Now a days, most available drug delivery system use bio-degradable, biocompatible and natural bio-polymer and are capable of rate controlled drug release. Now various research effort is being spent on oral sustained drug delivery system, these system being solid dosage form researches developed various sustained and controlled release form by entrapped the drug natural polymer and forming a gel [1]. Micro-beads are defined as monolithic sphere distributed the whole matrix as a molecular dispersion defined as the drug particle are dispersed into continuous phase of one or more than one miscible polymers [2]. Beads loaded the antacid to be useful for oral delivery to treatment of different type disease that is peptic ulcers and for the ulcerative colitis, carcinomas and infection of intestine [3]. The controlled systemic absorption specifically in the intestinal region offers interesting possibilities for the treatment of such an asthma, arthritis and inflammations. The sodium alginate is widely use in various disease in the form of controlled release dosage form due to its natural, biodegradable or mechanical and hydro-gel forming properties [4].

In present research work losartan potassium is used as model drug. Alginate is naturally polysaccharide polymer obtained from brown algae, consisting of two different unit, -d Mann uronic acid and -1- glucornic acid. Losartan potassium microbeads were prepared by ion tropic gelatin technique where the gelatin of natural anionic polysaccharide sodium alginate, the natural polymer are react with oppositely charged calcium ion, acting the cross linker or counter ion, to from immediately micro-beads. Various synthetic polymer used in preparation of micro-beads but some advantages and disadvantages are associated with it. Losartan potassium (LP) is a potent, highly specific angiotensin II type 1 (AT1) receptor antagonist [6] with antihypertensive activity [7]. It is readily absorbed from the gastrointestinal tract with oral bioavailability of about 33% [8] and a plasma elimination half-life ranging from 1.5 to 2.5 h [9].

MATERIAL AND METHODS:

Chemicals and reagents:

Losartan potassium was gifted from Viraj Pharmaceuticals, Mumbai. Losartan potassium (LP) is a potent, highly specific angiotensin II type 1 (AT1) receptor antagonist with antihypertensive activity. Sodium alginate is natural polymer powder is purchased from SD Lab, Mumbai. Calcium chloride is cross-linking agent is gifted from Unichem Lab, Pane. All other chemicals and reagents used were analytical grade unless otherwise indicated.

Preparation of microbeads:

Sodium alginate beads were prepared by dissolving 1.5gm, 2gm and 2.5gm sodium alginate in 50ml water. Then 100mg drug was added to sodium alginate solution and heat up to 10 minute. The above content was added drop wise into 100ml calcium chloride solution with stirring (250rpm) for 2hr. calcium chloride was employed as cross linking agent. The beads, thus formed were separated by filtration and washed with water and dried at room temperature [10] [11].

Selection of Natural Polymer:

On the basis of solubility study sodium alginate was selected as the polymer for loading of losartan potassium with calcium chloride is cross-linking agent [13].

Composition of Losartan Potassium SR Microbeads:

Table 1: Formulation Table of Losartan Potassium SR Microbeads

Ingredients (%)	Formulation Code
Ingredients (%)	F1	F2	F3
Losartan Potassium (mg)	100	100	100
Sodium alginate	1.50%	2%	2.50%
Calcium chloride	10%	10%	10%

Calibration curve of Losartan potassium:

Scanning of Losartan potassium by UV-spectrophotometer in phosphate buffer solution pH 6.8 Standard stock solution of Losartan potassium was prepared by dissolving accurately weighed 10mg of Losartan potassium in water in 100ml volumetric flask. The volume was then made up mark by using water, so as to get the solution of 100μg/ml [14].

Infrared study:

All the ingredients were studied for compatibility between them. For that purpose infrared spectra of individual ingredients were compared with infrared spectra of blended powder [15].

Differential Scanning Calorimetery (DSC):

The physical state of drug in the Losartan Potassium microspheres was analyzed by DSC. The thermograms of Losartan Potassium, Losartan Potassium microspheres with different polymers were obtained at a scanning rate of 10°C/min conducted over a temperature range of 25–350ºC, respectively [15].

Particle size:

Particle size of the alginate beads was determined using an optical microscope using mm compound microscope (OLYMPUS 01C). A standard stage micrometer was used to calibrate the optical micrometer [16].

