INTRODUCTION:

Multiparticulate drug delivery systems include microspheres and microcapsules, offering therapeutic as well as technological advantages. Microspheres generally of 1μm to 1000 μm.^1,2in size and consisting mucoadhesive polymer or as an outer coating microspheres. Microspheres, generally having the potential to be used for targeted and controlled release drug delivery; but addition to mucoadhesive properties the microspheres will be having additional advantages like increased absorption and enhanced bioavailability of the drugs because of high surface to volume ratio, a much more intimate contact with the mucus layer, specific targeting of drug to the absorption site can be achieved by attaching plant lectins, bacterial adhesions and antibodies, etc. on to the surface of the microspheres.

Mucoadhesive microspheres also can be made to stick to any mucosal tissue including those found in eye, cavity, urinary and alimentary canal, thus offering the benefits of both local as well as controlled release of drugs. Microspheres prepared with mucoadhesive as well as biodegradable polymers undergoes selective uptake by the M- cells of Peyer patches in gastrointestinal (GI) mucosa. This uptake mechanism has been used for the delivery of protein and peptide drugs, antigens for vaccination and plasmid DNA for gene therapy. Moreover, by keeping the drugs in close proximity to their absorption window in the GI mucosa. The mucoadhesive microspheres improve the absorption and oral bioavailability of many anti diabetic drugs. Most commonly used polymers to prepare floating microspheres are polycarbonate, hydroxy propyl methyl cellulose (HPMC), cellulose acetate, calcium alginate, eudragit S, chitosan etc. Now a day’s floating microspheres are considered one of most widely used buoyant systems because of their excellent floating properties. Most commonly used techniques in the preparation of microspheres are emulsion solvent evaporation and emulsion solvent diffusion method^5,6 Generally the drug release and floating properties are mainly depend on the type of polymer, type of plasticizer used and the solvent employed during the manufacturing process.

Diabetes is one of the major causes of death worldwide. repaglinide is used in the management of type II diabetes. It is commercially available in the form of conventional tablets.

Repaglinide is an oral anti hyperglycemic agent used for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). It belongs to the meglitinide class of short-acting insulin secretagogues, which act by binding to β cells of the pancreas to stimulate insulin release. Repaglinide induces an early insulin response to meals decreasing postprandial blood glucose levels. It should only be taken with meals and meal-time doses should be skipped with any skipped meal. The drug has a rapid onset and short duration of action. It has been used alone or in combination with other medications to treat type-2 diabetes. The half life of the drug is small i.e. 1 h. The absolute bioavailability of drug is 56%. The recommended dose range is 0.5mg to 4mg before meals, with a maximum daily dose of 16mg. The patient’s dose should be doubled up to 4mg with each meal until satisfactory glycemic control is achieved. At least one week should elapse to assess response after each dose adjustment. It requires frequent dosing to maintain therapeutic effect. Therefore, it would be beneficial to develop a drug delivery system which remains at the gastro intestinal tract for an extended period of time.

Microspheres encapsulated with anti-diabetic drug, increase the effectiveness and release of drug in control manner from polymer membrane and thereby maintain its concentration for longer duration. Due to its short acting action, fast clearance, enzymatic stability and absorption throughout GIT make repaglinide a suitable target for developing floating dosage form. The aim of the study was to increase the bioavailability and reduce the side effects of drug. Various polymers like ethyl cellulose and biodegradable acrylic polymers eudragit S-100 was used to achieve the controlled delivery of drug from polymer matrix and oil in water emulsion solvent evaporation technique was selected for formulation. The influence of various factors such as percentage yield, particle size, drug entrapment efficiency, floating properties and in-vitro release of the resulting microspheres were investigated. [3]

MATERIALS AND METHODS:

Materials:

The repaglinide drug was obtained as a gift sample from Macleods Pharmaceutical Limited Mumbai, India, ethyl cellulose, eudragit S-100, tween 80, sodium bicarbonate was purchased from Merck Specialities Private Limited, Mumbai. All other chemicals used in the research were of analytical grade.

