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PREPARATION AND CHARACTERIZATION OF FLOATING BIOADHESIVE MICROBEADS OF CLARITHROMYCIN

 

Clinical courses

About Authors:
*Shaikh T. K., Jadhav S.L., Gadhave M.V.
Department of Pharmaceutics,
VJSM’s Vishal Institute of Pharmaceutical Education and Research, Ale,
Pune (411412)
*taiyyab1234@gmail.com

Abstract-
Helicobacter pylori (H. pylori) are one of the most common pathogenic bacterial infections, colonizing an estimated half of all humans. It is associated with the development of serious gastro duodenal disease—including peptic ulcers, gastric lymphoma and acute chronic gastritis. H. pylori reside mainly in the gastric mucosa or at the interface between the mucous layer and the epithelial cells of the antral region of the stomach. The discovery of this microorganism has revolutionized the diagnosis and treatment of peptic ulcer disease. Most antibacterial agents have low minimum inhibitory concentrations (MIC) against H. pylori in culture.


Clarithromycin is readily soluble in the acidic environment of the stomach. In the intestine, where neutral to slightly alkaline pH conditions prevail; however, solubility of the active compound is low, which adversely affects absorption in the lower sections of the intestine due to this oral bioavailability of clarithromycin is low (50 to 55%). Local delivery of clarithromycin floating Microbeads are more beneficial for H. pylori infection in stomach because prolonged residence in stomach which could be possible with cytoprotective-Gastroretentive drug delivery system.Site of absorption of clarithromycin is upper part of GIT, hence delivering the drug through sustained release gastro retentive drug delivery reduces the size of dose.

REFERENCE ID: PHARMATUTOR-ART-1883

1.Introduction
Dosage forms that can be retained in stomach are called Gastroretentive drug delivery systems (GRDDS). GRDDS can improve controlled delivery of drugs that have an absorption window by continuously releasing the drug for a prolonged period of time before it reaches its absorption site, thus ensuring its optimal bioavailability1. Drugs having narrow absorption window are mostly associated with improved absorption at jejunum and ileum due to their enhanced absorption properties e.g. large surface area, or because of enhanced solubility in stomach as opposed to the more distal parts of the GIT2.

Approaches of development of dosage form for H.Pylori
Helicobacter pylori are one of the most common pathogenic bacterial infections, colonizing an estimated half of all humans. It is associated with the development of serious gastro duodenal disease - including peptic ulcers, gastric lymphoma and acute chronic gastritis. Current recommended regimes are not wholly effective and patient compliance, side-effects and bacterial resistance can be problematic. Drug delivery to the site of residence in the gastric mucosa may improve efficacy of the current and emerging treatments. Gastric retentive delivery systems potentially allow increased penetration of the mucus layer and therefore increased drug concentration at the site of action. Proposed gastric retentive systems for the enhancement of local drug delivery include floating systems, expandable or swellable systems and bioadhesive systems. Generally, problems with these formulations are lack of specificity, limited to mucus turnover or failure to persist in the stomach. Gastric mucoadhesive systems are hailed as a promising technology to address this issue, penetrating the mucus layer and prolonging activity at the mucus-epithelial interface. This review appraises Gastroretentive delivery strategies specifically with regard to their application as a delivery system to target Helicobacter.


1.1. Advantages Of Cytoprotective- Gastroretentive- Drug Syetem
Delivery Systemcytoprotective- Gastroretentive- Drug Delivery System greatly may improve pharmacotherapy of the stomach through local drug release leading to high drug concentrations at gastric mucosa (eradicating Helicobacter pylori from the submucosal tissue of the stomach), making it possible to treat stomach and duodenal ulcers, gastritis, and oesophagitis, reduce the risk of gastric carcinoma by antibiotics and administer non-systemic, controlled release antacid formulations (Calcium Carbonate).

1.2.Ideal Drug Candidates for Compounding In To Delivery System cytoprotective- Gastroretentive- Drug Delivery System


  • Drugs stable in gastric milieu.
  • Drugs having narrow absorption window.
  • Drugs to be used for gastro-duodenal local therapy.

1.3.Drugs incorporated into Delivery System cytoprotective-Gastroretentive- Drug Delivery System
Delivery System cytoprotective- Gastroretentive- Drug Delivery Systemcan be ideal carrier for drug loaded Microbeads, microcapsules, nanoparticles, vesicular systems, granules, coated granules, etc. Delivery System cytoprotective- Gastroretentive- Drug Delivery Systemcan be formulated in the form of tablet, capsule, chewable tablet, in-situ gel, suspention,or emulsion.

