Table 1. Evaluation parameters of various famotidine microcapsule formulations.

Formu-lations Code	Yield (%) (X±S.D.)	Particle Size D_g(µm) (X ± S.D.)		DEE (%) (X±S.D.)	Moisture Loss (%) (X±S.D.)		Circularity factor (s) (X±S.D.)
F1	35.2±0.02	74.1±0.01		43.7±0.03	19.52±0.019		1.00± 0.001
F2	45.2±0.05	148.4±0.03		59.2±0.05	5.59±0.031		1.00±0.002
F3	44.7±0.06	145.8±0.02		55.3±0.04	4.58±0.045		1.00±0.008
F4	81.2±0.06	114.3±0.01		56.8±0.04	15.03±0.062		1.08±0.004
F5	52.7±0.02	467.9±0.02		52.2±0.04	4.07±0.085		1.00±0.006
F6	51.7±0.04	278.3±0.02		49.6±0.04	3.51±0.068		1.00±0.002
F7	89.3±0.06	442.9±0.01		89.3±0.05	13.93±0.036		1.00±0.007
F8	85.3±0.07	457.3±0.03		84.2±0.05	28.54±0.055		1.00±0.004
F9	31.1±0.01	287.2±0.02		60.1±0.06	14.38±0.044		1.00±0.006
	ANOVA
	Source of Variation	SS	df	MS	F	P-value		F crit
	Between Groups	425095	4	106274	2.0868	0.02434		2.60597
	Within Groups	203706	40	5092.66
	Total	628801	44

Each values are represented as mean ± standard deviation (n=3). Standard error mean < 0.064.

Table 2. Loose surface crystal study and in vitro drug release kinetic studies of microcapsule formulations.

Formulation code	Surface drug content (%) (X±S.D.)			Zero order kinetics	First order kinetics		Higuchi square root equation		Korsmeyer-Peppas model
				Regression co-efficient (r)							n
F1	29.37±0.12			0.9803	0.9700		0.9779		0.987		0.169
F2	25.92±0.23			0.9801	0.9783		0.9788		0.954		0.405
F3	10.97±0.09			0.9911	0.9920		0.9936		0.923		0.922
F4	18.56±0.11			0.9841	0.9836		0.9899		0.967		0.187
F5	35.36±0.08			0.9679	0.9919		0.9939		0.979		0.483
F6	15.94±0.31			0.9779	0.9801		0.9893		0.934		0.520
F7	7.12±0.25			0.9943	0.9520		0.9917		0.913		0.223
F8	11.29±0.19			0.9745	0.9432		0.9898		0.943		0.720
F9	19.53±0.16			0.9789	0.9773		0.9679		0.999		0.402
ANOVA
SS		MS	df			F		P-value		F crit
2.13212		0.533	4			2.33		0.0465		2.57874
0.54104		0.012	45
Total -2.67315			49

Surface drug content value are represented as mean ± standard deviation (n=3). Standard error mean < 0.233.

Table 3. Accelerated stability studies of famotidine encapsulated microcapsule formulations

according to ICH guide lines.

Potency of various Formulations (%)
Weeks	Temp. (°C)	F1	F2	F3	F4	F5	F6	F7	F8	F9
Initial	RT	99.5	99.66	99.88	99.22	99.66	99.39	99.87	99.42	99.59
2	RT	99.4	99.05	99.84	99.01	99.40	99.36	99.82	99.39	99.46
	37±1	99.4	99.00	99.86	98.89	99.38	99.35	99.81	99.31	99.42
	60±1	99.3	99.68	99.83	98.83	99.37	99.28	99.85	99.29	99.45
4	RT	99.3	98.97	99.82	99.30	99.29	99.29	99.88	99.27	99.39
	37±1	99.3	98.91	99.80	98.87	99.24	99.27	99.83	99.26	99.36
	60±1	99.3	98.99	99.81	98.56	98.27	99.21	99.78	99.28	99.35
6	RT	99.28	98.87	99.78	98.97	99.21	99.24	99.76	99.28	99.28
	37±1	99.28	98.82	99.83	98.79	99.19	99.19	99.69	99.29	99.29
	60±1	99.29	98.91	99.81	98.50	99.18	99.18	99.68	99.23	99.27
8	RT	99.23	98.78	99.80	98.83	99.17	98.70	99.67	99.21	99.21
	37±1	99.21	98.45	99.80	98.69	99.16	98.16	99.68	99.20	99.24
	60±1	99.20	98.85	99.82	98.32	99.19	98.15	99.63	99.22	99.19

RT – Room temperature.