Methodology:

Analytical method development:

Calibration curve of repaglinide in 0.1N HCl:

Repaglinide pure drug (10mg) was dissolved in 10ml of methanol solution (stock solution 1). 1ml of solution was taken from stock solution 1 and made up the volume to 10ml with 0.1N HCl (100μg/ml) stock solution 2. From the stock solution 2, 1ml was taken and make up with 10 ml of 0.1N HCl (10μg/ml) gives stock solution 3. The above stock-2 solution was subsequently diluted with 0.1N HCl to obtain series of dilutions containing and 10,20,30,40 and 50μg/ml of solutions respectively. The absorbance of the above dilutions was measured at respective wavelength by using UV-Spectrophotometer considering 0.1N HCl as a blank. Then a graph was plotted by taking Concentration on X-Axis and absorbance on Y-Axis which gives a straight line linearity of standard curve was assessed from the square of correlation coefficient (R²) value.

Preparation of Floating microspheres:

The floating microspheres were prepared by solvent evaporation method (Harsh Bansal., et al., 2011). Drug, required polymer and other excipients were taken in different ratios as shown in table 1. Drug and excipients were dissolved in ethanol and dichloromethane (1:1). The obtained drug and polymer solution (aqueous phase) was poured slowly using syringe into 100 ml of water containing 5% V/V tween 80 (organic phase) The Preparation was stirred at 300 rpm for one hour. The obtained floating microspheres were filtered and dried overnight at room temperature.

Evaluation of formulated microspheres:

1. Micrometric studies^7,8

The obtained microspheres were evaluated for its micromeritic properties like bulk density (ρb), tapped density (^ρt), angle of repose (ɵ), Carr’s index and Hausner’s ratio.

a. Bulk density:

The bulk density (^ρb) was estimated by transferring the powder blend into a graduated cylinder. The bulk volume (Vb) and weight of the powder (M) were determined.

^ρb = M / Vb

b. Tapped density:

The measuring cylinder containing a known mass of powder (M) was tapped for a fixed time (100 ^{tapping). The minimum volume
(V}t)^{occupied in the cylinder as well as the weight of the blend was
measured. The tapped density (ρ}t^{) was estimated by using the following
formula.}

^ρt ^{= M / V}t

c. Carr’s compressibility index:

The simplest way to represent the free flow of powder is compressibility index, it was an indirect measure of size, shape, bulk density, surface area, moisture content and cohesiveness of the material.

^{Carr’s Index = ρ}b ^{- V}t ^{/ ρ}t ^*
100

^{Where ρ}b ^{= Bulk density V}t ^{= Tapped
density}

^{d. Hausner’s ratio}^:

Hausners ratio is an indirect index of ease of powder flow. It is calculated by the following formula:

Hausner’s ratio (H) = ^ρt/^ρb

^{Where ρ}t ^{= tapped density ρ}b ^{=
bulk density}

^{e. Angle of repose}^:

Angle of repose was determined using funnel method. The blend was poured through a funnel that can be raised vertically until a maximum cone height (h) is obtained. Radius of the heap (r) was measured and angle of repose (θ) was calculated using the following formula.

θ = tan^-1h/r

2. Drug excipients compatibility studies:

FTIR spectra of pure drug, optimized formulation were recorded using Perkin Elmer FTIR spectrophotometer, series 1600 which is calibrated with polystyrene using kbr dispersion method. (Sivaprasad.,et al., 2020) In this 2 – 4mg of drug sample was used. The resultant spectrum of the drug was compared with the reference spectrum of repaglinide.

3. Physical characterization:

SEM analysis:

The morphology of the microspheres was studied using a scanning electron microscope, SEM (Field emission microscope, JEOL, JSM-7600). The microspheres were prepared using a double adhesive tape stuck to an aluminium stab. Sprinkles of microspheres were applied on to the stub and dried overnight. They were then coated with gold under an argon atmosphere. (Kefilwe matlhola., et al., 2015)

4. Percentage yield:

The percentage yield was calculated to determine the total amount of product obtained from the raw materials. (Prabhu., et al., 2009)

Total amount of microspheres obtained

% yield: ------------------------------------------------- × 100

Total amount of polymer + Drug

5. Drug entrapment efficiency⁹

Microspheres equivalent to repaglinide dose were taken for evaluation. The amount of drug entrapped was estimated by crushing the microspheres. (Swamy P.V., et al., 2007)The powder was transferred to a 100 ml volumetric flask and dissolved in 10ml of methanol and the volume was made up to 100ml with 0.1N HCl. Kept it for sonication about 1 hour. Then solution was filtered through Whatmann filter paper and the absorbance was measured after suitable dilution spectrophotometrically at respective wavelength. The amount of drug entrapped in the microspheres was calculated by the formula mentioned above.

