INTRODUCTION:

The most commonly used formulations for applying drugs topically to the eye are the conventional solutions (eye drops) and eye ointment. Although they are the simplest form of drug delivery. They suffer from certain drawbacks, such as.

The eye drops on instillation causes reflex tearing where by most of the instilled dose gets washed out of the eye quickly, thereby reducing the effectiveness. The eye drops have to be therefore instilled several times in a day, which often lead to patient non-compliance. The ointment on the other hand, remain in contact with the eye for a much longer duration of time but because of its greasy nature causes blurring of vision, which limits its application at bed time only. Various other ophthalmic drug delivery system that are being evaluated include viscosity enhancers, gels, penetration enhancers, prodrugs, liposomes, nanoparticles, inserts, insitu-forming gels, oil-in-water emulsion, colloidal drug delivery system, microparticulates¹¹

Amongst these, the insitu gels are the simplest to fabricate and offer a significant increase in contact time with the eye when instilled^{1, 7}.Thus the focus of the present investigation was to prepare sol-gel drug delivery system using diclofenac potassium as drug model, Where it can be dropped as a solution into the conjunctival sac of the eye, upon contact with the lacrimal fluid, the polymer changes its conformation to form a gel. Studied the drug release, in vitro and efficacy study and compared it with conventional solution.

MATERIALS:

Diclofenac potassium: Brown and Burk pharmaceutical, Bangalore

Glycerin : Precision Scientific Co., Mumbai

Sodium chloride: Precision Scientific Co., Mumbai

Phenyl ethyl alcohol : Pal Pharmacy, Mumbai

Benzalkonium chloride: Qualigen Fine Chemicals, Mumbai.

Benzyl alcohol : Qualigen Fine Chemicals, Mumbai

Methyl paraben : Raula Pharma, Mumbai.

Propyl paraben : Raula Pharma, Mumbai.

Gelrite® : Sigma Co., Germany

Calcium chlorides dehydrate: Thomas Makers (chemicals), Mumbai.

EXPERIMENTAL:

1. PREPARATION OF FORMULATIONS: The formulation are briefly described here

A. 0.1% w/v diclofenac potassium conventional solution:

Solution containing different preservative base were prepared. diclofenac potassium along with other additives are added to preservative base, P^Hwas adjusted. The formulations were sterilized by membrane filtration and was filled and sealed in amber colored glass vials.

B. 0.1% w/v diclofenac potassium in-situ sol-gel 0.6% of gelrite was heated to a temperature of about 85⁰ C for 15 to 20 minutes and cooled with stirring. 0.5%v/v phenyl ethyl alcohol and 0.1%w/v diclofenac potassium were added and filter through 75µm filter cloth filled and sealed in amber colored glass vials. Autoclaved for 15-20 minutes at 121 ⁰C.

Table No.1: Formulation Table of Diclofenac Potassium Solution

Ingredients	F1	F2	F3	F4
Diclofenac potassium	0.1%	0.1%	0.1%	0.1%
Glycerin	0.5%	0.5%	0.5%	0.5%
Sodium chloride	0.9%	0.9%	0.9%	0.9%
Phenyl ethyl alcohol	0.5%	---	---	---
Benzyl alcohol	---	0.5%	---	---
Methyl paraben Propyl paraben	---	---	---	0.18% 0.02%
Benzalkonium chloride	---	---	0.01%	---

Table no 2. Formulation Table of Diclofenac Potassium with Gelrite

Ingredients	Quantities
Gelrite	6%
Diclofenac potassium	0.1%
Phenyl ethyl alcohol	0.5%
Aqueous vehicle	q.s

2. EVALUATION OF THE EYE DROPS:

A. Physicochemical Evaluation:

The ophthalmic solution was evaluated for the following parameters, visual appearance, pH, clarity, drug content. Visual inspection was carried out by observing the solution against fluorescent light. PH was checked using digital pH meter. Drug content was estimated by UV-spectrophotometer at 275nm.

B. Microbiological Evaluation¹³

The prepared eye drops were subjected to test for sterility as per the guidelines in I.P 1996.

3. DETERMINATION OF IN VITRO DRUG RELEASE PROFILE FROM THE FORMULATION.

The in vitro drug release was studied using a USP rotating paddle apparatus as reported by Rozier et al¹. 3ml of the solution was placed in a dialysis tube with cellophane membrane covered cells. Formulation was also tested without membrane covered cells. Simulated lacrimal fluid 7.4 (650ml) maintained at 37⁰C was used as the dissolution medium. The paddle speed was 50 rpm and aliquots were withdrawn at intervals of every hour for six hours. After each aliquot was withdrawn, it was replaced with an equal quantity of fresh medium. The amount of drug released was estimated by measuring the absorbance of the aliquots at 274nm.

