ISSN: 0975-8585

Research Journal of Pharmaceutical, Biological and Chemical

Sciences

Formulation and Evaluation of Clarithromycin loaded Mucoadhesive

Microspheres for Anti- Helicobacter pylori Effect

Venkateswaramurthy N*, Sambathkumar R, Perumal P

JKK. Nataraja College of Pharmacy. Komarapalayam. Namakkal District -[Link], India.

ABSTRACT

It is well documented that Helicobacter pylori infection can use chronic peptic ulceration and can increase
the risk of gastric denocarcinoma. The wide use of antibiotic therapies for H. pylori infection has also increased
the number of therapeutic failures. Recent data show a decreasing efficacy of these therapies worldwide. 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 exists. The purpose of this study was to design mucoadhesive
microspheres containing Clarithromycin as an anti-H. pylori agent to deliver the drug specifically to mucus layer
where [Link] resides and evaluate the effectiveness of the mucoadhesive microspheres for H. pylori eradication
therapy. Microspheres were prepared by using Eudragit RL100 as matrix and Carbopol 974P and Hydroxy propyl
methyl cellulose K4M as mucoadhesive polymer. The microspheres were prepared by emulsion solvent
evaporation technique. The prepared microspheres were evaluated with respect to the particle size, production
yield, encapsulation efficiency, shape and surface properties, mucoadhesive property, In-vitro drug release and
suitability for anti Helicobactor pylori effect. The preliminary results show great promise for this delivery strategy
in the treatment of H. Pylori infection.

Key words: clarithromycin, [Link], microsperes, mucoadhesive

*Corresponding author
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INTRODUCTION

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that causes one of the most common chronic
infections in humans. The infection produces chronic gastritis and predisposes to the development of peptic ulcer
]
disease [1,2] . Clarithromycin has highest rate of eradication of H. pylori in monotherapy in vivo [3], However,
some other reports and clinical trials indicate that the therapies cannot bring out complete eradication of H. pylori
and suggest that the therapeutic effect needs more investigation [4,5]. 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 exists [6,7]. Mucoadhesive microspheres highly suitable drug delivery system for [Link] eradication
because it specifically bind with mucus where [Link] resides and deliver the antibiotic for longer period. The
purpose of this study was to design mucoadhesive microspheres containing Clarithromycin as an anti-H. pylori
agent and to evaluate the effectiveness of the mucoadhesive microspheres for H. pylori eradication therapy.

MATERIALS AND METHODS

Materials

Clarithromycin was gigted by Ranbaxy laboratories Ltd, New Delhi, India, Hydroxypropyl methyl cellulose
K4M was obtained as gifts from Colorcon Asia Pvt. Ltd., Mumbai, India and Carbopol 974P was a gift from BF
Goodrich Co., Germany. Eudragit RL 100 was a gift sample from Microlabs, Bangalore. All other reagents and
chemicals used were of analytical grade.

Preparation of Microspheres

Microspheres were prepared by a solvent evaporation method. The solvent system acetone/liquid
paraffin was used. Agglomeration of microspheres was prevented by using 1% w/v Span80. Eudragit RL 100 was
used to form a matrix of microspheres and mucoadhesive polymer were chosen to produce mucoadhesion is
Carbopol 974P and Hydroxypropyl methyl cellulose K4M. Eudragit RL 100 was dissolved in acetone and weighed
quantity of Clarithromycin, Carbopol 974P and Hydroxypropyl methyl cellulose K4M were dispersed it. The total
volume of acetone was 12 ml. This homogeneous final dispersion was cooled to 5 °C and poured slowly with
stirring (700 rpm) into 80 ml of liquid paraffin containing 1% w/v span 80, which was previously also cooled to 5 °C.
The obtained emulsion was stirred at 40 °C for 40 min. The suspension of microspheres in liquid paraffin was
filtered and microspheres were washed by n-hexane and dried in vacuum at room temperature overnight.

Scanning electron microscopy

Scanning electron photomicrograph of Clarithromycin loaded mucoadhesive microspheres were taken. A

small amount of microspheres was spread on glass stub. Afterwards, the stub containing the sample was placed in
the scanning electron microscope (JSM 5610 LV SEM, JEOL, Datum Ltd, Tokyo, Japan) chamber. Scanning electron
photomicrograph was taken at the acceleration voltage of 20 KV, chamber pressure of 0.6 mm Hg, at different
magnification.