(F4)⁷, emulsification-ionic gelation method (F5)⁸, emulsion-evaporation method (F6)⁹, ionotropic gelation method (F7)¹⁰, coacervation phase separation method (F8)¹¹ and complex emulsion method (F9)¹², using famotidine as model drug and carbopol as rate controlling and mucoadhesive polymer in a fixed ratio of 1:2 w/w. Several batches were prepared and kept for further studies.

Percentage Yield¹⁰

The microcapsules were evaluated for percentage yield and percent drug entrapment. The yield was calculated as per the equation,

Percentage yield = Weight of microcapsule recovered X 100

Weight (drug + polymer)

Particle size measurement¹⁰

The size of the prepared microcapsules was measured by the optical microscopy method using a calibrated stage micrometer. Particle size was calculated by using equation,

X_g = 10 x [(n_i x log X_i) / N], Where, X_g is geometric mean diameter, n_i is number of particle in range, X_i is the mid point of range and N is the total number of particles. All the experimental units were analyzed in triplicate (n=3).

Drug entrapment efficiency (DEE)¹⁰

Drug loaded microcapsules (100 mg) were powdered and suspended in 100 ml water solvent system. The resultant

dispersion was kept for 30 mins for complete mixing with continuous agitation and filtered through a 0.45 µm membrane filter. The drug content was determined spectrophotometrically (UV-Visible-1700, Shimadzu, Japan spectrophotometer) at 265 nm using a regression equation derived from the standard graph (r²= 0.9978). The drug entrapment efficiency (DEE) was calculated by the equation, DEE = (Pc / Tc) X 100, Where, Pc is practical content, Tc is the theoretical content. All the formulations were analyzed in triplicate (n=3).

Scanning Electron Microscopy (SEM)¹³

The SEM analysis was carried out using a scanning electron microscope (LEO, 435 VP, U.K.). Prior to examination, samples were mounted on an aluminum stub using a double sided adhesive tape and making it electrically conductive by coating with a thin layer of gold (approximately 20 nm) in vacuum. The scanning electron microscope was operated at an acceleration voltage of 05 kV and resolution of 4000.

Percentage moisture loss¹⁴

The famotidine loaded microcapsules was evaluated for percentage moisture loss which sharing an idea about its hydrophilic nature. The microcapsules weighed (W₁) initially kept in desiccator containing calcium chloride at 37°C for 24 hours. The final weight (W₂) was noted when no further change in weight of sample was observed.

Moisture loss = [(W₁– W2)/ W2] X 100.

All the experimental units were studied in triplicate (n=3).

Determination of sphericity¹⁵

The particle shape was measure by computing circulatory factor (S). The tracing obtained from optical microscopy were used to calculate Area (A) and perimeter (P).

This will indicate the approximate shape of the prepared microcapsule calculated by the equation, S = P²/ 12.56 × A. All the experimental units were studied in triplicate (n=3).

Loose surface crystals study¹⁵

The Famotidine encapsulated microcapsules prepared were evaluated by loose surface crystal study to observe the excess drug present on the surface of microcapsules. From each batch, 100 mg of microcapsule was shaken in 20 ml of 0.1N HCl for 5 minutes and then filtered through whatman filter paper 41. The amount of drug present in filtrate was determined spectroscopically and calculated as a percentage of total drug content. All the experimental units were studied in triplicate (n=3).

