FORMULATION, DEVELOPMENT AND EVALUATION OF OFLOXACIN …
Transcript of FORMULATION, DEVELOPMENT AND EVALUATION OF OFLOXACIN …
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Gondkar et al. World Journal of Pharmacy and Pharmaceutical Sciences
FORMULATION, DEVELOPMENT AND EVALUATION OF
OFLOXACIN IN-SITU OPHTHALMIC GEL
S. B. Gondkar1*
, Pallavi R. Kute2 and R. B. Saudagar
3
1*
Department of Quality Assurance Techniques, R.G.Sapkal College of Pharmacy, Anjaneri,
Nashik-422213, Maharashtra, India.
2Department of Quality Assurance Techniques, R. G. Sapkal College of Pharmacy, Anjaneri,
Nashik-422213, Maharashtra, India.
3Department of Pharmaceutical Chemistry, R.G. Sapkal College of Pharmacy, Anjaneri,
Nashik-422213, Maharashtra, India.
ABSTRACT
Ofloxacin is a fluorinated 4-quinolone antibiotic which is used in the
treatment of ophthalmic ailments like infections, inflammations,
conjunctivitis, blepharitis, iritis, corneal ulcer etc. Commercially,
ofloxacin eye drop solution is available and it is quite easy for the
administration. However, the product has drawback of poor
bioavailability due to several factors such as tear production, non-
productive absorption, transient residence time, and impermeability of
corneal epithelium. In order to improve the bioavailability, residence
time and longer duration of action, an attempt was made to formulate
in-situ ophthalmic gel of Ofloxacin. In present study Carbopol 934 and
Sodium alginate were used as polymers. Carbopol 934 was used as a
pH sensitive polymer and Sodium alginate was used as mucoadhesive
polymer. All 9 prepared formulations were optimized by 32 factorial design. The prepared
formulations were evaluated for pH, clarity, viscosity, drug content, gel strength, bioadhesive
strength, in vitro drug release, antibacterial activity, isotonicity test, ocular irritancy test and
stability.
KEYWORDS: Ofloxacin, Carbopol 934, Sodium alginate, in-vitro drug release, antibacterial
activity.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 5.210
Volume 4, Issue 11, 800-822 Research Article ISSN 2278 – 4357
Article Received on
23 Aug 2015,
Revised on 15 Sep 2015,
Accepted on 03 Oct 2015
*Correspondence for
Author
S. B. Gondkar
Department of Quality
Assurance Techniques,
R.G.Sapkal College of
Pharmacy, Anjaneri,
Nashik-422213,
Maharashtra, India.
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INTRODUCTION
The field of Ocular drug delivery is one of the interesting and challenging endeavors facing
the pharmaceutical scientist.Eye is the most exclusive organ of the body and various drug
delivery systems are used to deliver drug into the eye.It is the sensory organ that converts
light to an electric signal that is treated and interpreted by the brain.Ocular disposition and
elimination of a therapeutic agent is dependent upon physicochemical, microbiological,
pharmaceutical properties and ophthalmic irritancy properties of ocular dosage forms as well
as the relevant ocular anatomy and physiology. To improve ocular drug contact time,
bioavailability and residence time, and to reduce the patient discomfort, frequency of dose, as
well as to slow down the elimination of the drug, there are significant efforts concentrating
towards newer drug delivery systems for ophthalmic administration. Development of newer,
more sensitive diagnostic techniques and novel therapeutic agents continue to provide ocular
delivery systems with high therapeutic efficacy. Conventional ophthalmic formulations like
solution, suspension, and ointment have many disadvantages which result into poor
bioavailability of drug in the ocular cavity. The specific aim of designing a therapeutic
system is to achieve an optimal concentration of a drug at the active site for the appropriate
duration. The various approaches that have been attempted to increase the bioavailability and
the duration of the therapeutic action of ocular drugs can be divided into two categories. The
first one is based on the use of sustained drug delivery systems, which provide the controlled
and continuous delivery of ophthalmic drugs. The second involves maximizing corneal drug
absorption and minimizing precorneal drug loss. Ideal ophthalmic drug delivery must be able
to sustain the drug release and to remain in the vicinity of front of the eye for prolong period
of time. Ophthalmic drug delivery is used only for the treatment of local conditions of the eye
and cannot be used as a portal of drug entry to the systemic circulation.
