Thursday, June 4, 2009

Formulation and Evaluation of Floating Chitosan Microspheres Containing Propranolol Hydrochloride


Fig. 1: Scanning electron photomicrograph of propranolol Hcl loaded chitosan-floating microspheres (batch F4)

Editor's Note: This article will be presented in two parts, the first of which features an introduction, materials and methods, including preparation of chitosan microspheres and in-vitro studies. The second part, which will be featured in the April/May issue of PFQ, will cover results and discussions, preliminary trials, factorial design and full model designs for drug entrapment efficiency and floating lag time and similarity factors. All references will also be listed.

SEVERAL APPROACHES HAVE BEEN PROPOSED TO RETAIN THE DOSAGE FORM IN the stomach. These methods include bioadhesive systems1, swelling and expanding systems and floating systems2,3. In fact, the buoyant dosage unit enhances gastric residence time (GRT) without affecting the intrinsic rate of emptying4. Unfortunately, Floating devices administered in a single-unit form [such as hydrodynamically balanced system (HBS)] are unreliable in prolonging the GRT owing to their 'all-or-nothing' emptying process and, thus, they may causes high variability in bioavailability and local irritation due to large amount of drug delivered at a particular site of the gastrointestinal tract (GIT) 5,6. In contrast, multiple unit dosage forms (e.g. microspheres) have the advantage that they pass uniformly through the GIT to avoid the vagaries of gastric emptying and provide an adjustable release, thereby, reducing the intersubject variability in absorption and risk of local irritation7. Recently, floating microspheres with a lower density than that of the GI fluids were adopted6. The floating chitosan microspheres were prepared by chemical denaturation method. Chemical denaturation involves denaturation of chitosan present in the inner phase of water/oil (w/o) emulsion. Denaturation is usually carried out using glutaraldehyde with continuous stirring8.

Chitosan is a biocompatible and biodegradable polysaccharide soluble in aqueous media of low pH and showing extremely low toxicity. It gives microspheres cross-linking with chemicals (e.g. glutaraldehyde, formaldehyde, and sulfuric acid)9,10. Many process parameters affecting characteristics of chitosan microspheres have been identified, and the significance of the effect has been established. It has been reported the irrespective of molecular weight, spherical chitosan microspheres are formed if the concentration of the chitosan solution is at least 1 percent w/v11.

Propranolol Hcl, an b-adrenoceptor antagonist that can acutely lower the blood pressure in human by blocking receptors non-selectively, is typically prescribed to treat hypertension, myocardial infraction, and cardiac arrhythmias. Its short biological half-life (3.9 � 0.4 hours) necessitates the need to administrate in two or three doses of 40 to 80 mg per day. The development of controlled-release dosage forms thus would clearly be advantageous. Researchers have formulated oral controlled-release products of propranolol Hcl by various techniques. Moreover, the site of absorption of propranolol Hcl is in the stomach. Dosage form that is retained in the stomach would increase the absorption, improve drug efficiency, and decrease dose requirements12. Thus, the aim of the present investigation was to characterize, optimize and evaluate floating microspheres of propranolol Hcl for oral controlled drug delivery.

In context of the above principles, a strong need was felt to develop a dosage form that delivered propranolol Hcl in the stomach and would increase in the efficiency of the drug, providing sustain action. Thus, an attempt was made in the present investigation to use chitosan as a low-density polymer and prepare floating propranolol Hcl microspheres. The microspheres were characteristised by in-vitro and iv-vivo tests and factorial design was used to optimize the variables.

Table 1: Results of preliminary trial batches.



Propranolol Hcl was obtained as gift sample from Mann Pharmaceutical Ltd (Mehsana, India). Chitosan (degree of deacetylation of 85 percent; intrinsic viscosity, 1390 mL/g in 0.30 M acetic acid/0.2 M sodium acetate solution; and viscometric molecular weight, 4.08 x 105 Da) was obtained as gift sample from Central Institute of Fisheries Technology (Cochin, India). Dioctyl sodium sulfosuccinate (DOSS) and petroleum ether were procured from Willson Laboratories (Mumbai, India) and S. D. Fine Chemicals Ltd (Mumbai, India), respectively. Glutaraldehyde, 25 percent in water and liquid paraffin were purchased from S. D. Fine Chemical Ltd (Mumbai, India) and Loba Chemicals Ltd. (Mumbai, India), respectively.

Fig. 2: (-�-) Represent theoretical dissolution profile of Propranolol HCl and (-?-) represent in vitro dissolution profile of Batch F4 in 0.1 N HCl.

Preparation of Chitosan Microspheres

Floating chitosan microspheres were prepared by chemical denaturation method. Chitosan was used as a polymer and was cross-linked using glutaraldehyde8. Two hundred milligrams of chitosan was dissolved in 20 ml of 1 percent w/v aqueous acetic acid solution. Fifty milligrams of drug was dissolved in the polymer solution. In batches P1 to P12 the polymer-to-drug ratio was kept constant at 4:113. F1 to F9 take 3:1, 4:1 and 5:1. The resultant mixture was extruded through a syringe (No. 20) in 100 ml of liquid paraffin containing 0.2 percent DOSS and stirring was carried out using a propeller stirrer (Remi, Mumbai, India) at 1000 rpm. After 15 minutes, glutaraldehyde (25 percent v/v aqueous solution) was added and stirring was continued. The volume of cross-linking agent varied in batches P1 to P12 from 0.5 to 2 ml Show in Table 1. In factorial design Batches F1 to F9, stirring speed and cross-linking time were kept 1000 rpm and 3 hours respectively. The polymer-to-drug ratio and volume of cross-linking agent varied in batches F1 to F9 as shown in Table 2. All other variables were used as mentioned in preliminary trial batches. Microspheres thus obtained were filtered and washed several times with petroleum ether (80:20) to remove traces of oil. They were finally washed with water to remove excess of glutaraldehyde. The micros-pheres were then dried at 40o C for 12 hours in hot air oven. The effect of formulation variables on characteristics of the microspheres is summarized in Table 1 and 2.