In vitro drug release¹⁶

In vitro drug release study was carried out in USP type-II dissolution test apparatus. Microcapsules were placed in basket of dissolution vessel containing 900 ml of 0.1N HCl maintained at (37±1) °C and stirred at 50 rpm. Aliquots of samples (5 ml) at an interval of 1 hour were withdrawn and filtered through a whatman filter paper. The samples were analyzed for famotidine content by UV-Visible spectrophotometer at 265 nm .All the experimental units were analyzed in triplicate (n=3).

In vitro drug release kinetic studies

Kinetic model had described drug dissolution from solid dosage form where the dissolved amount of drug is a function of test time. In order to study the exact mechanism of drug release from the microsphere, drug release data was analyzed according to zero order¹⁷, first order¹⁸, Higuchi square root¹⁹, Korsmeyer- Peppas model²⁰. The criteria for selecting the most appropriate model were chosen on the basis of goodness of fit test.

Mucoadhesion test by In vitro wash off method²¹

A piece of stomach mucosa (5 X 2cm) was taken from local slaughter house. It was mounted on to glass slides with adhesive. About 100 microcapsules were spread on to each wet rinsed tissue specimen and immediately thereafter the support was hung on the arm of a USP tablet disintegrating test machine. By operating the disintegrating test machine the tissue specimen was given a slow regular up and down movement in the test fluid at 37^oC taken in the vessel of the machine. At the end of every one hour up to 10 hours, the machine was stopped and number of microcapsules still adhering onto the tissue was counted.

Drug polymer interaction (FTIR) study¹³

IR spectroscopy was performed on Fourier transformed infrared spectrophotometer (840, Shimadzu, Japan). The pellets of drug and potassium bromide were prepared by compressing the powders at 20 psi for 10 min on KBr-press and the spectra were scanned in the wave number range of 4000-600 cm^-1. FTIR study was carried on pure drug, polymer and final formulations.

Accelerated Stability study^22,23

Stability studies were performed according to ICH guidelines. The microcapsule formulations were stored in hot air oven at room temperature, (37±1) °C and (60±1) °C for a period of 8 weeks. The samples were analyzed for drug content every two weeks by UV-Visible spectrophotometer at 265 nm.

Statistical Analysis²⁴

Statistical data analyses were performed using the one way ANOVA at 5 % level of significance (p < 0.05)and standard error mean (SEM).

Fig 1. Scanning electron photomicrograph of prepared microcapsules (F7) under lower resolution 5 KV X 200.

Fig 2. In vitro drug release profile of different famotidine microcapsule formulations.

RESULTS:

The composition of famotidine loaded mucoadhesive microcapsules in drug polymer ratio of 1:2 were designed and were prepared by different methods. The obtained microcapsules were found to be non aggregated. The percentage yield of all the formulations found in the ranges of 31.1 to 89.3 % as given in Table. The optical microscopy revealed that among all the microcapsule formulations, F4, F7 and F8 were opaque, discrete and spherical particles with smooth surfaces further confirmed by SEM study. The geometric diameter of the microcapsules lies in the ranges of 74.1 to 457.3 µm as represented in Table 1. The drug entrapment efficiency (DEE) of all the formulations was reported as high profile ranges from 43.7 to 89.3 %, which may be due to variation in method of preparation of microcapsules, as abridged in Table 1.

The shape of famotidine microcapsule as evidenced from the scanning electron photomicrograph shown in Fig 1. The percentage of moisture loss was tabularized in Table 1 and found in a range 3.51 to 28.54 %, which ensure the presence of diminutive water content. The minimum moisture content was observed with formulation F6. The famotidine loaded mucoadhesive microcapsules obtained having circularity factor very close to 1.00, which confirm their sphericity, as represented in Table 1. The microcapsules of formulation F7 is retaining minimum drug content at its surface as depicted in Table 2, which is explaining definition of microcapsule. The in vitro famotidine releases from microcapsules prepared by different methods were studied and represented in Fig 2. Few formulations were found to be release famotidine in a controlled manner for a prolonged period over 9 hours. To illustrate the kinetic of drug release from microcapsules, release data was analyzed according to different kinetic equations depicted in Table 2. Release data of all the formulations except F3, F5 and F6, followed zero order kinetic.