MATERIALS
Ofloxacin was obtained from Aarti Drugs Ltd.Thane, India as a gift sample. Carbopol 934
and Sodium alginate were purchased from LOBA chemie Pvt.Ltd.India.
Ultraviolet -Visible Spectroscopy
Determination of λmax in Distilled water
The UV spectrum of Ofloxacin was obtained using Ultraviolet Spectrophotometer (Jasco
V630). Accurately weighed 10 mg of the drug was dissolved in sufficient quantity of distilled
water and volume was made up to 100 mL to obtain a concentration of 100µg/mL.1mL of
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aliquot was withdrawn and volume was made up to 10 mL using distilled water to obtain the
concentration of 10µg/mL. The resultant solution was scanned from 200 to 400 nm and the
spectrum was recorded to obtain the value of maximum wavelength.
Preparation of Calibration curve in Distilled water
The stock solution of 100µg/mL was used to prepare different dilutions in the range of 2-
10µg/mL. The absorbance of resulting solutions were measured at 287 nm using distilled
water as blank by UV-visible spectrophotometer.
Compatibility Study
Fourier Transform Infrared Spectroscopy
Compatibility study was carried out by using Fourier Transform Infrared Spectrophotometer
(Cary 630, Agilent Technologies, USA). FTIR study was carried on pure drug and physical
mixture of drug and polymers. Physical mixtures were prepared and samples were kept for 1
month at 40 ºC. The infrared absorption spectrum of Ofloxacin and physical mixture of drug
and polymers were recorded over the wave number 4000 to 650 cm-1
.
Formulation and Development of In-situ Ophthalmic Gel
The quantities of drug and other ingredients were weighed as per following table and
formulations were prepared in following manner:
Cleaning of glassware and container
All the glassware‟s were washed with distilled water and then sterilized by drying at 160°C
for 1 hr in hot air oven.
Preparation of solution ‘A’
Accurately weighed quantity (0.3 gm) of the Ofloxacin was dissolved in 100 mL distilled
water. The sodium metabisulphite was added to above mixture with continuous stirring.
Preparation of polymer dispersion ‘B’
Solution „A‟ was divided into 2 equal volumes, Carbopol 934 and sodium alginate were
added to each of these volumes respectively and was allowed to hydrate for 12 hours to
produce a clear solution/ dispersion
Sterilization of ophthalmic formulation
Prepared solutions were autoclaved at 1210 C for 15 min.
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Aseptic filling to container
The formulation was aseptically transferred to previously sterilized glass vials and sealed.
Formulation optimization
32 full factorial design was applied to the formulation that showed the satisfactory results. To
see the effect of concentration of variables Carbopol 934 and Sodium alginate on various
responses like % drug release and antibacterial activity. For the Carbopol 934 lower, middle
and higher level were 0.3, 0.4 and 0.5 gm respectively. Similarly for the Sodium alginate
lower, middle and higher level were 0.4, 0.5, 0.6 gm respectively. Composition of all batches
is shown in table no.1.
Table no.1: Composition of formulation batches as per 32 factorial design
Formulation code
Ingredient (%)
F1
F2
F3
F4
F5
F6
F7
F8
F9
Ofloxacin (w/v) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3
Carbopol 934 (w/v) 0.3 0.4 0.5 0.3 0.4 0.5 0.3 0.4 0.5
Sodium alginate (w/v) 0.4 0.4 0.4 0.5 0.5 0.5 0.6 0.6 0.6
Sodium Metabisulphite (w/v) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
Distilled water (q.s.) (ml) 100 100 100 100 100 100 100 100 100
Evaluation of ophthalmic gel
Physical parameter
Clarity
The formulations were visually checked for the clarity.
pH
pH of each formulation was determined by using Digital pH meter (Digital pH meter 335).
This was previously calibrated by pH 4 and pH 7. The pH values were recorded immediately
after preparation.
Rheological study
Viscosity
The rheological properties of solution & gels were determined by the Brookfield viscometer;
type DV-II + PRO using spindle no.61 and 63.Viscosity of the formulations were taken at
two different pH i.e. at pH 6 and at pH 7.4 with varying shear rate.
