INTRODUCTION:
Experimental evidence has shown that intravenous (i.v.) or intragastric (i.g.) administration of unfractionated heparin enhances gastric ulcer healing in rats1,2. However, anti-ulcer doses of heparin also produce an anticoagulant effect when the drug is given by I.V. injection. Interestingly, oral administration of unfractionated heparin increases ulcer healing without affecting the coagulation function of the animal2. It is traditionally known that oral administration of the large molecule of heparin is ineffective as an anticoagulant because of poor absorption in the gastrointestinal tract. It is possible that oral unfractionated heparin may be partially degraded in the gastrointestinal tract, such that fragments of unfractionated heparin may contribute to ulcer healing in the stomach when the drug is taken orally3.
In order to localize the heparin at site on the gastric epithelium for improving the efficacy of anti-ulcer activity a mucoadhesive microspheres composed of Carbopol 934P and Ethyl Cellulose have been developed.
MATERIALS AND METHODS:
Materials:
Heparin was a kind gift from M. Petitou (Sanofi, France), Carbopol 934P was a gift from BF Goodrich Co., Germany. Ethyl cellulose was purchased from CDH chemicals, New Delhi, India. All other reagents and chemicals used were of analytical grade.
Preparation of Microspheres:
Microspheres were prepared by a solvent evaporation method. The solvent system acetone/liquid paraffin was used. Agglomeration of microspheres was prevented by using 1% w/v Span80. Ethyl cellulose was used to form a matrix of microspheres and Carbopol 934P were chosen as a mucoadhesive polymer produce mucoadhesion (Table 1). Ethyl cellulose and Heparin was dissolved in acetone and weighed quantity of Carbopol 934P were dispersed in it. The total volume of acetone was 12 ml. This homogeneous final dispersion was cooled to 5°C and poured slowly with stirring (700 rpm) into 80 ml of liquid paraffin containing 1% w/v span 80, which was previously also cooled to 5°C. The obtained emulsion was stirred at 40°C for 40 min. The suspension of microspheres in liquid paraffin was filtered and microspheres were washed by n-hexane and dried in vacuum at room temperature overnight.
Scanning electron microscopy:
Scanning electron photomicrograph of Heparin loaded mucoadhesive microspheres were taken. A small amount of microspheres was spread on glass stub. Afterwards, the stub containing the sample was placed in the scanning electron microscope (JSM 5610 LV SEM, JEOL, Datum Ltd, Tokyo, Japan) chamber. Scanning electron photomicrograph was taken at the acceleration voltage of 20 KV, chamber pressure of 0.6 mm Hg, at different magnification. The photomicrograph of batch F6 is depicted in Fig. 1.
Particle size measurement:
The prepared microspheres were sized by using a Malvern 2600 Laser Diffraction Spectrometer. The size of the microspheres was determined in n-hexane as a non-dissolving dispersion medium and the particles were suspended mechanically by magnetic stirring during the measurement.
Table 1: Formulation composition of mucoadhesive microspheres of Heparin
|
S. No |
Ethyl Cellulose (% w/v) |
Carbopol 934P (% w/v) |
Heparin (mg) |
|
1 |
2.5 |
1.0 |
500 |
|
2 |
5.0 |
1.0 |
500 |
|
3 |
7.5 |
1.0 |
500 |
|
4 |
5 |
0.5 |
500 |
|
5 |
5 |
1.5 |
500 |
|
6 |
5 |
2 |
500 |
Fig.1.SEM photograph of Heparin loaded mucoadhesive microspheres
Determination of drug encapsulation efficiency:
To determine the total drug content of microspheres a known amount of microspheres were ground to fine powder. Accurately weighed (50mg) grounded powder of microspheres were soaked in 50 ml of distilled water and sonicated using probe sonicator for 2 h. The whole solution was centrifuged using a tabletop centrifuge to remove the polymeric debris. Then the polymeric debris was washed twice with fresh solvent (water) to extract any adhered drug. The clear supernatant solution was filtrated through a 0.45 μm syringe filter then analyzed for Heparin content colorimetrically by following method. Aliquots (500 mL) of each aqueous sample were reacted with 4.5mL of the Azure II solution (0.01mg/mL) at room temperature and assayed in triplicate at 530 nm by UV-VIS spectroscopy4.
In vitro evaluation of mucoadhesiveness:5
A strip of goat intestinal mucosa was mounted on a glass slide and accurately weighed mucoadhesive microspheres in dispersion form was placed on the mucosa of the intestine. This glass slide was incubated for 15 min in a desiccators at 90% relative humidity to allow the polymer to interact with the membrane and finally placed in the cell that was attached to the outer assembly at an angle 450. 0.1.HCL (pH 1.2), previously warmed to 37 ± 0.50 C, was circulated to the cell over the microspheres and membrane at the rate of 1 ml/min with the help of pump. Washings were collected at different time intervals and microspheres were separated by centrifugation followed by drying at 50 C. The weight of microspheres washed out was taken and percentage mucoadhesion was calculated by
where Wa = weight of microspheres applied; W1 = weight of microspheres leached out.
In Vitro Drug Release Studies:
In vitro Heparin release rate was tested using basket apparatus on a dissolution tester (Model ZRS-8, Tianjin University Precision Instrument Factory). The test conditions were as follows: A sample of microspheres equivalent to 25 mg of Heparin were suspended in 900 ml of pH 7.8 phosphate medium with the temperature maintained at 37± 0.50C. The rotating rate of the basket was adjusted to 100 rpm. At different intervals, 10 ml of samples were withdrawn and filtered through a 0.8-Am cellulose acetate membrane filter. The equivalent volume of the medium with the same temperature was added to the dissolution vessel. The amount of heparin determined by colorimetrically4 (Fig.-2).
Fig.2 Effect of Ethyl cellulose and Carbopol 934P on the in vitro release of Heparin pH 7.8 phosphate medium.
RESULTS AND DISCUSSION:
The mucoadhesive microspheres of heparin prepared in this study were well-rounded spheres with the size ranging approximately from 151 to 287 µm. The study of in vitro bioadhesion revealed that all the batches of prepared microspheres had good bioadhesive property ranging from 81±1.145% to 93±1.421%. On increasing the mucoadhesive polymer concentration, the bioadhesive property of the microspheres also increased. The formulation F6 showed the highest bioadhesive property (93±1.421%). These studies suggest that the spherical matrix of microspheres can interact with mucosubstrate on the surface of the stomach, and adhere to mucosa more strongly and could stay in stomach for prolong period for more effective anti ulcer activity. (Table 2).