Fig 3. Mucoadhesion measurement of various famotidine microcapsules by in vitro wash-off test.

Fig 4. FTIR spectrum of pure drug (Famotidine).

Fig 5. FTIR spectrum of famotidine microcapsule formulation (F7).

The drug release of all the formulations except F1 and F9, following Higuchi square root kinetic model. The Table 2 showed that all the formulations release the drug by diffusion following Fickian (n<0.5) transport mechanism except the formulation F3, F6 and F8, which follow non-Fickian (n>0.5) transport mechanism. The wash-off test for mucoadhesion for microcapsule formulations were represented Fig 3. All the formulations except F3, F6 and F8, are showing good mucoadhesion property. The microcapsule formulation F7 is showing maximum mucoadhesion property. The spectrums of formulation (F7) was compared with the standard spectrum of famotidine to confirm drug polymer interaction by using FT-IR spectrophotometer and principal peaks appeared at 1638, 1535, 1290, 781 cm^-1, identified in both pure drug and the formulation, as depicted in Fig 4 and 5. The accelerated stability studies were performed according to ICH guidelines for 8 weeks and the results were found to be stable in varying temperature, as represented in Table 3. Statistical verification with one way ANOVA method attested the fact that all the data were found to be significant at 5 % level of significance (p < 0.05).

DISCUSSIONS:

The generalized microparticulation protocol depends on, choice of ingredient, successful method of preparation of microcapsules and optimization at every preparative steps. The yields were found to be satisfactory except F1, F2, F3 and F9. The formulation F7 is showing maximum yield. The formulation F1 is showing minimum particle size and formulation F8 is showing maximum particle size. The maximum DEE was shown by formulation F7. The shape of famotidine microcapsule as evidenced from the scanning electron photomicrograph was spherical and uniformly distributed. The presence of diminutive water content which can be due to the involvement of water in process method and hydrophilic property of mucoadhesive polymer. But the lessen proportion of water obtained indicates its proper drying and instant hardening of microcapsule due to quick gelation occurred between calcium chloride and sodium alginate facilitate the storage behavior of the formulations. The sphericity result confirms that microcapsules are of spherical in shape. The loose surface crystal studies lend a hand to estimate the excess amount of drug attached on the surface of microcapsules after a successful drug entrapment. Among all the formulations, microcapsules of formulation F7 were found to release famotidine in a controlled manner with constant fashion over extended period of time. The formulations except F3, F5 and F6, showing a constant release profile, independent of formulation variations. The drug release of all the formulations except F1 and F9, have a diffusion controlled release pattern which is dependent on method of microcapsules preparation. The mucoadhesion result explaining that the method of preparation for microcapsule is also playing an important role in fixing of mucoadhesion property. The FT-IR spectrophotometer showed that there is no drug and polymer interaction.

CONCLUSION:

The formulation F7containing drug: polymer ratio 1:2 prepared by ionotropic gelation method was found to be the best microcapsule formulation, regarding all the properties evaluated in order to achieve objective of this study. The novel formulation design facilitated the optimization and successful development of famotidine microcapsule formulations. Our data concluded that ionotropic gelation method is the best method for designing of famotidine muoadhesive microcapsule, which may be an effective strategy for the development of easy, reproducible and cost effective method to prove its potential for safe and effective controlled for oral drug delivery therapy.

ACKNOWLEDGEMENTS

Authors wish to thank Nicholas Piramol India limited, for providing gift sample of Famotidine. We are also thankful to the staff of Central Drug Research Laboratory, Lucknow and regional research laboratory (RRL), Bhubaneswar, Orissa, for providing important information during study. Author wish to thanks Corel Pharma Ltd., Ahmadabad, for providing carbopol-934 for this study.

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Received on 18.04.2009 Modified on 20.06.2009

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009,;Page 732-737