Formulation, Characterization and In Vitro Evaluation of Mucoadhesive Microspheres of Clarithromycin and Omeprazole

 

Aijaz A. Sheikh¹*, K.R. Biyani¹, N.M. Gawai ¹, Fauziya Firdos¹ and Sonali D. Ingole²

¹ Department of Pharmaceutics, Anuradha College of Pharmacy, Chikhli, Maharashtra, India,

² Department of Pharmaceutics, P.W College of Pharmacy, Yavatmal, Maharashtra, India,

*Corresponding Author E-mail: aijazpsd@gmail.com

ABSTRACT:

The objective of the present investigation was to formulate and evaluate the mucoadhesive microsphere of clarithromycin and omeprazole using hydroxyl propyl methyl cellulose K4M, hydroxyl propyl methyl cellulose K100M and carbopol 971p. Microspheres were prepared by non-aqueous emulsification solvent evaporation method using liquid paraffin as a cross linking agent. The prepared microspheres were evaluated for micromeritic properties.  Batch CF5 and OF5 shows the highest adhesion property i.e. 08 hrs and 7.55 hrs respectively. Likewise batch CF5 and OF5 also shows highest % drug entrapment i.e. 82% and 96.26% respectively and declared as an optimized batches. These batches were filled in 0 size capsules and evaluated for weight variation, content uniformity and % cumulative drug release.

 

 

 

INTRODUCTION:

Mucoadhesion is topic of current interest in the design of drug delivery system. The term ‘mucoadhesive’ is commonly used for materials that bind to the mucin layer of a biological membrane. Mucoadhesive drug delivery system   utilizes the property of bioadhesion of certain polymers which become adhesive on hydration and can be used for targeting a drug to a particular region of the body for extended periods of time^{1, 2, 3}. Mucoadhesive drug delivery systems promises several advantages that arise from localization at a given target site, prolonged residence time at the site of drug absorption and an intensified contact with the mucosa increasing the drug concentration gradient^{4, 5}. Hence, uptake and consequently bioavailability of the drug is increased and frequency of dosing reduced with the result that patient compliance is improved^{6 7}. Helicobacter pylori (H.pylori) are a gram‐negative bacillus responsible for one of the most common infections found in humans worldwide⁸. H.pylori causes gastric diseases, such as peptic ulcer, gastric mucosa associated lymphoma. One reason for the incomplete eradication of H.pylori is probably due to the short residence time of antimicrobial agents in the stomach so that effective antimicrobial concentration cannot be achieved in the gastric mucous layer or epithelial cell surfaces where H. pylori exists⁹.

 

The main objective of this work is to formulate and evaluate the mucoadhesive microspheres containing clarithromycin and omeprazole as an anti‐H. pylori agent that will remain in vicinity of absorption site for prolonged period of time.

 

MATERIAL AND METHODS:

Clarithromycin and omeprazole was a kind gift from Medley Pharmaceutical Ltd., Daman, India. Carbopol 971p, HPMCK 4M, HPMC K100M were supplied by Lubrizol Pvt. Ltd., Mumbai. All other chemicals were of analytical grade and were purchased from Concept Pharma, Aurangabad, Maharashtra, India.

 

 

Formulation of microspheres:

The microspheres were prepared by non-aqueous emulsification solvent evaporation method. Briefly, clarithromycin and polymers were mixed in ethanol and dichloromethane. The slurry was introduced in to 200 ml of liquid paraffin while being stirred at given rpm by mechanical stirrer for given time to allow the solvent to evaporate completely and the microspheres were collected by filtration. The microspheres were washed repeatedly with petroleum ether (40 –50 ^o C) until free from oil. The collected microspheres were dried for 1hr at room temperature and subsequently stored in desiccators over fused calcium. The same procedure was followed for preparation of omeprazole microsphere.

 

 

Table 01: Formulation of microspheres

Batches	CF1	CF2	CF3	CF4	CF5	CF6	OF1	OF2	OF3	OF4	OF5	OF6
Clarithromycin (mg)	300	300	300	300	300	300	---	---	---	---	---	---
Omeprazole (mg)	---	---	---	---	---	---	300	300	300	300	300	300
Carbopol 971p (mg)	300	300	150	150	150	150	300	300	150	150	150	150
HPMC  K4M (mg)	---	---	150	150	---	---	---	---	150	150	---	---
HPMC  K100M (mg)	----	----	----	----	150	150	----	----	----	----	150	150
Ethanol (ml)	36	36	36	36	36	36	36	36	36	36	36	36
Dichloromethan (ml)	36	36	36	36	36	36	36	36	36	36	36	36
Liquid paraffin (ml)	200	200	200	200	200	200	200	200	200	200	200	200
RPM	1000	750	1000	750	1000	750	1000	750	1000	750	1000	750
Time (min)	45	60	45	60	45	60	45	60	45	60	45	60

 

 

Evaluation of microspheres:

1)       Particle size determination

Particle size was determined by using an optical microscope under regular polarized light, and the mean particle size was calculated by measuring 50-100 particles with the help of a calibrated ocular micrometer^{1, 2}.

