Formulation and In Vitro Evaluation of Periodontal Films
Containing Metronidazole
Biswajit Basu1*, Kevin Garala1, Manojkumar Tyagi2, G.L. Prabhushankar2,
P. R. Sathesh Babu2
1Department of Pharmaceutics, Atmiya Institute of Pharmacy,
2Department of Pharmaceutics,
ABSTRACT
Local delivery devices are
designed to deliver the drug locally into periodontal pocket. Metronidazole is
a nitroimidazole used to treat protozoal
infections. For local delivery, metronidazole films
were prepared by solvent casting technique using ethyl cellulose, hydroxy propyl methylcellulose
and eudragit RL-100 with dibutylphthalate
and polyethylene glycol 400 as plasticizers. FTIR and UV spectroscopic methods
revealed no interaction between metronidazole and
polymers. The films were evaluated for their thickness uniformity, folding
endurance, weight uniformity, content uniformity, tensile strength, and surface
pH. Data of in-vitro release from films were fit to different equations
and kinetic models to explain release kinetics. Hixon-Crowell,
Higuchi, and Korsmeyer-Peppas models were used to fit
the in-vitro release data. Formulation F6 released 94.18% of drug at the end of 120 h, was
considered as best formulation. Short-term stability study revealed that drug
content decreased in various films was ranging from 1.361 to 2.209%.
KEYWORDS: Metronidazole; periodontal pocket;
periodontal films; local delivery; in-vitro release.
INTRODUCTION:
Periodontitis is a disease associated with
periodontium in which irreversible step of loss of
attachment of teeth occurs.1 The effective
use of antimicrobial agent for the treatment of periodontal disease requires an
adequate drug concentration at the site of action and a means to maintain that
level for a sufficient duration to allow the agent to act. Topical applications like mouthwashes,
dentifrices and gels have been successfully tried in controlling the microbial
plaque. Topical agents follows an exponential concentration profile while blood
and crevicular fluid levels remains at zero, initial
salivary concentration reach levels 20 to 50 times bactericidal levels,
following expectoration, salivary drug level rapidly fall to approximately one
tenth of their initial concentration. Topical agent fail to penetrate deep into
periodontal pockets, hence their effectiveness is limited to supragingival areas. So to overcome all these limitations
various controlled drug delivery systems, administrating therapeutic levels of
antibacterial agents directly into periodontal pocket have been tested as a way
to minimize total body dosage and resulting side effects and to maintain
therapeutic drug levels in the gingival crevicular
fluid.2,3
Metronidazole is a nitroimidazole, used to treat protozoal
infections. It is bactericidal to anaerobic organisms and is believed to
disrupt bacterial DNA synthesis. Metronidazole is not the drug of choice for
treating Actinobacillus actinomycetemcomitans
infections. However, it is effective against Actinobacillus actinomycetemcomitans
when used in combination with other antibiotics.4,5 Metronidazole is
also effective against anaerobes such as Porphyromonas gingivalis and Prevotella intermedia.6 The dose recommended for the treatment of
anaerobic infection is 500 mg oral; i.v every 6-8
hours for seven days. Systemic treatment for periodontitis
by the oral route using oral tablets requires a total of about 5250 to 7000 mg
of metronidazole. However, non-systemic treatment for
periodontitis with the slow release composition uses
only about 2.4 mg per periodontal pocket.