Estimated % of drug content in microspheres

Entrapment:-------------------------------------------- × 100

efficiency Theoretical % drug content

6. in- vitro evaluation of floating ability:

Floating microspheres (equivalent to 2mg) were dispersed in 100ml of 0.1 N Hydrochloric acid solution (pH 1.2) to simulate gastric fluid at 37°C. The mixture was stirred with a paddle at 50rpm and after 12 hr, the layer of buoyant microspheres (Wf) was pipetted and separated by filtration simultaneously sinking microsphere (Ws) was also separated. Both microspheres type were dried at 40°C overnight. Each weight was measured and buoyancy was determined by the weight ratio of the floating microspheres to the sum of floating and sinking microsphere.

Buoyancy (%): W_f / (W_f+W_s) × 100

Where Wf and Ws = the weights of the floating and settled microspheres respectively. All the determinations were made in triplicate.

7. In- vitro wash-off test for microspheres:

The mucoadhesive property of the microspheres is evaluated on goat intestinal mucosa by using phosphate buffer, as per monograph.(Bhubani Nayak., et al., 2009) Weighed microspheres were distributed onto the wet rinsed tissue specimen and later the slides were hung onto the arm of a USP tablet disintegrating test machine with suitable support at 37^oC. The weight of the microspheres released at different time intervals was measured. The % mucoadhesion was estimated by the below mentioned formula,

Wa-W1

%Mucoadhesion: ------------------ × 100

Wa Where,

Wa is the weight of microspheres applied

W1 is the weight of microspheres released

7. In- vitro drug release studies:

The dissolution study of floating microspheres was performed over a time period of 12 hrs using USP type I (Basket) Dissolution Testing Apparatus (Lab India) 900ml of 0.1N HCl was used as dissolution medium agitated at 100 RPM, at temperature of 37⁰± 0.5⁰C. Sink conditions were maintained during the study. 5ml samples were withdrawn at 1 hour time interval, passed through 5µm membrane filter and the samples were analyzed by UV visible Spectrophotometer at the wavelength 237nm. (Chowdary K.P.R., et al., 2004)

8. Release kinetics studies:

Release data of optimized formulation was subjected to different mathematical models to determine the release mechanism of the microspheres: Zero order (% cumulative drug release vs. time), first order (log % drug release vs. time), Higuchi model (% cumulative drug release vs. square root of time) and Peppas exponential equation (log % drug release vs. log time). and regression coefficient (r²) values were calculated. (Bhubani Nayak., et al., 2009)

RESULTS AND DISCUSSION:

Construction of calibration curve of repaglinide

Fig:1. Calibration curve of repaglinide in 0.1N HCl at 237 nm.

Micromeritic studies:

The flow properties of repaglinide mucoadhesive microspheres were estimated by studying their tapped density, bulk density, carr’s index, hausner’s ratio and angle of repose. All the batches of microspheres were found to be free flowing with good flow properties as shown in the Table no 2.