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Measurement of the gel strength
A sample of 50 g of the gel was put in a 50 ml graduated cylinder. A weight of 14.33 g was
placed on the gel surface. The gel strength, which is an indication for the ophthalmic gel at
physiological temperature, was determined by the time in seconds required by the weight to
penetrate 5 cm into the gel. All measurements were performed in triplicate (n=3). The
apparatus used for measuring gel strength is shown in Fig. 1
Fig. 1: Gel strength measuring device (A) Weights (B) Device
(C) Graduated cylinder (D) Gel
Bioadhesive Strength[71]
“Detachment force is the force required to detach the two surfaces of mucosa when a
formulation is placed in between them”. The detachment force was measured by using a
modified physical balance (A). A fresh goat corneal membrane was obtained from local
slaughter house. A section of fresh cornea was cut from the goat eye and washed with saline
solution.
i) Fabrication of equipment
The equipment was fabricated by us in the laboratory as shown in fig. A double beam
physical balance was taken, both the pans were removed. The left pan was replaced with a
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brass wire, to which was hanged a teflon disc (D), also locally fabricated. The dimensions are
2 cm height and include an expanded cap of diameter 3.8 cm and thickness 2 cm. Another
teflon disc of 2 cm height and 1.5 cm diameter was placed right below the suspended disc
upon the base of the balance. The right pan (B) was replaced with a lighter pan so that, the
left pan weighs 5.20 gm more than the right pan. The lower teflon block was intended to hold
the mucosal tissue (E) of goat corneal membrane and to be placed in a beaker containing
simulated tear fluid pH 7.4.
ii) Measurement of adhesion force
Goat corneal membrane was obtained commercially; the cornea was collected into a sterile
container containing sterile buffer solution of pH 7.4. The corneal membrane brought was
stored in a refrigerator until use.
The following procedure was used for all the test formulations using the above equipment.
The goat corneal membrane was removed from refrigerator and allowed to attain equilibrium
with ambient conditions in the laboratory. The goat corneal membrane was carefully excised,
without removing connective and adipose tissue and washed with simulated tear fluid
solution. The tissue was stored in fresh simulated tear fluid. Immediately afterwards the
membrane was placed over the surface of lower teflon cylinder (E) and secured. This
assembly was placed into beaker containing simulated tear fluid pH 7.4 at 37 ± 2°C.
From each batch, some quantity of gel was taken and applied on the lower surface of the
upper teflon cylinder. The beaker containing mucosal tissue secured upon lower cylinder (E),
was manipulated over the base of the balance so that, the mucosal tissue is exactly below the
upper cylinder (D). The exposed part of the gel was wetted with a drop of simulated tear
fluid, and then a weight of 10 gm was placed above the expanded cap, left for 10 minutes.
After which the gel binds with mucin. The weight was removed. Then slowly and gradually
weights were added on the right side pan till the gel separates from the mucosal surface/
membrane.
The weight required for complete detachment is noted (W1) (W1-5.20gm)) gives force
required for detachment expressed in weight in grams. Procedure was repeated for two more
times. Average was computed and recorded.
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iii) Calibration of test equipment
Initially, a gel from the same batch was taken ten times and individual force required for
complete detachment was noted and SD was calculated.
iv) Force of adhesion (N)
Bioadhesive strength= (Bioadhesive strength/1000) × 9.81
Bond strength (N/m2) = Force of adhesion (N)/ Surface area of disk (m2)
The Modified Apparatus for Bioadhesive study is shown in Fig. 3
Fig. 2: Modified bioadhesion test apparatus (Fabricated) A-Modified physical
apparatus, B-Right pan, C- Weights, D-Upper teflon disc, E-Lower teflon disc, F-
Corneal membrane, G-Simulated tear fluid.
Drug Content
The drug content was determined by taking 1 ml of the formulation and diluting it to 100 ml
with distilled water.Aliquot of 5 ml was withdrawn and further diluted to 25 ml with distilled
water.Ofloxacin concentration was determined at 287 nm by using UV-Visible
spectrophotometer (JASCO V-630).
