FORMULATION, DEVELOPMENT AND EVALUATION OF OFLOXACIN …

www.wjpps.com Vol 4, Issue 11, 2015.

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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,


3Department of Pharmaceutical Chemistry, R.G. Sapkal College of Pharmacy, Anjaneri,


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



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Right eye after 24hr Left eye after 24h

Right eye after 48hr Left eye after 48 hr


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.

REFERENCES

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Pharma Science Monitor:An International Journal of Pharmaceutical Sciences., 2011;

2(3): 1-25.

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review, International Journal of Scientific Research and Reviews, 2014; 3(2): 260-273.

3. Sireesha Satya D, Prabha Suria K, Prasanna Muthu P.,Advanced approaches and

evaluation of ocular drug delivery system,American Journal of Pharmatech Research,

2011; 1(4): 72-92.

4. Pawar et al. Formulation,development and evaluation of aqueous injection of poorly

soluble drug made by novel application of mixed solvency concept.International Journal

of Drug Delivery., 2013; 5(2): 152-166.

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Cefixime by first and ratio first derivative UV spectrophotometry.Chronicles of Young

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Cengage learning., 2007; 38.

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study.Scholars Research Library, Der Pharmacia Lettre., 2012; 4(5): 1541-1546.

9. Patel et al.Design and characterization of ofloxacin mucoadhesive in situ

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13. Charde et al.Development and evaluation of Herbal formulation for the treatment of

Acne.International Journal of Pharmaceutical Sciences and Research., 2014; 5(6): 2250-

2260.

14. Verma Lekhraj, Sakir Mohammad, Singh Navdeep, Mehra Ritu, Mehan

Sidharth.Development of phase change solutions for ophthalmic drug delivery based on

ion activated and pH induced polymers.International Journal of Pharma Professional‟s

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15. S.S.P.Hiremath et al.Formulation and evaluation of a novel in situ gum based ophthalmic

drug delivery system of Linezolid.Sci Pharm., 2008; 76: 515-532.

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Distribution of Drug Products.Pharmaceutical Technology., 2004: 69-73.

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FORMULATION, DEVELOPMENT AND EVALUATION OF OFLOXACIN …