As patients with the most
advanced cases of periodontitis may have about 20
diseased pockets, and if the metronidazole films are
placed in each of these pockets, then the total dosage of metronidazole
would be about 50 mg. This total dosage is less than 1% of the total systemic
(oral) dose and reflects a substantial safety factor over systemic treatment
with oral metronidazole. A preferred dosage for a
periodontal pocket, is one in which each pocket receives about two mg of metronidazole, preferably in a slow release form that
releases from 0.01 to 0.2 mg per day for each day that the polymeric device
remains in the periodontal pocket.7
MATERIALS AND METHODS:
Methods:
Preparation of film containing metronidazole:
Periodontal films were prepared by solvent casting
technique. Glass moulds were used for casting the films. Ethylcellulose,
Eudragit RL-100 alone and in combination with hydroxy propyl methyl cellulose
were dissolved in chloroform and alcohol mixture with dibutyl
phthalate as a plasticizer in a beaker using magnetic stirrer to get different
concentration of polymeric solutions. Into these solutions metronidazole
of required concentration was added. After complete mixing, the solution was
poured into a clean glass mould placed on a horizontal plane. The solvent was
allowed to evaporate slowly by inverting a glass funnel with a cotton plug in
the stem of the funnel was placed on the mould at room temperature for 24 h.
After complete evaporation of solvent, cast film was obtained. Inverted funnel
was continuously kept on the mould to control drying rate. The prepared cast
films were lined with butter paper and stored in a dessicator.
To accommodate different variables, batches of cast films were prepared. The
compositions of films are given in Table 1.
Evaluation of the films:
Formulated films were subjected to the preliminary
evaluation tests. Films with any imperfections, entrapped air, or differing in
thickness, weight or content uniformity were excluded from further studies.
Thickness uniformity of the films: The thickness of each film
was measured using screw guage (thickness tester) at
different positions of the film and the average was calculated.
Uniformity of weight of the films: Film (size of 1 cm2) was taken from different areas of
film. The weight variation of each film
was calculated.
Tensile strength of the films: Tensile strength of the films was determined with
Universal strength testing machine. The sensitivity of the machine is 1 gram.
It consists of two load cell grips; the lower one is fixed and the upper one is
movable. The test film of specific size (4 ´ 1 cm2) was fixed
between these cell grips and force was gradually applied till the film breaks.
The tensile strength of the film was taken directly from the dial reading in
kilograms.
Drug content uniformity of films: Film
(size of 1 cm2) was taken from different areas of film and placed in
a 10 ml volumetric flask; 10 ml of ethyl alcohol was added and kept aside till
the film dissolve completely. From this solution, 1 ml was pipette out and
diluted to 10 ml with double distilled water. The absorbance of the solution
was measured at 320.4 nm. The polymer solution without drug serves as a blank.
In case of HPMC film, combination of water and alcohol is used to dissolve the
film.
Folding endurance: As described by Kevin et al., the folding endurance of the films was
determined by repeatedly folding one film at the same place till it broke or
folded up to 300 times, which is considered satisfactory to reveal good film
properties. The film was folded number
of times at the same place without breaking gave the value of the folding
endurance.8 This
test was done on all the films for five times.
Surface pH: Periodontal
films were left to swell for 1 hour on the surface of the agar plate, prepared
by dissolving 2% (w/v) agar in warmed double distilled water under stirring and
then pouring the solution into the petridish to
gelling / solidify at room temperature. The surface pH was measured by means of
pH paper placed on the surface of the swollen film. The mean of three readings
was recorded.9
Viscosity: Aqueous solutions containing
both polymer and plasticizer were prepared in the same concentration as that of
films.
In-vitro drug release: In-vitro drug release was performed by taking 1 cm2 of
periodontal film in a vial containing one ml of double distilled water. One ml
of double distilled water was withdrawn from 1st to 5th
day, every day and immediately replaced with one ml of fresh double distilled
water.10 The
drug content was estimated by measuring the absorbance after suitable dilution
at 320.4 nm.