 

2)       Tapped Density:

The sample of about 10 g of powder is carefully introduced into a 25 ml graduated cylinder.  The cylinder was dropped at 2 second intervals onto a hard wood surface 100 times from a height of 1 inch. It was calculated by using equation given below,

                D_o = M / Vp

Where,

D_o = bulk density

M = weight of samples in grams

Vp = final tapped volumes of granules in cm³

 

3)       Angle of repose:

The Angle of repose (θ) i.e. flow property of the microspheres which measures the resistance to particle flow was  calculated as,

                tan θ  =  2H / D

Where, 2H / D is the surface area of the free standing height of the microspheres heap that is formed after making the microspheres flow from the glass funnel ^{10, 11, 12}.

 

 

Table 02: Micromeritics studies of clarithromycin microspheres. (S.D. n=3)

Batches	Average particle size(mm) S.D. n=3	Tapped density (g/cm3)	Angle of repose (q)
CF1	369.13 (11.2)	0.47 (0.08)	30 o 21
CF2	324.65 (15.6)	0.48 (0.008)	29 o 74
CF3	312.05 (11.4)	0.48 (0.004)	29 o 74
CF4	281.46 (7.8)	0.47 (0.008)	27 o 21
CF5	256.16 (8.4)	0.49 (0.008)	30 o 21
CF6	252.16 (11.4)	0.49 (0.004)	30 o 12

 

Table 03: Micromeritics studies of omeprazole microspheres. (S.D. n=3)

Batches	Average particle size(mm) S.D. n=3	Tapped density (g/cm3)	Angle of repose (q)
OF1	325.65 ( 15.4)	0.49 (0.008)	30. o 21
OF2	312.05 (11.4)	0.47 (0.008)	27 o 21
OF3	312.13 ( 8.46)	0.48 (0.004)	29 o 74
OF4	270.81 (14.4)	0.47 (0.004)	28 o 76
OF5	281.46 (7.8)	0.46 (0.003)	26 o 21
OF6	256.16 (8.8)	0.48 (0.004)	27 o 21

 

4)       Adhesion property

A freshly cut of 5 cm long piece of pig intestine obtained from a local abattoir within 1 hr of killing the animal was cleaned by washing with isotonic saline solution. An accurate weight of microspheres was placed on mucosal surface which was attached over a polyethylene plate that fixed in an angle of 40º relative to the horizontal plane, and 0.1NHCl warmed at 37ºC was peristaltically pumped at a rate of 5 ml/min over the tissue. The duration for completely washing of microspheres from pig intestine was recorded and averaged from five determinations ^{13, 14}.

 

Table 0 4: Adhesive time study of clarithromycin microspheres

Batch   code	Adhesive Time in hrs
CF1	6
CF2	5.4
CF3	6.5
CF4	6.45
CF5	8
CF6	7.35

 

Figure 01: Adhesive time study of clarithromycin microspheres

Table 0 5: Adhesive time study of omeprazole microspheres

Batch   code	Adhesive Time in hrs
OF1	6.1
OF2	5.45
OF3	6.45
OF4	6.55
OF5	7.55
OF6	7.2

 

Figure 0 2: Adhesive time study of  omeprazole microspheres

 

5)       Drug entrapment efficiency

Microspheres equivalent to 20 mg of the drug clarithromycin were taken for evaluation. The amount of drug entrapped was estimated by crushing the microspheres and extracting with aliquots of 0.1N HCl repeatedly. The extract was transferred to a 100 ml volumetric flask and the volume was made up using 0.1N HCl. The solution was filtered and the absorbance was measured after suitable dilution spectrophotometrically at 282 nm against 0.1NHCl as a blank. The amount of drug entrapped in the microspheres was calculated by the following formula,

                                      Calculated drug concentration

% Drug entrapment  =  ------------------------------------ x 100

                                     Theoretical drug concentration

 

Same procedure was repeated for omeprazole microspheres and absorbance was measured at 305nm.

 

Table 06: Drug entrapment efficiency of clarithromycin (S.D. n=3)

Batch No.	Drug entrapment efficiency
	Drug Content	% Drug entrapment of clarithromycin
CF1	40.9 (0.40)	68.0
CF2	41.8 (0.52)	69.8
CF3	43.8(1.1)	73.0
CF4	45. 0 (0.7)	75.0
CF5	54.66 (1.5)	82.0
CF6	46.8 (2.0)	78.0

 

Table 07:  Drug entrapment efficiency of omeprazole (S.D. n=3)

Batch No.	Drug entrapment efficiency
	Drug Content	% Drug entrapment Of omeprazole
OF1	49.5 (0.60)	51.0
OF2	50.5 (1.3)	65.0
OF3	52.8 (0.45)	70.0
OF4	55.3(0.60)	73.0
OF5	89.12 (1.4)	96.26
OF6	57.0 (1.6)	75.0

6)       Formulation of capsule single unit dosage form of optimized batch:

Capsules size was selected according to the volume of microsphere needed to deliver the drug dose. The contents were filled in the tarred capsule, which were then reweighed. Care was taken to fill the capsule completely in order to maintain a uniformity of weight. The optimized batch containing clarithromycin and omeprazole was evaluated for general appearance, weight variation, content uniformity and in-vitro drug release studies.