In-vitro Drug Release Study
In vitro release study of the formulated ophthalmic in-situ gel was carried out by using
diffusion cell through egg membrane as a biological membrane. Diffusion cell with inner
diameter 24mm was used for the study. 1 mL formulation was placed in donor compartment
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and Freshly prepared 100 mL artificial tear fluid ( sodium chloride 0.670g, sodium
bicarbonate 0.200g, calcium chloride dehydrated 0.008g, potassium chloride 0.248g,distilled
water q.s 100mL.) was placed in receptor compartment. Egg membrane was mounted in
between donor and receptor compartment. The position of the donor compartment was
adjusted so that egg membrane just touches the diffusion medium. The whole assembly was
placed on the thermostatically controlled magnetic stirrer. The temperature of the medium
was maintained at 37°C ± 0.5°C. 1mL of sample was withdrawn from receiver compartment
after 30 min, 1, 2, 3, 4, 5, 6, 7 & 8 hrs and same volume of fresh medium was replaced. The
withdrawn samples were diluted to 10mL in a volumetric flask with distilled water and
analyzed by UV spectrophotometer at 287.0 nm.
Antibacterial Activity
An agar diffusion method was used for the determination of antibacterial activity of
formulations. Standard Petri dishes (9 cm diameter) containing medium to a depth of 0.5 cm
were used. The sterility of the lots was controlled before use. Suspension was prepared by
suspending 1-2 colonies of Staphylococcus aureus from 24hr cultures in Nutrient agar
medium into tubes containing 10 mL of sterile saline. The tubes were diluted with saline. The
inoculum (0.5mL) was spread over the surface of agar and the plates were dried at 35°C for
15 min prior to placing the formulation. The bores of 0.5 cm diameter were prepared and 2
drops of formulation (0.3 % w/v) were added in the bores. After incubation at 35°C for 24
hrs, the zone of inhibition around the bores was measured.
Isotonicity Evaluation
The formulations were mixed with few drops of diluted blood on a slide. The diluted blood
was prepared by using Grower‟s solution and Slide was observed under microscope at 45x
magnification. The shape of blood cells were compared with standard marketed ophthalmic
formulation.
Test for Sterility
Method: The sterility test was carried out as per IP (2014) method. The three medium were
taken for this test i.e. fluid thioglycolate medium, artificial fluid thioglycolate medium and
soyabean casein medium. The three set were prepared each set containing three tubes of each
medium. The first set was a negative control for this sterile media is used, second set was a
positive control for this sterilized media inoculated with Staphylococcus aureus (MH1714)
was used and third set was a test. The 1mL sterile optimized formulation was taken and this
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formulation was diluted with 100mL sterile water for injection, from this 5mL test solution
was added in each medium. The formulation was incubated for not less than 14 days at 20-
250C in the fluid thioglycolate medium and at 20-25
0c in soyabean casein digest medium to
find out growth of bacteria in formulation.
Ocular Irritancy Test
The Test conditions for ocular irritancy test are shown in Table no.
Table no.2: Test conditions for ocular irritancy test
Test Conditions
Method for test Draize test
Strain of rabbit New Zealand White Albino
Weight of rabbit 2-3 kg
Volume of formulation instilled 50 µL
Left eye of rabbit Sterile water for injection
Right eye of rabbit Optimized formulation (F5)
The optimized formulation was used for eye irritancy study. The protocol was approved by
Institutional Animal Ethics Committee.
The Modified Draize Eye Irritation: The 01 Albino rabbit weighing 2-3 kg was used.
According to the draize test, the amount of formulation was applied to the eye is 50 µl was
placed into the lower cul-de-sac. The evaluation of ocular lesions was made at 1, 4, 24, 48,
72hrs, and 1 week after administration. 3 day washing period with saline was carried out. The
sterile formulation was instilled twice a day for a period of 7 days, and a cross over study
carried out. The rabbit was observed periodically for redness, swelling, watering of the eye.