Drug entrapment efficiency:

Accurately weighed amount of Losartan Potassium F1, F2, and F3 beads were kept in 100mlof USP phosphate buffer solution of pH 6.8 and kept for 24 hours. The solution was filtered and an aliquot following suitable dilution was assayed spectrophotometrically (UV-VIS Spectrophotometer, Thermo Spectronic UV 1) for Losartan Potassium at 205.3nm [17]. The drug entrapment efficiency was determined using the following relationship:

Experimental drug content

Drug entrapment efficiency=-------------------------- X100

Theoretical drug content

Drug content:

Drug content was determined by taking 100 particles per batch. An amount equivalent to 100mg of Losartan Potassium was weight and dissolved in phosphate buffer pH 6.8 in 100ml volumetric flasks. From these suitable dilution was prepared to get 10ug/ml and the solution was analyzed at 206 nm using a UV spectrophotometer [17].

Actual drug content in beads

Drug content (%) =---------------------------- X100

Theoretical drug content

In vitro drug release studies:

Losartan Potassium In vitro drug release from Losartan Potassium was performed using USP Apparatus I in 900 mL of buffer pH 1.2 for 2hrs and pH 6.8 for remaining time period stirred at 37°C and 50rpm maintaining sink conditions. The accurately weighed Losartan Potassium microbeads were enclosed in a sieve, placed in the basket, and processed for dissolution testing. All the Losartan Potassium microbeads stayed in the basket during 12-h dissolution testing (i.e., no particles diffused out of the sieve). Dissolution samples (5mL) were withdrawn at regular intervals (0, 1, 2, 4, 6, 8, 10 and 12h) using an auto sampler with replacement of equal volumes of fresh medium [18] [19]. The samples were filtered through a 0.45-μm filter and analyzed spectrophotometrically at 205nm in triplicate. Drug concentration was calculated using a calibration curve.

The data obtained in the in-vitro dissolution study was grouped according to two modes of data treatment as follows:

1. % Drug release Vs time in h.

2. Cumulative percentage drug release vs. time in h.

Swelling behavior of sustained release microbeads:

Swelling ratio was studied by measuring the percentage water uptake by the beads. About 50 mg prepared beads take in 0.1 N HCl (pH 1.2). Beads were removed from their respective swelling media after 8 h and weighed after drying the surface water using filter paper. The water uptake was calculated as the ratio of the increase in prepared placebo beads were accurately weighed and placed in 100ml of phosphate buffer (pH 6.8) and weight of beads after swelling to the dry weight [20] [21].

Swollen weight-initial weight

Swelling ratio =------------------------------------ X100

Initial Weight

RESULTS AND DISCUSSION:

Overlay Spectra of Losartan Potassium:

The overlain spectrum of both drug was recorded (Fig.1) 205.30nm (λ max of LOS) were selected for further study (Figure 1).

Figure 1: Overlay Spectra of Losartan Potassium

Procedure for Calibration curve of Losartan potassium in PBS pH 6.8.:

An absorption maximum of losartan potassium was found to be at 205.3nm. Prepare Losartan potassium standard stock solution (100μg/ml) in phosphate buffer pH 6.8 as dissolution media. From this solution, aliquots of 0.2, 0.4, 0.6, 0.8 and 1.2ml were transferred to the series of 10 ml volumetric flasks and final volume is made with water, so as to get drug concentrations of 2-12.0μg/ml [22]. The absorbance of these drug solutions were estimated at 205.3nm (Figure 2). This procedure was performed in triplicate to validate the calibration curve. Absorbance of solution increases with concentration. The results are shown in following table.

Regression and Optical characteristics of LOS

Table 2: Regression and Optical characteristics of LOS

Parameters	Value For LOS
Beer’s law limit (µg/mL)	2-12
Correlation Coefficient (r)	0.996
Slope	0.082
Intercept	0.030

Table 3: Sectrophotometric Data of Calibration Curve

Conc. (μg/ml)	Absorbance(Ǻ)			Mean
2	0.198	0.192	0.193	0.19433
4	0.365	0.362	0.36	0.36233
6	0.502	0.498	0.504	0.50133
8	0.699	0.702	0.7	0.70033
10	0.878	0.875	0.881	0.878
12	0.99	0.996	0.998	0.99467

Figure 2: calibration Curve of LOS [y = 0.082 xs + 0.030 (R² = 0.996)]

FTIR study:

The infrared absorption spectrum of losartan is shown in Figure 3. The spectrum was obtained in a KBr pellet using Shimadzu FT-IR infrared spectrophotometer (model 8400S) equipped with IR Solution software, version 1.0, over the range of 667.25–3189.68 cm^-1 at a resolution of 4 cm^-1. The OH absorption band appeared at 3189 cm^-1 while C] N absorption band appeared at 1565 cm^-1.