Table: 2 Micromeritic property of floating microspheres of repaglinide

Formulation code	Mean partical size	Bulk density (gm.cm³)	Tapped density (gm./cm³)	Hauseners ratio	Carrr’s index	Angle of repose
F1	385.15±1.08	0.36±0.04	0.44±0.01	1.22±0.01	18.18±0.03	27.00±1.93
F2	451.84±2.07	0.41±0.05	0.47±0.01	1.14±0.02	12.76±0.02	26.02±1.80
F3	493.24±2.43	0.40±0.01	0.48±0.02	1.2±0.01	16.66±0.03	26.56±1.43
F4	455.22±2.52	0.35±0.02	0.40±0.05	1.14±0.05	12.5±0.06	27.72±1.89
F5	471.52±2.05	0.40±0.07	0.47±0.04	1.17±0.03	14.8±0.04	30.88±2.78
F6	477.5±2.15	0.44±0.01	0.50±0.02	1.13±0.02	12±0.05	26.56±1.68
F7	381.55±2.54	0.32±0.06	0.39±0.08	1.21±0.04	11.13±0.01	28.49±1.71
F8	473.9±2.16	0.39±0.01	0.45±0.02	1.15±0.06	13.33±0.04	26.80±1.68
F9	482.12±2.21	0.41±0.05	0.48±0.07	1.17±0.01	14.5±0.08	27.11±1.59
F10	401.51±1.84	0.37±0.07	0.44±0.06	1.18±0.02	15.90±0.5	28.04±1.08

All the values were represented as mean ± standard deviation (n=3)

Physical characterization¹³:

The mucoadhesive microspheres of repaglinide prepared by solvent evaporation method found to be spherical, discrete and free flowing. The microspheres were uniform in size with size range of 385 to 493 µm. The SEM photograph shown in Figure 4 indicates that the microspheres were spherical in nature.

Fig: 4. SEM image of repaglinide optimized formulation

Percentage yield, in- vitro buoyancy and entrapment efficiency

The percentage yield, in- vitro buoyancy and drug entrapment efficiency of the repaglinide mucoadhesive microspheres were estimated and were shown in Table no 3.

Table: 3 Percentage yield, in-vitro buoyancy and entrapment efficiency of floating microspheres of repaglinide

Formulation code	Percentage yield	*In vitro* buoyancy (%)	Entrapment Efficiency (%)
F1	86.19±0.28	74.69±0.97	81.62±1.72
F2	94.19±0.48	85.34±1.29	90.57±1.94
F3	96.87±0.54	91.87±0.62	92.59±2.01
F4	93.08±0.29	93.56±1.03	91.81±2.47
F5	97.48±0.57	95.81±2.11	95.62±2.07
F6	84.05±0.39	70.42±1.36	79.68±1.46
F7	89.17±0.43	81.57±0.84	83.74±1.67
F8	92.74±0.82	88.36±1.40	89.16±2.05
F9	94.64±0.55	90.51±1.10	91.42±1.85
F10	92.78±0.15	92.67±1.27	90.63±1.15

All values represented as mean ± standard deviation (n=3)

The percentage yield ranges from 84.1% to 97.5%. The in-vitro buoyancy was found to be from 74.7% to 95.8%, while the drug entrapment efficiency was ranges from 79.7% to 95.6%.

In- vitro wash-off test for microspheres¹¹:

Since the F5 formulation shows higher percentage yield, in-vitro buoyancy and drug entrapment efficiency, it was considered for in-vitro wash-off test for microspheres. The test was carried out for a period of 4 hours. The results obtained were shown in table no 4. The optimized formulation F5 shows the 79% mucoadhesion at the end of 4 hours.

Table: 4 in-vitro wash-off test for the optimized formulation F5

Formulation Code

After 1 hr

After 2 hr

After 3hr

After 4 hr

In-vitro drug release and its kinetics^10,12:

The in-vitro drug release data for all the formulations of repaglinide in 0.1N HCl were estimated and these were shown in Table 5 and 6

Table: 5 In-Vitro drug release data of repaglinide microspheres F1 to F5

Time (hr)	% Cumulative drug release
Time (hr)	F1	F2	F3	F4	F5
0	0	0	0	0	0
1	21.09	18.42	14.36	9.56	5.21
2	39.64	33.08	22.42	17.43	14.36
3	53.34	45.14	39.44	26.71	24.19
4	79.27	59.23	47.54	34.92	32.84
5	79.29	71.36	58.63	42.64	42.96
6	79.26	84.36	64.43	56.19	50.11
7	-	91.34	73.32	69.81	58.26
8	-	91.36	84.01	78.64	64.41
9	-	91.31	90.14	86.48	71.65
10	-	-	91.08	94.61	79.29
11	-	-	90.51	94.08	84.39
12	-	-	-	94.51	96.51