Ageing: Optimized
medicated films were subjected to stability testing. Films were placed in a glass beaker lined
with aluminium foil and kept in a humidity chamber
maintained at 40 + 2oC and 75 + 5% RH for 1
month. Changes in the appearance and
drug content of the stored films were investigated after storage. The data presented were the mean of 3 determinations.11
Table 1:
Composition of different formulations containing metronidazole
|
Ingredients |
Film Code |
||||||
|
F1 |
F2 |
F3 |
F4 |
F5 |
F6 |
||
|
Metronidazole
(mg) |
120 |
120 |
120 |
120 |
120 |
120 |
|
|
Ethyl
cellulose (mg) |
* |
* |
440 |
460 |
460 |
480 |
|
|
Eudragit RL 100 (mg) |
460 |
440 |
* |
* |
20 |
* |
|
|
HPMC
(mg) |
20 |
40 |
40 |
20 |
* |
* |
|
|
Dibutyl phthalate (ml) |
* |
* |
0.2 |
0.2 |
0.15 |
0.15 |
|
|
PEG
400 (ml) |
0.1 |
0.1 |
* |
* |
* |
* |
|
|
Alcohol
(ml) |
15 |
15 |
2 |
2 |
2 |
2 |
|
|
Chloroform
(ml) |
* |
* |
13 |
13 |
13 |
13 |
|
* No
ingredient is added
Table 2: Physicochemical characteristics of periodontal films containing metronidazole
|
FC |
TN (mm)
|
WU (mg) |
TS (kg) |
CU
(%) |
FE |
|
|
Dummy films |
Drug loaded films |
|||||
|
F1 |
0.225 |
33.801 |
4.466 |
5.033 |
85.612 |
> 250 |
|
F2 |
0.24 |
34.231 |
3.000 |
4.133 |
87.067 |
> 250 |
|
F3 |
0.28 |
34.712 |
1.720 |
2.700 |
85.219 |
> 250 |
|
F4 |
0.268 |
32.590 |
2.133 |
3.066 |
83.292 |
> 250 |
|
F5 |
0.215 |
33.352 |
2.866 |
3.800 |
82.977 |
> 250 |
|
F6 |
0.246 |
32.338 |
2.300 |
3.666 |
82.368 |
> 250 |
FC is film code (F1,
F2, F3, F4, F5, and F6
are formulations). TN, WU, TS, CU, and FE are thickness, weight uniformity,
tensile strength, content uniformity, and folding endurance, respectively. Each
value is an average of three determinations.
Evaluation of films:
The
physicochemical properties of metronidazole
periodontal films are presented in Table 2.
Thickness uniformity: All the films have uniform
thickness throughout with the standard deviation of ± 0.018 μm
(n = 3).
Weight uniformity: Drug loaded films (1 x 1 cm2)
were tested for uniformity of weight. The weight was found to be uniform in the
prepared batches with standard deviation of ± 0.856 mg per film (n = 3)
Tensile strength: The tensile strength of
drug-loaded films was higher than dummy films (Table 2). This is justified
because dissolved metronidazole strengthened the
bonding of polymer chains. The tensile strengths of films were in the order of
F1 > F2 > F5 > F6 > F4
> F3.
Content uniformity: The
results of content uniformity indicated that the drug was uniformly dispersed. Recovery was possible to the tune of 82.36
to 87.06
% for
formulations F1 to F6 (Table 2). The drug content
analysis of the prepared formulations had shown that the process employed to
prepare the films in this study was capable of giving films with a uniform drug
content and minimum batch variability.
Folding endurance: Films did not show any cracks
even after folding for more than 250 times.
Hence it was taken as the end point.
Folding endurance did not vary when the comparison was made between
dummy films and drug-loaded films (Table 2).
Table 3: In-vitro release
profile of metronidazole films from F1 to F6
|
Time (h) |
Percentage of
drug release |
|||||
|
F1 |
F2 |
F3 |
F4 |
F5 |
F6 |
|
|
24 |
86.430 |
89.858 |
71.330 |
66.242 |
67.783 |
55.23 |
|
48 |
93.675 |
96.757 |
90.690 |
82.450 |
80.154 |
77.54 |
|
72 |
97.770 |
98.909 |
95.760 |
92.150 |
91.486 |
90.20 |
|
96 |
- |
- |
96.818 |
93.245 |
93.135 |
92.50 |
|
120 |
- |
- |
- |
- |
94.314 |
94.18 |
Viscosity: The
viscosities of the solutions were ranges from 12.10 to 33.70 cps for films F1 to F6.