1.4.Disadvantages of Gastroretentive systems
There are certain situations where gastric retention is not desirable. and slow release of such drugs in the stomach is unwanted.. Furthermore, other drugs such as Isosorbide dinitrate, that are absorbed equally well throughout the GI tract will not benefit from incorporation into a gastric retention system.

1.5.Development Of Floating Microbeads
Floating Microbeads are gastro-retentive drug delivery systems based on non-effervescent approach. Hollow Microbeads are in strict sense, spherical empty particles without core. These Microbeads are characteristically free flowing powders consisting of proteins or synthetic polymers, ideally having a size less than 200 micrometer. Solid biodegradable Microbeads incorporating a drug dispersed or dissolved throughout particle matrix have the potential for controlled release of drugs3. Gastro-retentive floating Microbeads are low-density systems that have sufficient buoyancy to float over gastric contents and remain in stomach for prolonged period. As the system floats over gastric contents, the drug is released slowly at desired rate resulting in increased gastric retention with reduced fluctuations in plasma drug concentration.

1.6.Mechanism Of Floating Microbeads
When Microbeads come in contact with gastric fluid the gel formers, polysaccharides, and polymers hydrate to form a colloidal gel barrier that controls the rate of fluid penetration into the device and consequent drug release. As the exterior surface of the dosage form dissolves, the gel layer is maintained by the hydration of the adjacent hydrocolloid layer. The air trapped by the swollen polymer lowers the density and confers buoyancy to the Microbeads. However a minimal gastric content needed to allow proper achievement of buoyancy4,5. Hollow Microbeads of acrylic resins, eudragit, polyethylene oxide, and cellulose acetate; polystyrene floatable shells; polycarbonate floating balloons and gelucire floating granules are the recent developments.

2. EXPERIMENATAL WORK

Materials -

Name of chemicals

Supplied /Gifted by

Clarithromycin

Biochem Pharmaceuticals Daman, India

HPMC

Research Lab.

Calcium carbonate

Research Lab.

Sodium Alginate

Research Lab.

Glacial acetic acid

Research Lab.

Calcium chloride

Research Lab.

Hydrochloric acid

Research Lab.

A.Preparation of HPMC Alginate bead
The sodium alginate beads were prepared by ionotropic gelation method. Sodium alginate beads formed as alginate undergoes ionotropic  gelation by sodium ions  developed from the reaction of  alginate and acetic acid.

a) Preparation of Dope Solution:
SodiumAlginate was dissolved in distilled water at specific concentration; the solution was stirred thoroughly after drug added.

b) Preparation of Coagulation Solution:
Homogeneous aqueous solution of HPMC and calcium chloride in various ratios were prepared. The gelation medium was prepared by dissolving chitosan in 1% glacial acetic acid. Solutions mixed for 2 hr’s before use and pH of Coagulation fluid adjusted 4.5±0.1.The homogenous alginate solution was extruded using a 22G syringe needle into the coagulation/ gelation medium. The distance between the edge of the needle and the surface of the gelation medium was about 10 cm. The gel beads formed were left in the solution with gentle stirring for specific time at room temperature to be cured. After beads were collected, washed with distilled water twice and oven-dried subsequently (40 °C).[6]

3. PREPARATION OF CLARITHROMYCIN FLOATING MICROBEADS
The weighed amount of sodium alginate was dissolved in water. The amount of sodium alginate was added in various percentages (2.0%-4.0%), stirred for 1 hr. using mechanical stirrer (Remi equipments, Mumbai). The weighed amount of Clarithromycin was dispersed in to polymeric solution and stirred for 30 min. and then allowed to stand in sonicator till the removal of entrapped air bubbles. After sonication, this solution was added dropwise from the distance 5 cm. using syringe fitted with needle (22G) in to coagulation fluid 200 ml consisting of calcium chloride solution (3%) and chitosan (0.5-1.5) dissolved in 1% acetic acid in 250 ml beaker kept on magnetic stirrer at room temperature. Beads were left for curing for specified time and after curing, beads were collected by filtration and washed twice with distilled water and allowed to dry at 40?C for 24 hrs. The dried beads were weighed and stored for further evaluation.[6,7]

Prepared beads were evaluated for bead size, sphericity of beads, % entrapment efficiency, % drug content, % swelling index and % drug release.

4. EVALUATION OF BEADS
4.1. Particle size and shape:[8] 
About 100 beads were randomly picked up thrice and their sizes of dried beads were measured by using Stage micrometer. Shape of bead was observed by visual observation.