Particle size measurement

The prepared microspheres were sized by using a Malvern 2600 Laser Diffraction Spectrometer. The size
of the microspheres was determined in n-hexane as a non-dissolving dispersion medium and the particles were
suspended mechanically by magnetic stirring during the measurement.

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Degradation of clarithromycin in pH 1.2 [8]

The degradation rate of the antimicrobial agent at pH 1.2 was examined by reported method with slight
modification. A known amount of clarithromycin was added to the medium, which was preheated at 37ºC±0.2°C,
to make a final concentration of 10.0 μg/ml. An aliquot of the medium was withdrawn at predetermined time
intervals and neutralized with a NaOH solution before being quantified by HPLC. Then the solution was filtrated
through a 0.45 μm syringe filter then analyzed for clarithromycin content by reversed-phase high performance
liquid chromatography (RPHPLC) method using a mobile phase consisting of acetonitrile–aqueous 0.05 M
phosphate buffer solution of pH 4.0 (40:60 v/v). The apparatus used for HPLC analysis was an Agilent 1100
quaternary pump, with a variable wavelength detector, thermostatted autosampler and column thermostat. A
Hypersil ODS C18 column (250mm×4.6mm ID, 5 μm, Thermo, UK) was fitted with a Phenomenex guard column
packed with octadecyl C18 (Phenomenex, USA).The column temperature was maintained at 40°C and flow rate of
1ml/min. The The concentrations of the parent drug remaining were analyzed by RP-HPLC assay. The degradation
of clarithromycin was assumed to follow pseudo-first order kinetics, which is described by the following equation:

C=Coe−kt

In which C is the concentration of clarithromycin remaining at time t, Co is the initial concentration of

clarithromycin, and k is the pseudo-first order degradation rate constant. The half-life (t1/2) of clarithromycin was
determined from the pseudo-first order degradation rate constant. Degradation rate constant used to correct the
drug release data obtained in acidic media.

Determination of drug encapsulation efficiency

To determine the total drug content of microspheres a known amount of microspheres wereground to
fine powder. Accurately weighed (50mg) grounded powder of microspheres weresoaked in 50 ml of distilled water
and sonicated using probe sonicator for 2 h. The whole solution was centrifuged using a tabletop centrifuge to
remove the polymeric debris. Then the polymeric debris was washed twice with fresh solvent (water) to extract
any adhered drug. The clear supernatant solution was filtrated through a 0.45 μm syringe filter then analyzed for
Clarithromycin content by high performance liquid chromatography and the conditions for the HPLC assay were
the same as before.

Vitro Drug Release Studies

Release of Clarithromycin from the microspheres was studied in 0.1N HCL (900 mL) using a USP XXIII
paddle method Dissolution Rate Test Apparatus ( Dissco 2000, Labindia) with a rotating paddle stirrer at 50 rpm
and 37° ± 1°C. A sample of microspheres equivalent to 25 mg of Clarithromycin was used in each test. Samples of
dissolution fluid were withdrawn through a filter (0.45 μm) at different time intervals and were assayed for drug
release by high performance.

In- vitro evaluation of mucoadhesivenes [9]

A strip of goat intestinal mucosa was mounted on a glass slide and accurately weighed mucoadhesive
microspheres in dispersion form was placed on the mucosa of the intestine. This glass slide was incubated for 15
min in a desiccator at 90% relative humidity to allow the polymer to interact with the membrane and finally
0
placed in the cell that was attached to the outer assembly at an angle 45 . Phosphate buffer saline (pH 6.4),
0
previously warmed to 37 ± 0.5 C, was circulated to the cell over the microspheres and membrane at the rate of 1
ml/min with the help of pump. Washings were collected at different time intervals and microspheres were
0
separated by centrifugation followed by drying at 50 C. The weight of microspheres washed out was taken and
percentage mucoadhesion was calculated by

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Percentage mucoadhesion = Wa - Wl X 100

Wa
where Wa = weight of microspheres applied; W1 = weight of microspheres leached out.
iquid chromatography. The drug release experiments were conducted in triplicate (n = 3).