The score point was recorded as per the chart given in Table no
Table no.3: Score rating for eye irritancy study
Stability studies
Test conditions for stability study are shown in Table no.4
Sr.no. Score Rating
1 0 None
2 1 Slight
3 2 Mild
4 3 Moderate
5 4 Severe
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Test Conditions
Duration of study: 3 months
Temperature conditions: 40±2ºC
Relative humidity conditions: 75±5%
Frequency of testing the samples: 30 days
The formulation was evaluated mainly for its physical characteristics like appearance/clarity,
pH, viscosity and drug content after 3 months.
RESULTS AND DISCUSSION
Determination of λmax of Ofloxacin in distilled water
Fig. 3: UV-visible spectrum of Ofloxacin in distilled water
The UV spectrum of Ofloxacin solution (10μg/mL) exhibited wavelength of absorbance
maximum at 287.0 nm. This is near to the reported value. However, keeping in mind the
probable concentrations likely to be encountered while carrying out In-vitro release studies
and considering the predicted theoretical λmax involved, the working λmax was decided as
287.0 nm.The spectrum of Ofloxacin is shown in Fig.
Calibration Curve of Ofloxacin in distilled water
The calibration curve (Fig.) was found to be linear in the concentration range of 2-10μg/mL
(Table no.5) having coefficient of regression value R2 =0.9823 and Slope y = 0.0648x.
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Absorbance’s of different concentrations of Ofloxacin in distilled water
Sr.no. Concentration
(µg/mL) Absorbance
1 2 0.1401
2 4 0.2878
3 6 0.3456
4 8 0.5247
5 10 0.6546
Fig.4: Calibration curve of Ofloxacin in distilled water
Infra-Red Spectrum
Infra- red spectrum of Ofloxacin is shown in Fig. The major peaks observed and
corresponding functional groups are given in Table no. Infra-red spectrum shows peak
characteristic of structure of Ofloxacin.
Fig 5: FTIR spectrum of Ofloxacin
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3043.889
2785.818
1836.890
1709.343
1619.406
1546.151
1521.434
1449.0671395.246
1347.725
1287.759
1239.254
1198.027
1144.552
1113.937
1052.9881009.503
978.150
952.977
877.973
850.356
827.270
802.878
779.701
744.989
709.350667.940
Mixture PK
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
102
100
98
96
94
92
90
88
86
84
82
Wavenumber
%T
ransm
ittance
Fig. 6: Fourier Transform Infra-red of drug with polymer
Evaluation of Ophthalmic Gel Formulation
Physical parameter
Clarity
On careful visual inspection against dark and white background, all the prepared ophthalmic
gel formulations were found to be free from any suspended particulate matter. The prepared
formulations are shown in fig. 7
Fig.7: Prepared formulations
pH:
Table no.5 pH values of formulations
Sr. No Formulation code Observed pH (±S.D.)
1 F1 6.50 ± 0.01
2 F2 6.52± 0.01241
3 F3 6.54 ± 0.02549
4 F4 6.56 ± 0.1322
5 F5 6.8 ± 0.060
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6 F6 6.58± 0.1258
7 F7 6.62± 0.00565
8 F8 6.65 ± 0.01732
9 F9 6.68± 0.03605
Rheological study
Viscosity
The viscosity of formulations at pH 6 and at pH 7.4 is shown in Table no.and respectively.
The Viscosity profile of formulations at pH 6 and at pH 7.4 is shown in Fig.and respectively.
Table no.6: Viscosity of formulations at pH 6
Fig.8: Viscosity profile of formulations at pH 6
Table no.7: Viscosity of formulations at pH 7.4
rpm
Viscosity (cp) at pH 6
Formulation code
F1 F2 F3 F4 F5 F6 F7 F8 F9
5 45.77 185.2 256.4 510.9 557.9 608.9 770.5 825.3 900.2
10 40.79 177.6 235.6 485.3 509.5 587.9 639.6 749.9 845.8
15 36.59 172.6 215.3 450.7 490.1 585.2 558.8 550.4 745.9
20 33.59 165.5 196.2 385.8 379.2 487.3 480.6 368.2 635.3
25 13.80 156.2 185.5 205.2 255.1 385.4 316.7 226.4 550.8
rpm
Viscosity (cp) at pH 7.4
Formulation code
F1 F2 F3 F4 F5 F6 F7 F8 F9
5 98.3 426 757 837.9 885.4 1080 2008 2586 3767
10 85.2 394 700 730.2 767.8 1064 1948 1548 2747
15 76.7 345 650 610.4 615.9 1045 1800 1104 2320
20 55.99 295 545 588.2 539.9 1032 1739 903.8 2082
25 23.99 226 475 550.6 508.7 1024 1650 791.8 1929
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Fig.9: Viscosity profile at pH 7.4
Measurement of the Gel Strength
The gel strength of ophthalmic in-situ gel formulation is shown in Table no.8
Table no.8: Gel strength of formulations
Sr. No Formulation code Gel strength (sec) (±S.D.)