4.2. Percentage drug content and % entrapment efficiency:[9]
An accurately weighed quantity of 10 mg beads were taken and crushed in mortar with pestle and dissolved in 10 ml of phosphate buffer. The resultant suspension was centrifuged 3600 rpm for 30 min. The solution was sonicated for 2-3 hrs. using sonicator (Citizen). The resultant dispersion was filtered through Whattman’s filter paper (No.041) and analysed at 270 nm using UV spectrophotometry (Shimadzu, 1800). The experiments were done in triplicate and results were calculated.

A) Percentage Drug Content:
The % drug content was calculated by formula:

% Drug content = DW/TW × 100  

Where, DW – amount of drug found in total dried beads,
TW - Total weight of dried Beads.

B)  % Entrapment Efficiency:
The encapsulation efficiency was calculated according to the following relationship.

% Entrapment efficiency= AQ/TQ × 100

Where, AQ- actual amount of drug found in the beads,
TQ- Theoretical amount of drug found in the beads.

4.3. Equilibrium Swelling studies:[8,9,10]
The accurately weighed dried  beads were placed in USP dissolution apparatus II containing 900ml, phosphate buffer (pH 6.8) maintained at 37±20C and allowed to swell upto constant weight. The beads were removed, blotted with filter paper, and changes in weight were measured. The experiments were carried out in triplicate. The degree of swelling (Swelling index) was then calculated from the formula,

                          (Wg –Wo)
Swelling index =  -------------    × 100
                             Wo

Where, Wo is the initial weight of beads and Wg is the weight of beads at equilibrium swelling in the medium.

4.4. In vitro Dissolution Studies:[9,11]
In vitrodissolution studies were performed for all the formulation using USP XXIII Dissolution test apparatus II (paddle type). An accurately weighed sample of beads containing 150 mg ofClarithromycin drug was dropped into 900 mL of 0.1 N HCl pH1.2 maintained at a temperature of 37ºC ± 0.5ºC and stirred at a speed of 50 rpm. At different time intervals, a 5 mL aliquot of the sample was withdrawn and the volume was replaced with an equivalent amount of plain dissolution medium kept at 37ºC.  After 2 hr’s Same procedure was done into phosphate buffer 6.8 pH. The collected samples were filtered using Whattman’s filter paper (No.041) and analyzed at λmax 270 nm using a UV-visible spectrophotometer against 0.1N HCl and phosphate buffer 6.8 PH as blank. The results expressed were the mean of three experiments.

Sr. No.

Specification

Standard values

1

Apparatus

USPXIII  dissolution apparatus II

2

Speed

50 rpm

3

Volume of media

900 ml

4

Dissolution Media used.

0.1 N HCL solution,

phosphate buffer 6.8 pH

5

Stirrer

Paddle type.

6

Aliquot taken at each time interval of 1 hr

5 ml.

7

Temperature

37+ 0.5o C.

8

λ max

270 nm

Table No. 4.3. Dissolution test details for dissolution of beads:

4.5. Surface morphology of the beads:[8,9]
The external surface morphology and their internal cross section image of optimized batch of beads were obtained by scanning electron microscope (SEM, Philips XL20, Holland) under vacuum. The samples for SEM were prepared by mounting dried beads on a double adhesive tape stuck to an aluminium stub. The stubs were then coated with platinum to a thickness of about 10 Å under an argon atmosphere using a gold sputter module in a high-vacuum evaporator. Afterwards, the stubs containing the coated samples were placed in the scanning electron microscope (JSM-6360A, JEOL and Tokyo, Japan) chamber. The samples were then randomly scanned and photomicrographs were taken at the acceleration voltage of 10 kV.

Fig. SEM MORPHOLOGY OF CLARITHROMYCIN MICROBEADS

4.6.Determination of thermal behaviour by Differential scanning Calorimetry (DSC):[9]
The differential scanning calorimetric analysis of optimized batch wascarried out using Differential Scanning Calorimeter (Mettler Toledo). Sample of optimized batch wasplaced in a platinum crucible and the DSC thermograms were recorded at a heating rate of 10ºC/min in the range 30ºC to 300ºC. Nitrogen gas was purged at the rate of 30 ml/min. to maintain inert atmosphere.

Fig. DSC Study of Clarithromycin and CL Microbeads

Result of DSC study shows the CL shows thermal peak at 220 oC and the microbeads formulation also given the same characteristic peak at 220.31oC indicates no endothermic degradation of CL in the formulation.

4.7. FTIR:
FTIR study ofClarithromycin, chitosan polymer, and Clarithromycinloaded optimized batch of chitosan were carried out using FTIR Spectrophotometer (FTIR-8400S,).  Placebo beads were crushed in mortar and IR was recorded by potassium bromide dispersion technique in which crushed placebo beads and potassium bromide was placed in sample holder and IR was recorded using FTIR Spectrophotometer (FTIR-8400S).