Table: 6 In-Vitro drug release data of repaglinide microspheres F1 to F10

Time (hr)	Cumulative % Drug Release
Time (hr)	F6	F7	F8	F9	F10
0	0	0	0	0	0
1	30.23	23.74	20.71	18.65	15.74
2	51.65	42.18	35.43	25.41	24.91
3	69.45	58.65	49.54	37.68	35.11
4	75.34	74.87	58.42	48.36	42.91
5	75.06	82.65	67.54	60.18	54.37
6	75.18	82.59	75.43	72.11	63.58
7	-	82.41	84.15	80.55	75.19
8	-	-	83.67	80.14	83.08
9	-	-	84.01	80.08	82.69
10	-	-	-	-	82.71
11	-	-	-	-	-
12	-	-	-	-	-

From the diffusion profiles it was evident that all the formulations (F1- F10), showed a cumulative percentage release of 75.34% to 96.51%, based on the dissolution profiles, the F5 formulation was considered to be the optimized formulation, which showed the highest cumulative percentage release of 96.51%. And it was found that as the polymer concentration was increases the release rate was also increases. The percentage of drug release of the optimized formulation F5 was found to be initially 5.21% at 1 h and drug release increased to 96.51% at 12 h.

The in- vitro release data was subjected to various kinetic models to predict its drug release kinetic mechanism. All the kinetic models are shown in Fig.5 and the results are shown in Table no.7. In the table R² value is correlation value, k is rate constant and n is release exponent. To explain the mechanism of drug release, korsmeyer-peppas equation was used. Value of slope (n) was calculated and the values were tabulated and it was found to be 1.157, which indicates anomalous non-fickian diffusion i.e. coupling of diffusion as well as erosion, which indicates that the drug release is sustained by more than one process.

Fig: 5 Drug Release kinetic plots for optimized formulation-F5.

Table: 7 Interpretation of R2 values and the rate constants of release kinetics of nanoparticles.

Model	R²	K	n
Zero order	0.996	8.161	-
First order	0.969	-0.066	-
Higuchi	0.991	34.96	-
Korsmeyer-Peppas	0.988	-	1.157

CONCLUSION

Floating microspheres of repaglinide were prepared by novel oil-in-water emulsion solvent evaporation technique, using various biodegradable polymers such as ethyl cellulose and eudragit S-100 in order to retain drug in body for longer period of time. repaglinide was insoluble in water and its half life is short i.e. 1 h so it requires frequent dosing before meals and thereby imposing side effects. Hence this drug requires a novel gastro retentive drug delivery system which can provide an extended period of time in stomach and improve oral bioavailability. Floating microspheres were characterized for its floating ability, compatibility study, particle size and shape, drug content, in- vitro drug release and entrapment efficiency.

Major advantages of this system include ease of preparation, good floating ability, high encapsulation efficiency and sustained drug release over several hours. From this study it was concluded that formulation of floating microspheres of repaglinide offers prolonged gastric residence time and continuous release of the medication over an extended period of time thus oral bioavailability of the drug and subsequent efficacy was also improved.

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Received on 04.09.2020 Modified on 08.11.2020

Research J. Pharm. and Tech 2021; 14(11):5673-5679.

DOI: 10.52711/0974-360X.2021.00986

Formulation code	Drug (mg)	Ethyl cellulose (mg)	Eudragit S 100 (mg)	Sodium Bicarbonate (mg)	DCM (ml)	Ethanol (ml)
F1	100	50	-	500	5	5
F2	100	100	-	500	5	5
F3	100	150	-	500	5	5
F4	100	200	-	500	5	5
F5	100	250	-	500	5	5
F6	100	-	50	500	5	5
F7	100	-	100	500	5	5
F8	100	-	150	500	5	5
F9	100	-	200	500	5	5
F10	100	-	250	500	5	5

Table: 1 Formulation of floating Microspheres

Hausner’s ratio (H) = ρt/ρb

3. Physical characterization:

Hausner’s ratio (H) = ^ρt/^ρb