From the spectra of Losartan potassium and physical mixture of Losartan potassium, it was observed that all characteristic peaks of Losartan potassium were present in the combination spectrum, thus indicating compatibility of the Losartan potassium and polymer.

Figure 3: FTIR of Losartan Potassium

Differential Scanning Calorimetery (DSC):

Figure 4: DSC of Losartan potassium

The pure drug Losartan Potassium shown as an endothermic peak at 260.91oC. The peak neither is nor shifted in the case of DSC of the Losartan Potassium microspheres formulation containing Losartan Potassium

Evaluation parameters for Losartan Potassium SR Microbeads:

Table 4: Evaluation parameters for Microbeads

Parameter	Number of Batch
Parameter	F1	F2	F3
Particle size (mm)	0.61	0.72	0.77
Drug entrapment efficiency (%)	73.06	76.02	79.06
Drug content (%)	12.05	16.63	18.2
Swelling behavior of sustained release microbeads (ratio)	156	208	267

The result of particle size of all formulation is depicted in table. All formulation of Losartan potassium micro beads showed the particle size between 0.61 to 0.77mm. The lowest particle size was obtained with formulation F1 i.e. 0.61mm. The higher particle size was obtained with formulation F3 i.e. 0.77mm the result also suggest that increasing concentration of sodium alginate in formulation does not on particle size. The % entrapment efficiency of all formulation showed in between range of 73.05 - 79.06. The lowest % entrapment efficiency was obtained in formulation F1 and highest was found to be formulation F3. The result demonstrated that effect on % entrapment efficiency with in decreasing concentration of sodium alginate. The % entrapment efficiency study suggests that the higher particle leads to maximum % entrapment efficiency of some of the formulation of micro beads.

The result concluded that the size of the beads can affected on encapsulating capacity of micro beads. The % drug content of entire formulation of sodium alginate based of Losartan Potassium was showed in following table. All the formulation of micro beads produced the optimum drug content in the range of 12.05 – 18.20 %. The minimum drug content was found to be formulation F1 and highest in formulation F3.

The swelling ratio of entire formation of sodium alginate based microbeads of Losartan Potassium was shown in following table. All the formulation of microbeads produced the optimum swelling ratio in the range 156-267. The minimum swelling ratio of formulation F1 and higher swelling ratio of formulation F3 is observed (Figure 6 and 7).

Figure 5: Swelling behavior at 0th hr.

Figure 6: Swelling behavior after 12th hr.

In-vitro Drug Release Studies:

The release profile of all batches of microbeads was studied using phosphate buffer (pH 6.8). The comparative in-vitro drug release curves as shown in Figure2. From the results observed that, the drug releases of F 1, F 2 and F 3 formulations is found to be 50.2% to 98.52%, 49.21% to 98.23% and 43.21% to 98.52% respectively shown in Table 4. Among all formulations, F 3 was show maximum drug release i.e. 98.51% after 12 hr. It was observed that extent of drug release is attributed to the increase in amount of sodium alginate. Percent drug release profile is shown in figure 8.

Table 7: In-vitro Drug Release Data

Time (hr)	Percent drug release at time (hr)
Time (hr)	F1	F2	F3
0	0	0	0
1	50.2	49.2	43.21
2	55.1	54.21	51.23
4	75.58	63.02	60.54
6	88.04	72.71	69.58
8	98.52	77.15	74.21
10	-	98.23	89.72
12	-	-	98.51

Figure 3: Release profile of Losartan potassium SR Microbeads

Stability studies:

Stability studies of the optimized formulation (F3) under accelerated storage conditions as per ICH guidelines did not reveal any degradation of the drug and changes in the in vitro release profiles of the optimized formulation after storage for 6 months were statistically insignificant as compared to the refrigeration control sample (ANOVA, p > 0.05).

CONCLUSION:

In conclusion, the present study reveals the characteristics of Losartan Potassium micro beads formulation. The ion tropic gelatin can be used in producing Losartan Potassium micro beads. The formulation variable, drug loading, polymer concentration, cross linking agent, stirring speed and curing time influenced the mean particle size, entrapment efficiency, drug content and swelling ratio characteristics of prepared micro beads.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of YSPM’s Yashoda Technical Campus, Faculty of Pharmacy, Wadhe Satara for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 10.03.2020 Modified on 05.04.2020

Asian J. Res. Pharm. Sci. 2020; 10(3):153-157.

DOI: 10.5958/2231-5659.2020.00029.6