 

Table 08: Formulation of capsule of optimized   batch

Batches	Microsphere equivalent to drug dose(mg)	Capsule size
CF5	20	0
OF5	20	0

 

7)       Evaluation of capsule:

a)       General appearance:

The capsules were visually evaluated for texture, pinholes, deformity, and appearance.

 

Table 09:  General appearance of capsule

Evaluation parameter	Observation
Texture	Fine and smooth
Pinholes	None
Deformity	None
Appearance	Satisfactory

 

b)       Weight variation:

This test was conducted on 20 capsules. Each capsule was individually weighed to find out weight variation. Average weight was calculated.

 

Table 10: Weight variation test   (S.D. n=3)

Average weight (mg)	Permitted limit (mg)	Range of all capsule (mg)	Permitted limit of  variation	Pass/ fail
650.43 (1.25)	682.95-617.91	631.43-660.45	±.5%	Pass

 

c)       Content uniformity test:

10 capsules were accurately weighed, remove the hard gelatin shells and powder equivalent to 20mg of drug clarithromycin and dissolved it in 1N NaOH. Drug content was calculated by measuring absorbance at wavelength 282.00 nm. Same procedure is followed for omeprazole at wavelength 305.00 nm.

 

Table 11:  Content uniformity test

Batches.	Amount of drug (mg) (n= 10)	% Drug content
CF5	224.13(2.2)	89.65
OF5	18.52 (1.3)	92.6

 

d)       In-vitro drug release:

The release rate of drug clarithromycin and omeprazole from capsule was determined using USP dissolution testing apparatus I (Basket type). The dissolution test was performed using 900 ml of 0.1 N HCl, at 37 ± 0.5ºC and 100 rpm. A sample (5 ml) of the solution was withdrawn from the dissolution apparatus at various sampling time 0 min, 1hr, 2hr, 3hr, 4hr and so on), and the samples were replaced with fresh dissolution medium to avoid sink condition. The samples were filtered through Whatman filter paper no. 41. Absorbance of these solutions was measured at 282 nm for clarithromycin and 305.0 nm for omeprazole.

 

Table 12:  Cumulative drug release of optimized batches of capsule   (S.D. n=3)

Time (hrs)	Capsule of CF5 batch	Capsule of OF5 batch
0	00.00	00.00
1	24.01(0.28)	34.31(0.19)
2	42.23(0.26)	43.87(0.37)
3	51.42 (0.42)	52.829(0.51)
4	58.92 (0.30)	58.09(0.42)
5	59.33 (0.44)	65.8(0.58)
6	66.42 (0.53)	81.89(0.73)
7	69.06 (0.63)	91.87(0.82)
8	91.36 (0.83)	95.69(0.93)

 

Figure  03: Cumulative release of  CF5 and OF5 Batches

 

RESULT AND DISCUSSION:

The present study was carried out to develop mucoadhesive drug delivery system in the form of microsphere dosage form in combination of clarithromycin and omeprazole. The prepared microspheres of clarithromycin and omeprazole gave good micromeritic results. The various batches have the average particle size in the range of 252mm to 370 mm. The tapped density was found to be in range of 0.47-0.49 g/cm³ and angle of repose in between 27^o 21 -30^o 21. Microparticles which prepared under optimized conditions i.e., agitation speed 1000 rpm and stirring time 45 min were found to be spherical in shape, uniform in size and exhibited a smooth surface. CF5 and OF5 batches showed highest adhesion property and % drug entrapment. Depending up on these results batches CF5 and OF5 were optimized and selected for further studies. The optimized batch of capsules showed fine and smooth texture with satisfactory appearance and passes the weight variation test. The % drug content for CF5 and OF5 batches were 89.65 % and 92.6% respectively (Table 11). The triplicate study in-vitro dissolution was carried out in 0.1 N HCl. The CF5 and OF5 batches showed 91.36% and 95.69% cumulative drug release in 08 hrs of study (Fig.03).

 

CONCLUSION:

Mucoadhesive microsphere of clarithromycin and omeprazole were prepared successfully by using non-aqueous emulsification solvent evaporation method. The method followed was economical to get reproducible microspheres.

 

REFERENCES:

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Received on 15.07.2011          Modified on 09.08.2011

Accepted on 22.08.2011         © RJPT All right reserved