INTRODUCTION:

The treatment of an acute disease or chronic illness has been mostly accomplished by delivery of drugs to patients using various conventional drug delivery systems. Many drug delivery systems have been designed by various researchers to modulate and release a drug over an extended period of time. Studies of the controlled release of drugs for their extended and safe use have recently become an important field of research. Polymers play an important role in the formulation of drug products. Many attempts have been made to develop functional microcapsule in which permeability is controlled by temperatures, pH, light, electric fields etc. A major advantage of drug release from this formulation is that it is largely independent of pH, food and other physiological parameters¹, and it is possible to modulate the release characteristics by optimizing the properties of the drug, polymer and cross-linking agent.^2,3

Encapsulation Mechanism:^4-18

Alginic acid can be either water soluble or insoluble depending on the type of the associated salt. The salts of sodium are soluble, whereas the salts of Poly valent cations e.g. calcium, are water insoluble.

Thus, polyvalent cations are responsible for the cross-linking of both different polymer molecules and different parts of the same polymer chain. The process of gelation involves the exchange of calcium ion for sodium ions. The first step in the internal phase when the alginate solution containing biological materials or drug is dispersed into small drops. Secondly, the droplets are solidified by gelling or membrane formation at the droplet surface. It has been suggested that the cross-links were caused either by simple ionic bridging of two carboxyl groups on adjacent polymer chains via calcium ions or by chelation of single calcium ions by Hydroxyl and carboxyl grouped on each of a pair of polymer chains. During gelling the concentration of divalent cation have large impact on the gel network. (Fig.-1)

The objective of the present study is to design, develop and optimize an alternate approach for having the loading dose and maintenance dose in the formulation of drug in a biodegradable polymer beads¹⁹. This approach is a convenient method where the core material without any coating gives a sustained release. If coated with polymer will give beads having prolonged rate of release. The polymer based drug delivery system provides a means of controlling drug levels in an efficient manner²⁰.

2Na (alginate) + Ca^{++ --------} Ca (alginate)² + 2Na⁺

Alginate beads have been found stable in a range of organic solvents and are therefore, in contrast to other hydrogels, potentially useful in application involving entrapment of enzymes, microorganisms and drugs.

Isosorbide dinitrate, a vasodialator used in the treatment of angina and heart failure (antihypertensive) has a half life of about 45-60 minutes, 20 minutes and 4 hour when administered by sublingual, intravenous and oral route of administration respectively²¹. The usual dose of isosorbide dinitrate acute angina is 2.5 to 10 mg sublingually and for long term angina is 30 to 120 mg taken very few times a day. Thus it was thought that developing a sustained release calcium alginate beads of isosorbide dinitrate would eliminate name of the problem such as patient compliance and fluctuation in systemic dry concentration, which is associated with conventional dosage forms²². Hence calcium alginate beads containing drug were prepared by encapsulation procedure, with different cross-liking agents to investigate the release variables²³.

MATERIALS AND METHODS:

Isosorbide dinitrate was obtained from M/S Cipla Ltd., Mumbai , sodium alginate was procured from Fisher Inorgancis and Aromatic Ltd., Chennai. Formaldehyde (LR Grade) and Gluteraldehyde (LR Grade) were purchased from S.D. Fine Chem Ltd., Mumbai. All other materials used in the study were of analytic grade. The major pieces of equipment employed in the study encompassed: Remi stirrer (Remi Instruments), Research Centrifuge (Remi Instruments), USP Dissolution Apparatus XXIII, UV Spectrophotometer (UV160 A Shimadzu) and UV Chamber.

Methods:

(a) Standard Curve for Isosorbide Dinitate

The analytic method employed is based on the reduction of a portion of nitrate of the compound to nitrite upon alkali hydrolysis. This resulted nitrite is determined Spectrophotometrically with a high degree of sensitivity through Diazotization and Coupling procedure.

(i) Preparation of Primary Stock Solution:- Analytical Standard curves of Isosorbide was prepared in normal for comparing further investigation.²²For preparation of standard curve drug equivalent to 100mg was weighed accurately and was dissolved in 50% ethanol and made up the volume, resulted in drug concentration of 1mg/ml.

(ii) Preparation of Secondary Stock Solution:- From the above stock solution 10ml is taken and made upto100ml with 50% Ethanol, which gives a concentration of 100µg/ml.