1 F1 0.50 ± 0.03
2 F2 0.59 ± 0.01
3 F3 0.86 ± 0.07
4 F4 1.05 ± 0.05
5 F5 1.30 ± 0.08
6 F6 1.50 ± 0.43
7 F7 1.50 ± 0.41
8 F8 2.30 ± 0.12
9 F9 2.50 ± 0.04
Drug Content
The Drug content of formulations is shown in Table no.9
Table no.9: Percent drug content of ophthalmic in-situ gel
Formulation Code Drug content (%) (±S.D.)
F1 99.89 ± 0.05
F2 99.22 ± 0.03
F3 98 ± 0.1024
F4 99.66 ± 0.05
F5 101.5 ± 0.04
F6 101.8 ± 0.08
F7 100.05 ± 0.05
F8 100.2 ± 0.05
F9 98 ± 0.10
Bioadhesive Strength
The detachment force of formulation is shown in Table no.10
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Table no.10: Detachment force of formulations
Formulation Code Detachment force (Newton)(±S.D.)
F1 0.1898 ± 0.35
F2 0.2019 ± 0.2797
F3 0.2424 ± 0.3618
F4 0.3507 ± 0.1769
F5 0.4393 ± 0.3357
F6 0.4869 ± 0.1791
F7 0.5077 ± 0.3465
F8 0.6407 ± 0.3061
F9 0.6457 ± 0.2705
In-vitro Drug Release Study
The In-vitro drug release study of formulation is shown in Table no.11
Table no.11: Cumulative drug release of formulations
Cumulative Drug Release (%) (±S.D.)
Time in
(Hrs) F1 F2 F3 F4 F5 F6 F7 F8 F9
0 0 0 0 0 0 0 0 0 0
30 min 40.12±0.015 33.34±
0.014
30.49±0
.013
10.35±0.
012
15.47±
0.011
9.08±
0.010
8.29±0.0
13
11.20±0.0
10
10.11±0.
011
1 60.49±0.008 48.21±
0.004
50.57±0
.005
20.18±0.
002
30.48±
0.002
25.18±
0.003
18.21±0.
003
22.14±0.0
04
21.45±0.
002
2 76.34±0.007 61.37±
0.005
65.45±0
.005
30.16±0.
008
38.95±
0.006
34.56±
0.004
24.12±0.
004
39.12±0.0
03
28.61±0.
002
3 85.98±0.006 72.18±
0.003
79.34±0
.006
42.89±0.
003
47.13±
0.004
41.89±
0.003
31.45±0.
006
45.34±0.0
06
33.23±0.
001
4 98.56±0.004 86.31±
0.005
89.38±0
.008
56.82±0.
001
59.48±
0.001
50.89±
0.002
40.56±0.
004
51.32±0.0
03
40.78±0.
006
5 98.57±0.003 99.1±0.
002
97.5±0.
004
64.28±0.
009
70.56±
0.009
62.38±
0.002
50.19±0.
006
60.24±0.0
01
45.15±0.
002
6 98.58±0.002 99.15±
0.001
97.51±0
.002
79.67±0.
005
81.16±
0.005
75.87±
0.001
61.23±0.
004
68.21±0.0
02
59.21±0.
001
7 98.59±0.001 99.18±
0.003
97.54±0
.005
99.26±0.
006
90.12±
0.002
82.34±
0.003
74.87±0.
001
75.33±0.0
05
68.28±0.
004
8 98.60±0.001 99.2±0.
006
97.60±0
.004
99.29±0.
004
100.05
±0.003
92.02±
0.005
87.28±0.
