all groups were anesthetized by intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10mg/kg) and placed individually in a plastic rat holder with several openings from one of which the animal tail was taken out. The tail was cleaned properly with water-wetted cotton. Then incision (10mm long and 1.5mm deep) was made with a scalpel between 8 and 9cm from the tip of the tail (Figure 1). The bleeding time was assessed at intervals of 15s and compared with the control group21-23.

 

Evaluation of In vivo anticoagulant activity of dipyridamole cocrystal matrix tablets using clotting time model:

Wistar rats were divided into three groups (n=6). Group I was considered as the control group and was administered with distilled water at a dose of 1ml/kg, group II was administered with the suspension of plain DYP in distilled water at a dose of 50mg/kg orally while group III was administered with the suspension of powdered tablets from the optimized batch of Matrix tablets of DYP cocrystals at the dose of 50mg/kg orally. After one hour, the rats of all groups were anesthetized by intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10mg/kg) and blood was withdrawn into the capillary tube through retro-orbital route to fill 3/4th of the capillary tube. Clot formation was checked after every 30 seconds by breaking a piece of a capillary tube and slightly stretching apart the two ends of the broken capillary tube. The time at which a thread- like structure called fibrin extends between the two ends of the capillary tube is noted down as clotting time24,25.

 

Statistical analysis:

The results were expressed as Mean+SEM (n=6). Comparison between the groups was made by one-way analysis of variance (ANOVA) followed by Tukey’s Kramer Multiple Comparison test using Instat Graph Pad software (version-3).

 

RESULTS AND DISCUSSION:

Evaluation of pre-compression parameters of dipyridamole cocrystal matrix tablets:

Bulk density was found in the range of 0.29±0.012 to 0.363±0.014g/cm3, tapped density between 0.346±0.014 and 0.514±0.023g/cm3, using the above two density data, Hausner's Ratio and Compressibility Index (CI) were calculated. The powder blends of all formulations with Hausner's ratio <1.25 indicated better flow properties. The compressibility index was found to be between 0.01 and 0.22% and the compressibility and flowability data indicated an excellent flowability of all powder blends. The better flowability of all powder blends was also evidenced from the angle of repose (in the range of 25.66±2.1 to 30.45±1.49) which is below 40 θº, indicating good flowability as per the previously documented studies26,27.

 

Evaluation of post-compression parameters of dipyridamole cocrystal matrix tablets:

%Drug content of the tablets was assayed spectrophotometrically at 282nm. The DYP quantity in different trial batches varied between 93.4% and 99.90%. Tablet weights varied between 262 and 285mg, hardness between 5±0.28 and 7±0.45kg/cm2 (average kg/cm2), thickness between 4.29±0.03 and 5.54 ±0.03mm (average 4.5mm) and friability ranged from 0.04% and 0.14% (average 0.9%) 28,29.

 

In-vitro dissolution study of dipyridamole cocrystal matrix tablets:

The in vitro release profile indicated that Batch (F4) was the most promising formulation as the extent of drug release from this formulation was high as compared to other formulations which are suitable for sustained release drug delivery system as per the literature30. The in vitro drug release studies in Stomach pH conditions were carried out in 0.1 N HCl (pH 1.2) for the first 4 hrs. and after in 6.8 phosphate buffer till 12hrs. The Batch F4 shows 101% release in 12hrs.

 

Stability study of dipyridamole cocrystal matrix tablets:

All physical parameters of the optimized batch of tablets were found to be within the standard range when kept under accelerated stability conditions during the stability studies indicating that the formulation showed good stability 3132.

 

Acute oral toxicity test of dipyridamole cocrystal matrix tablets:

The results showed that DYP cocrystals matrix tablets were found to be safe without any mortality and morbidity up to the dose of 2000mg/kg.

 

In vivo anticoagulant activity:

Effect of DYP and DYP cocrystals matrix tablets on bleeding time:

Untreated animals (control group) showed a mean bleeding time of 51secs while the animals treated with test drug suspensions showed comparatively increased mean bleeding time. However, as compared to plain DYP (mean bleeding time – 190 secs), the DYP tablets (mean bleeding time- 241 secs) exhibited more significant (P<0.001) efficacy in enhancing the bleeding time which correlated well with pharmacokinetic data (Figure 1). Hence, we could conclude that the developed formulation exhibited better anticoagulation activity than plain drug suspension by improving the oral bioavailability of DYP33,34.

 

Figure 1: Comparative bleeding time in control untreated, DYP treated and DYP cocrystals matrix tablet treated group

Results are expressed as mean + SEM. Comparison between the groups was made by one-way analysis of variance (ANOVA) followed by Tukey’s test*, #-P<0.05, **, ##-P<0.01, ***, ###-P<0.001; *- DYP and DYP cocrystals Matrix tablets treated groups against control untreated group; #- DYP tablet treated group against DYP treated group.

 

Effect of DYP and DYP cocrystals matrix tablets on clotting time:

The clotting time was assessed after 2 hours of treatments (DYP cocrystal matrix tablets and plain drug suspension). Compared with the control group, all treatments significantly (P<0.001) prolonged the clotting time. Untreated animals (control group) showed a mean clotting time of 1.49 mins while the animals treated with test drug suspensions showed comparatively increased mean clotting time. However, as compared to plain DYP (mean clotting time - 2.71mins), the DYP Tablets (mean clotting time - 3.8mins) exhibited more significant (P<0.01) efficacy in enhancing the bleeding time which correlated well with pharmacokinetic data (Figure 2). Hence, we could conclude that the developed formulation exhibited better anticoagulation activity than plain drug suspension by improving the oral bioavailability of DYP35-37.

 

Figure 2: Comparative clotting time in control untreated, DYP treated and DYP cocrystals matrix tablet treated group

Results were expressed as mean + SEM. Comparison between the groups was made by one-way analysis of variance (ANOVA) followed by Tukey’s test *, #-P<0.05, **, ##-P<0.01, ***, ###-P<0.001; *- DYP and DYP Cocrystal Matrix Tablets treated groups against control untreated group; #- DYP Cocrystal Matrix Tablets against DYP treated group

 

CONCLUSION:

The present study concluded that the pH-dependent solubility and short absorption window of DYP was overcome by formulating it in cocrystal form and converting it into IR tablet formulation. The formulation showed significant improvement in the rate of dissolution, absorption and bioavailability of DYP, which in turn improved its In vivo anticoagulant activity by virtue of enhanced cutaneous bleeding time and clotting time. Thus, the improvement in pharmacokinetic and pharmacodynamic parameters of DYP would enhance the efficacy and increase patient compliance.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

This study was supported by the School of Health Sciences and Technology, Dr. Vishwanath Karad, MIT, WPU, Kothrud, Pune- 411038, Maharashtra, India by providing the required experimental facilities.

 

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Received on 03.09.2021            Modified on 10.03.2022

Accepted on 23.07.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(7):3104-3108.

DOI: 10.52711/0974-360X.2023.00510