(iii) Preparation of solutions of Different Concentrations:- Aliquots of 0.5ml, 1.0ml,1.5ml, 2.0ml, 2.5ml and 3.0ml were withdrawn from the secondary stock solution and diluted to 10ml to give concentration of 50to 300µg of the drug respectively. Added 5ml of 2N Sodium Hydroxide and heated it for 10 minutes. The tubes were then cooled at room temperature and 6ml of Hydrochloric acid, 1ml of sulphanillic acid and 1ml of N-(1-napthyl)-ethylene diamino chloride solution. The full purple colour appeared within five minutes. After 10 minutes, absorbance of solution in each tube measured at 540nm against reagent blank prepared in similar manner without the addition of drug.

Similar estimations were also done using phosphate buffer of pH 1.2, 4.5 and 7.5. Values compiled and tabulated in Table 1.

Table 1 :- Mean Absorbance Readings of Isosorbide dinitrite

Sl. No.	Concen tration (mg/ml)	Absorbance at 540nm
Sl. No.	Concen tration (mg/ml)	Normal	pH 1.2	pH 4.5	pH 7.5
1	0.0	0.00	0.00	0.00	0.00
2	5.0	0.0491	0.0511	0.0248	0.1476
3	10.0	0.0992	0.0998	0.0508	0.2887
4	15.0	0.1541	0.1455	0.0759	0.4355
5	20.0	0.2038	0.1933	0.1025	0.5843
6	25.0	0.2554	0.2453	0.1282	0.7261
7	30.0	0.3056	0.2945	0.1543	0.873

Table 2: Data for dissolution studies % Drug Release

Time in Hours	% Drug Release
	Formulation A			Formulation B			Formulation C
	pH 1.2	pH 4.5	pH 7.5	pH 1.2	pH 4.5	pH 7.5	pH 1.2	pH 4.5	pH 7.5
0	00.00	00.00	00.00	00.00	00.00	00.00	00.00	00.00	00.00
1	17.52	18.00	22.99	18.13	22.60	26.51	32.14	38.42	46.11
2	27.85	22.41	29.23	26.54	29.87	34.44	39.42	42.14	48.14
3	39.05	29.12	47.97	37.87	32.18	40.37	41.32	56.14	52.10
4	45.43	40.95	51.95	44.12	40.38	43.15	54.89	64.41	68.32
5	51.34	54.45	67.99	57.63	45.41	52.31	69.49	67.90	77.13
6	62.75	63.18	72.16	61.01	63.49	65.14	76.24	68.41	84.47
7	68.17	72.15	78.18	64.32	65.14	69.87	77.14	72.59	85.15

(b) Formulation:²⁴

Sodium alginate solution was introduced drop wise into a solution of calcium chloride which was kept in stirred condition using a Remi-stirrer at moderate speed. The sodium alginate drops were converted to calcium alginate beads by entrapment method as mentioned earlier.

Morphology: The formation of good quality beads with a spherical shape depends mainly on the viscosity of drug / polymer solution. When viscous liquids are ‘dropped’ they tend to cause sperulation i.e., to produce beads with an attached satellite sphere. This was observed for solution of sodium alginate of concentrations 4% w/v. In case of solution with concentration 1% w/v of sodium alginate, tailing of spherules was formed. A series of trials were conducted after which concentration of 2% w/v of sodium alginate was selected.

Formulation A: Drug incorporated calcium alginate beads

For calcium alginate beads a trial was conducted by ensuring the concentration of sodium alginate as 1% w/v, 2% w/v, 3% w/v, 4% w/v and , 2% w/v, 3% w/v, 4% w/v, 5% w/v for calcium chloride. Good spherical free flowing beads were obtained when the concentration of sodium alginate were 2% w/v and calcium chloride 5% w/v which was finalized as formulation A.

Both empty and drug containing beads were prepared using cross-linking agents with minimum amount and the dried weight was obtained, whereby the total required amount of drug to be incorporated was calculate.

Formulation B and C: Drug Incorporated modified calcium alginate beads.

To select the concentration of support (i.e. sodium alginate) with gluteraldyhyde and formaldehyde, a trial was conducted by fixing the concentration of cross-linking agents.