Table 2: 32 Full Factorial Design Layout

Assay of Propranolol Hcl

Propranolol Hcl estimated by Ultraviolet visible (UV/Vis) spectrophoto-metric method (Shimadzu UV-1601 UV/Vis double beam spectrophotometer, Kyoto, Japan). Aqueous solution of propranolol Hcl was prepared in 0.1 N Hcl (pH 1.2) and absorbance was measured on UV/Vis spectrophotometer at 289 nm (USP XXVII). The method was validated for linearity, accuracy, and precision. The method obeys Beer's Law in the concentration range was 10 to 50 �g/ml. When a standard drug solution was analyzed repeatedly (n=3), the mean error (accuracy) and relative standard deviation (precision) were found to be 0.84 percent and 1.2 percent, respectively.

Fig. 3: Percent inhibition of heart rate after oral administration of Propranolol HCl (-?-) and it's chitosan-floating microspheres (-�-) in rabbits.

Determination of Mean Particle Size

The particle size of microspheres was determined by using optical microscopy Method14. A small amount of dry microspheres was suspended in purified water (10 ml). A small drop of suspension thus obtained was placed on a clean glass slide. The slide containing chitosan microspheres was mounted on the stage of the microscope and ferret's diameter of at least 100 particles was measured using a calibrated optical micrometer (Labomed CX RIII, Ambala, India). The average particle size of micros-pheres of batch P1 to P12 and Batches F1 to F9 are depicted in Table 1 and 2, respectively.

Determination of Drug Entrapment Efficiency

Efficiency of drug entrapment for each batch was calculated in terms of percentage drug entrapment (PDE) as per the following formula:

PDE = (practical drug loading / theoretical drug loading) x 100

Theoretical drug loading was determined by calculation assuming that the entire drug present in the chitosan solution used gets entrapped in microspheres and no loss occurs at any stage of preparation of microspheres. Practical drug loading was determined by tacking a 20 mg of accurately weighed microspheres were crushed in a glass mortar-pastel and the powdered micros pheres were suspended in 10 ml of 0.1 N Hcl (pH 1.2). After 24 hours the solution was filtered and the filtrate was analyzed for the drug content. The drug entrapment efficiency for batches P1 to P12 and F1 to F9 are reported in Table 1 and 2, respectively.

In-vitro Buoyancy studies

The in-vitro buoyancy was determined by floating lag time method 15. The fix quantity of chitosan microspheres were placed in 100 ml beaker containing 0.1 N Hcl. Note the time required for the microspheres to rise to the surface and float was determined as floating lag time. The floating lag time of batches F1 to F9 is depicted in Table 2.

Scanning Electron Microscopy

A scanning electron photomicrograph of drug-loaded floating chitosan microspheres was taken. A small amount of microspheres was spread on glass stub. After wards, the stub containing the sample was placed in the scanning electron microscope (JSM 5610 LV SEM, JEOL, Datum Ltd, Tokyo, Japan) chamber. The scanning electron photomicrograph was taken at the acceleration voltage of 20 Kv, chamber pressure of 0.6 mm Hg, original magnification x 800. The photomicrograph is depicted in Figure 1.

In-vitro Drug Release Study

The drug release study was carried out using USP XXVI basket apparatus (Electrolab, TDT-06T, Mumbai, India) at 37 � 0.5 oC and at 100 rpm using 900 ml of 0.1 N Hcl (pH 1.2) as a dissolution medium (n=3) as per USP XXVI dissolution test prescribed for propranolol Hcl tablet (USP XXVI). Microspheres equivalent to 40 mg of propranolol Hcl were used for the test. 10 ml of sample solution was withdrawn at predeterrmined time intervals, filtered through a 0.45 � membrane filter, dilute suitably and analyzed spectrophotometrically. Equal amount of fresh dissolution medium was replaced immediately after withdrawal of the test sample. Percent drug dissolved at different time intervals was calculated using the Beer Lambert's equation. The time required for 80 percent (t80) drug to release was calculated using weibull equation16. The average values of t80 for the batches F1 to F9 are mentioned in Table 2. The percentage drug release of batch F4 is shown in Figure 2.

Comparison of Dissolution Profile

The similarity factor (�2) given by SUPAC guidelines for modified release dosage form was used as a basis to compare dissolution profile. The dissolution profiles are considered to be similar when �2 is between 50 to 10017,18. The dissolution profiles of products were compared using a similarity factor (�2).

This similarity factor is calculated by following formula:

When n is the number of dissolution time and Rjand Tjare the reference and test dissolution values at time t. �

The authors can be reached through Dr. Jayvadan K. Patel, of the Department of Pharmaceutical Technology, S. K. Patel College of Pharmaceutical Education and Research at Ganpat University (Gujarat, India) at 0091-2762-286082

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