Fig. IR SPECTRUM OF CLARITHROMYCIN

4.8. Drug release profile and model fitting:
The release data were evaluated by using Disso MS-Excel. The dissolution data for Beads was apply for various drug release kinetic models like Zero order, First order, Matrix and Korsemeyer Peppas model was determined.

4.9. Stability studies:
The stability studies for beads were done by keeping the sample beads from optimized batches for 2 months. The beads were filled in capsule and sealed in aluminium packaging coated inside with polyethylene and kept in humidity chamber maintained at 40?C temperature and 75% relative humidity for 2 months. At the end of 2 months the sample were analysed  for different parameters like physical appearance, % drug content, % entrapment efficiency, % swelling ratios and % drug release studies.

4.FORMULA DEVELOPMENT:-

FORMULATION

F1

F2

F3

F4

F5

F6

F7

F8

F9

Carithromycin(mg)

250

250

250

250

250

250

250

250

250

Sod. Alginate(%)

2

2

2

3

3

3

4

4

4

HPMC K4M (%)

1

1.25

1.5

1

1.25

1.5

1

1.25

1.5

Chitosan (%)

0.5

1

1.5

0.5

1

1.5

0.5

1

1.5

CaCO3 (%)

1

1

1

1

1

1

1

1

1

5.DRUG RELEASE PROFILE OF MICROBEADS:-

Time
(hr)

F 1 %
Release

F 2 %
Release

F 3 %
Release

F 4 %
Release

F 5 %
Release

F 6 %
Release

F 7 %
Release

F 8 %
Release

F 9 %
Release

0

0

0

0

0

0

0

0

0

0

0.5

18.45

16.4

17.3

18.32

16.33

17.12

16.49

18.25

18.5

1

36.19

31.94

32.76

33.54

31.93

33.24

30.1

33.76

33.51

2

54.78

49.53

50.3

51.35

49.51

51.25

48.41

52.5

52.8

3

61.43

58.41

59.43

60.67

59.3

61.6

58.73

62.74

62.1

4

68.53

65.91

65.87

66.12

65.9

67.45

64.5

66.29

66.32

5

78.95

76.45

75.87

76.32

74.5

75.2

73.78

75.32

75.34

6

87.49

87.42

87.45

87.41

86.94

87.1

86.34

88.12

88.43

7

96.86

96.13

95.7

94.76

94.12

94.98

94.34

93.43

94.23

8

99.34

97.39

97.34

96.98

96.42

97.12

97.95

98.1

98.2

References-
1.Ross and Wilson “Text book of anatomy and physiology”
2.P.S. Rajinikanth , J. Balasubramaniam, B. Mishra,  “Development and evaluation of a novel floating in situ gelling system of amoxicillin for eradication of Helicobacter pylori”, International Journal of Pharmaceutics 335 (2007) 114–122
3.Chickering DE , Jacob JS. and Matho WE. Reactive Polymers 1995;(25):189-206.
4.Seng CH.J Pharm Sci 1995;74(4):399-405.
5.Chawla C, Gupta P, Koradia V, Bansal AK, Gastroretention: A Means to Address Regional Variability in intestinal drug Absorption. Pharmaceutical technology, 2003;27(2):50-68.
6.Anil K., Willem F. Stevens Chitosan–alginate multilayer beads for controlled release of ampicillin 290 (2005) 45–54.
7.Anal, A., Bhopatkar, D., Tokura, S., Tamura H., Stevens, 2003. Chitosan–alginate multilayer beads for gastric passage and controlled intestinal release of protein. Drug Dev. Ind. Pharm. 29, 713–724.
8.Anandrao R. Kulkarni, Kumaresh S. Soppimath, Tejraj M. Aminabhavi. Ontrolled release of diclofenac sodium from sodium alginate beads crosslinked with glutaraldehyde Elsevier science 74(1999)29-36.
9.Srinatha A., Pandit J. Ionic Cross-linked Chitosan Beads for Extended Release of Ciprofloxacin: In vitro Characterization. Eur. J. Pharm. Biopharma. 54 (2004), 252–263.
10.George p., Nikolaos B., Swelling studies and in vitro release of verapamil from calcium alginate and calcium chitosan beads. Sci 323(2006)34-42.
11.Fatemeh A., Sayeh M., Maryam I., Masoud S., Farid D., In vitro evaluation and modification of pectinate gel beads containing trimethyl chitosan, as a multi-particulate system for delivery of water-soluble macromolecules to colon, Sci Dir., 61(2005)39-51

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