Calcium chloride 5% w/v and different concentration such as 1% w/v, 2% w/v, 3% w/v for sodium alginate and 2% w/v, 3% w/v, 4% w for gluteraldehyde and formaldehyde were prepared. A spherical free flowing dried beads were obtained when the concentration of sodium alginate were 2% w/v with gluteraldehyde and formaldehyde both concentration of 0.2% v/v for formulation B and C respectively. Along with the cross-linking agents, drug was incorporated with each formulation having a concentration of 100-120 mg.

Micrometric studies were done to evaluate the flow properties and particle size distribution.

(c ) Drug Content Estimation:

The drug content estimation in the cal;cium alginate beads were carried out for three randomly picked up samples and then mean drug content in respective formulations were calculated. An accurately weighed amount of the beads before drying, approximating the weight of the required dried beads were taken and pulverized with the solvent combination of ether and 50% ethanol (1:1) for complete extraction for 24 Hrs. This was centrifuged for 30 minutes at 100 rpm. The clear solution was spotted for ascending thin layer chromatography with solvent system as Toluene : Methanol : water ( 4:3:0:5)²² along with pure drug dissolved in ethanol used as the standard, which was examined under UV light in a UV chamber to ensure its quality. The Rf values of formulation were compound with the standard.

The spectrophotometric absorbance of the filtrate was measured at 540 nm after appropriate dilution and the drug content determined. Percentage entrapment efficiency was computed from the ratio of assayed drug content to that of drug amount incorporated in the beads.

( d ) In-vitro release studies:

In vitro drug release studies of prepared beads were carried out in triplicate for all the formulation, employing USP XX III Apparatus 1 (rotating basket method) at 100 rpm and 37 ± 0.5⁰ C using phosphate buffers (pH 1.2, 4.5, 7.5). An aliquot of the samples were periodically withdrawn at suitable time intervals and the volume replaced with respective fresh dissolution medium. The samples were analyzed spectrophotometerically at 540 nm and the percentage drug release was calculated from the absorbance data which have been tabulated in Table 2. In vitro drug release profile of Isosorbide dinitrite contained prepared beads is shown in Fig:2. The plots of amount of drug released Vs time were found to be linear indicating that the drug release mechanism might be of diffusion type proposed by Higuchi²⁴(Fig-3-5).

RESULTS AND DISCUSSION:

Calcium alginate beads is one of the most commonly applied micro particles which may be formulated into tablets or capsules to obtain a desirable sustained action formulation. The drug incorporated beads were modified using cross-linking agent like gluteraldehyde and formaldehyde. The pre-formulation studies were carried out by thin layer chromatography to find out its drug incorporation along with the polymer used and compared for drug entrapment efficiency with that of standard drug. The Rf values of the formulation were compared with that of the standard which revealed negligible interaction of drug with that of other ingredients used. From the drug content estimation it was clear that all the formulations showed uniformity in drug content.

The dissolution studies shows that the calcium alginate beads containing the drug had a good controlled release pattern of drug in the release kinetics. When the cross linking agent was added, an even enhance controlled release was evident from the drug release data. The phosphate buffer had an overall influence of the release of drug from the polymer. The beads containing formaldehyde as cross-linking agent gave a maximum drug release of 85% in pH 7.5. The results were tabulated collectively in Table 2 and the comparative graphical evaluation in Fig: 2,3 and 4.

Effective drug release was found for all the formulation in phosphate buffer pH 7.5. The swelling nature of beads was found more enhanced in basic buffer because the sodium alginate is originally an acidic polymer. Futher study with respect to varying concentrations of different cross-linking agents can be carried out.

CONCLUSION:

The method of preparation of drug loaded beads of Isosorbide dinitrite was found to be simple and reproducible. The sustained release of Isosorbide dinitrite from such beads finally packed in hard gelatin capsules will help to improve the therapeutic efficacy and patient compliance by redusing the dose and frequency of dosing of the drug. This study also shows that the polymer calcium alginate could be used as carrier for Isosorbide dinitrite for Micro-encapsulation. Studies can be further carried out by incorporating basic polymer such as chitosan which would enhance the controlled drug invitro availability of the drug in acidic pH. Further investigation can be carried to establish invivo release patterns.

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Received on 16.08.2010 Modified on 04.09.2010

Research J. Pharm. and Tech. 4(5): May 2011; Page 719-724