Table 1: Formulation composition of mucoadhesive microspheres of Clarithromycin

Formulation Code Eudragit RL 100(%w/v) Carbopol 974P(%w/v) HPMC K4M* (%w/v)

F1 3 1.0 1.0
F2 5 1.0 1.0
F3 7 1.0 1.0
F4 5 0.5 0.5
F5 5 0.75 0.75
F6 5 1.5 1.5
HPMC = Hydroxypropyl methyl cellulose

Table. 2. Physico-chemical characteristics of the Clarithromycin loaded mucoadhesive

microspheres

Drug Entrapment
Formulation Mean Particle Mucoadhesion
[Link] (%) ±S.D ( n=3)
code size (µm) (%) ±S.D* ( n=3)

1 F1 173 82± 1.13 83±2.178

2 F2 225 88±1.77 86±1.856
3 F3 337 90±2.11 87±1.775
4 F4 203 90±1.82 82±1.145
5 F5 274 86±1.61 91±0.987
6 F6 306 82±2.21 93±1.421
S.D = Standard deviation

Fig 1. SEM photograph of microsphere (Formulation F6)

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100

Cumulative percentage Release

80

60

40

20 F1
F2
F3
0
0 2 4 6 8 10 12
Time (h)

Fig.2 Effect of EudragitRL100 on the inv release of Clarithromycin in 0.1 N Hcl

100
Cumulative percentage release

80

60

40
F4
F5
20
F6
0
0 2 4 6 8 10 12
Time(h)

Fig.3 Effect of Mucoadhesive polymers on on the In-vitro release of Clarithromycin in 0.1 N HCL

RESULTS AND DISCUSSION

The mucoadhesive microspheres Clarithromycin prepared in this study were well-rounded spheres
with the size ranging approximately from 17 to 337 µm. The study of In-vitro bioadhesion revealed that all the
batches of prepared microspheres had good bioadhesive property ranging from 82±1.145 % to 93±1.421%. From
the result of the In-vitro release test, the effect of Eudragit RL100 concentration on Clarithromycin release from
were observed a significant decrease in the rate and extent of drug release was observed with the increase in
polymer concentration in microspheres and could be attributed to increase in the density of the polymer matrix
and also increase in the diffusional path length which the drug molecules have to traverse. Similarly, the effect of
mucoadhesive polymers concentration on release properties of Clarithromycin were also studied. An increase in

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mucoadhesive polymers concentration caused retardation in drug release from the microspheres because of an
increase in the viscosity of polymer solution and formation larger size microspheres. The release of Clarithromycin
from these batches were characterized by an initial phase of high release (burst effect) followed by a second phase
of moderate release. In-vitro studies clearly indicates that the prepared formulations possess good bioadhesive
properties. These properties enable the microspheres to adhere to the gastric mucosal surface and stay in stomach
for prolonged periods and could ensure the stability of Clarithromycin in gastric environment, which eventually
resulted in better eradication of H. pylori than the conventional dosage forms.

REFERENCES

[1] Blaser MJ. Helicobacter pylori and gastric diseases. Br Med J 1998;316:1507–10.
[2] Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med 2002;347:1175–86.
[3] K.C. Myung, S. Hongkee, C. Hoo-Kyu. Int J Pharm 2005;297:172–9.
[4] Lin CK, Hsu PI, Lai KH. J Gastroenterol 2002;34:547–51.
[5] Kawabami E, Ogata SK, Portorreal AC. Gastroenterol 2001;38: 203–6.
[6] Cooreman MP, Krausgrill P, Hengels KJ. Antimicrob Agents Chemother 1993;37:1506–9.
[7] Atherton JC, Cockayne A, Balsitis M, Kirk GE, Hawley CJ, Spiller RC. Gut 1995;36:670– 4.
[8] Zhepeng L., Weiyue L., Lisheng Q., Xuhui Z., et al. J Control Rel 2004;81,327-34.
[9] Jain SK, Chourasia MK, Jain AK. Jain RK. Drug Deliv 2004;11: 113–22.

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