Viscosity of the solution of film F6 was highest when compared to
others, because of the presence of Ethyl cellulose.
In vitro
release studies: In vitro release studies performed using double distilled water showed an initial
burst release (Figure 1), which is expected to kill most of the periodontal
organisms, followed by controlled release, sufficient to inhibit the growth of
the micro-organisms.
Periodontal film made of ethyl cellulose (F6)
is better than others because the extent of release was maintained for about 5
days. All the formulations showed initial burst release and controlled release
in later phases, as shown in Figure 1. Higher drug release from films F1,
F2, and F3 showed 97.7%, 98.9%, and 96.8% respectively.
The higher drug release from these films was possible because of the formation
of more pores and channels due to presence of higher HPMC content. As HPMC act
as resorbable carriers, it dissolved readily during in
vitro drug release. The cumulative amounts of drug released from the films
are shown in (Table 3).
Eudragit RL100 being an inert
polymer, solvent penetration into the film was rate limiting factor for the
release of the active principle. At the beginning of the process, the active
substance at and near the surface of the film dissolves quickly. When the
dissolution process advances, there is a greater resistance to the penetration
of the solvent in the inside of the matrix film, due to the non-hydrophilicity of the polymer and the decreasing length of
the solvent front. The drug, easily accessible by water immediately dissolves
and diffuses from the interface between the film surface and surrounding media
after which diffusion process slows down. In-vitro
release studies showed that the drug release was more sustained in case of film
F6 followed by F5 > F4 > F3 >
F2 > F1. The regression values of films F1
to F6 are higher with first order and therefore the release kinetics
followed first order from all films.
Hixon Crowell cube root law and
Higuchi’s models were applied to test the release mechanism. The R2
values are higher for Higuchi’s model compared to Hixon
Crowell cube root law for all the films. Hence metronidazole
release from all the films followed diffusion rate controlled mechanism. According to Korsmeyer-Peppas
model, the release mechanism from films
F1 to F4 followed case II transport (n > 1). However, films F5 and F6 followed
Non-Fickian diffusion or anomalous behavior (a value
of slope between 0.5 and 1).
Fig. 1: In-vitro release profile of metronidazole from film F1 to F6
Fig. 2: a) IR spectrum of Metronidazole pure, b) IR
spectrum of metronidazole and ethyl cellulose
mixture, c) IR spectrum of metronidazole and eudragit RL-100 mixture, d) IR spectrum of metronidazole and HPMC 47 cps mixture
Ageing: Films
that were placed in humidity chamber for
short time stability studies were withdrawn every week and analysed for their drug content. Percentage drug present in
the films were determined spectrophotometrically. Decrease in the drug content
from the films ranged from 1.361to 2.209%. It
was found that the drug loss is less though the films were stored for one
month. The films were also observed for their appearance and texture. These
properties did not change in films during the period of study. Periodontal films
containing metronidazole using ethyl cellulose, HPMC,
and eudragit RL100 polymers showed satisfactory
characteristics without being drastically influenced by ageing.
CONCLUSION:
On the basis of
in vitro characterization it was
concluded that metronidazole could be incorporated in a slow release device for the treatment of periodontitis. Periodontal films consisting
of ethyl cellulose, the bioadhesive polymer HPMC, and
rate-controlling polymer of Eudragit RL100 with dibutyl pthalate and PEG-400 as
plasticizers demonstrated sustained and controlled release of the drug. The
drug remained intact and stable in the periodontal films during storage, with
no significant chemical interaction between the drug and the excipients.
Further, detailed investigation is required to establish in-vivo efficiency of these films.
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Received on 07.07.2009
Accepted on 12.09.2009
© A & V Publication all right reserved
Research Journal of Pharmaceutical
Dosage Forms and Technology.
1(3): Nov. – Dec. 2009, 240-243