007
83.49±0.0
01
79.82±0.
003
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Fig.10: In-vitro drug release profile of formulations
Antibacterial Activity
The result of antibacterial activity of formulations is shown in Table no.12
Table no.12: Zone of inhibition and % efficacy of formulations
Sr.no Formulation
Code
Staphylococcus aureus
Maximum Zone of
Inhibition (mm) % Efficacy
1 Standard value 28 100
2 F1 24.33 ± 0.5 90.89
3 F2 25.33 ± 0.05 91.28
4 F3 25.66 ± 0.05 92.24
5 F4 26 ± 0.01 93.14
6 F5 27.33 ± 0.005 98.60
7 F6 26.66 ± 0.06 94.18
8 F7 26.33 ± 0.5 95.45
9 F8 27 ± 0.005 96.34
10 F9 25.66 ± 0.01 97.12
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Fig.11.: Zone of inhibition of formulations
Fig.12: Surface response plot showing effect of Carbopol 934 and sodium alginate on
drug release.
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Fig.13: Surface response plot showing effect of Carbopol 934 and sodium alginate on
antibacterial activity.
Isotonicity Evaluation
The shape of blood cells, blood cells with Ofloxacin formulation F5 and blood cells with
Oflox as a marketed formulation are shown in Fig.14
a. Blood cells b. Blood cells with Ofloxacin Formulation (F5)
c. Blood cells with Oflox as marketed formulation
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Test for Sterility
There was no appearance of turbidity and hence no evidence of bacterial growth when
optimized formulation was incubated for 14 days at 30ºC to 35
ºC in case of fluid thioglycolate
medium and at 20ºC to 25ºC in case of soyabean-casein digest medium. The preparations
examined, therefore, passed the sterility test.
Ocular Irritancy Test
Right eye after 30 min Left eye after 30 min
Right eye after 1hr Left eye after 1hr
Right eye after 4hr Left eye after 4hr
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Right eye after 24hr Left eye after 24h
Right eye after 48hr Left eye after 48 hr
Right eye after 72hr Left eye after 72hr
Right eye after 1 week Left eye after 1 week
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Stability study
Accelerated Stability study of optimized F5 formulation at temperature (40ºC±2ºC) shown in
Table no.13
Table no.13: Stability study of Optimized formulation (F5)
Sr.No. Observations Before Stability testing After Stability testing
1 month 2 months 3 months
1. Clarity Clear Clear Clear Clear
2. Visual Appearance Transparent Transparent Transparent Transparent
3. pH 6.8 6.8 6.81 6.82
4. Drug content 101.5% 101.5% 101.4% 101.3%
CONCLUSION
The following conclusions can be drawn from present study-
1) Preformulation evaluation study has shown the identity & purity of Ofloxacin.
2) Infrared spectroscopy studies of Ofloxacin alone and there physical mixture with
Carbopol 934 and Sodium alginate revealed that, Ofloxacin is compatible with all the
polymers used.
3) The clarity of the prepared formulations was found satisfactory.
4) pH of all the formulations was found to be in between the ophthalmic pH range (6.5-8.5),
which is in tolerable range in contact with ocular tissues.
5) The viscosities of all the formulations were greatly affected by concentration of Carbopol
934 and sodium alginate.
6) Gel strength and Bioadhesive strength of formulations resembles to the viscosity results.
7) Drug content of all the formulations was found to be in between 98-101.8% which was in
acceptable range.
8) The release kinetics results obtained indicate that formulation containing 0.4 % w/v
Carbopol 934 and 0.5 w/v sodium alginate showed highest release i.e.100.05% after 8 hrs.
which indicates that the formulation have shown prolonged release. This optimized
formula was also confirmed by design expert 7.0.0 optimization software.
9) Antibacterial activity study shows comparable results with standard value but out of 9
formulations F5 shows better % efficacy i.e. 98.60 which indicate the dependency of
antibacterial activity with drug release from formulation.
10) The optimized formulation has shown the maintenance of tonicity.
11) The optimized formulation has passed sterility test.
12) The optimized formulation showed no ocular irritancy.
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13) The optimized formulation F5 showed good stability and no change in any physical
characteristics over a 3 months period.
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