Table No. 3: Physicochemical Evaluation of Diclofenac Potassium Solution

Tests	F1	F2	F3	F4
Visual appearance	+	+	+	+
Clarity	+	+	+	+
pH	6.25	6.25	6.50	6.52
Test for sterility	--	--	--	--
Drug content in mg mean±S.D*	49.87± 0.011	49.80± 0.08	49.81± 0.012	49.50± 0.14
% drug content	99.74	99.6	99.62	99

S.D*= standard deviation N =3

4. RHEOLOGICAL STUDIES¹

The rheological properties of sol-gel were measured using a Brookfield viscometer LVD++ model. The measurements were made by using disc spindle number 2 at 35⁰C. This temperature was maintained through out the experiment, by placing the beaker containing 200ml solution in a large beaker of about 1000ml containing water maintained at 38⁰C

FIGURE: 1

Table No. 4a: In Vitro Diffusion Profile of Diclofenac Potassium Sample Formulation with Cellophone Membrane Covered Cells

% Drug release
Sl. No	Average mean ± S.D*
1	23.66±0.536
2	31.57±0.211
3	36.22±0.143
4	41.08±0.320
5	44.60±0.110
6	48.19±0.416

S.D*= standard deviation N =3

Formulation with simulated lacrimal fluid was used for the study, measurements were made at different rpm like 10, 20, 30, 50, 60, 100 and viscosities were noted at respective rpm values. To mimic the situation, where the gelrite solution upon ocular instillation is diluted with the available tear fluid and the gelation is induced by supply of electrolytes. The solution prepared in de-mineralized water were mixed with simulated tear fluid, in the proportion 25:7 (application volumes 25ml normal volumes of tear fluid in the eye 7µl)

Table No. 4b: In Vitro Diffusion Profile of Diclofenac Potassium Reference Solution with Cellophone Membrane Covered Cells

% Drug release
Sl. No	Average mean ± S.D*
1	32.48±0.665
2	38.02±0.774
3	43.68±0.108
4	52.97±0.108
5	58.08±0.909
6	63.28±0.900

S.D*= standard deviation N =3

5. GELATION OF GELLAN GUM WITH DIVALENT CATION:

Composition of simulated lacrimal fluid

Sodium chloride 0.67g

Sodium bis carbonate 0.20g

Calcium chloride dihydrate 0.008g

Water 100g

Preparation of calcium chloride solution: 100mg of calcium chloride in 10ml of demineralized water.

Table No. 4c: In Vitro Diffusion Profile of Diclofenac Potassium Sample Formulation without Cellophone Membrane Covered Cells

% Drug release
Sl. No	Average mean ± S.D*
1	28.82±0.536
2	39.36±0.211
3	56.56±0.143
4	63.22±0.320
5	82.68±0.110
6	88.50±0.416

S.D*= standard deviation N =3

Preparation of series of concentration:

A series of concentration ranging from 10µl, 20µl, 40µl, 60µl, 80µl, and also 100µl, 200µl, 400µl, 600µl were prepared. This concentration was taken in 10ml vials and to this 10ml of formulation was added.

6. BIOLOGICAL EVALUATION: Biological evaluation includes the test for eye irritation and test for efficacy of the formulation.

Table No. 4d: In Vitro Diffusion Profile of Diclofenac Potassium Reference Solution without Cellophone Membrane Covered Cells

% Drug release
Sl.no	Average mean ± S.D*
1	99.67±0.122

S.D*= standard deviation N =3

A. Eye irritation test:

The eye irritation potential for the prepared eye drops was assessed by the Draize test², on albino rabbits, 3 rabbits weighing between 2-2.5 kg were used for the study. The eye drops were installed in one of the eye of each rabbit for 2 day as per the following schedule.

Day 1: 1 drop of the formulation was dropped and observed for 2 hours; left for 2 hours.Again, after total 4 hours, 2 drops of formulation was added and observed.

Day 2: 3 drops and 4 drops were given and observed for 2 hours. The contra lateral eye received normal saline in the same dosing schedule. The degree of irritation was assessed and scored as proposed by Drazie²

B. EFFICACY STUDY²:

This study was carried out on albino rabbits, weighing between 2-2.5 kg, 3 rabbits were used for the study. Inflammation was induced in both eyes of the rabbits using 25µl turpentine liniment I.P and the ability of the formulation under test for the faster recovery from the induced inflammation was assessed.²

The rabbit’s eye received eye wash with water for injection I.P To the right eye and left eye of each rabbit 25µl of turpentine liniment I.P was instilled with the help of micro pipette into each eye and left over night. This is done to balance the inflammation stress on both the eye. The next day to the left eye one drop of the formulation was instilled where as the right eye served as control.

FIGURE: 2

Table No 5: Viscosity Table Containing Gelrite In 7.4 Ph Simulated Lacrimal Fluid

RPM	Viscosity (CPS) mean± S.D*
10	423±0.208
20	325±0.216
30	283±0.163
50	232±0.141
60	227±0.182
100	117±0.216

S.D*= standard deviation N =3

After every 7 hours again 1 drop was given and scoring was done until the eye returned to its normal level.

RESULTS AND DISCUSSION:

The result of the evaluation of the prepared eye drops are:

Physicochemical evaluation: Table no1and 2 shows the values of the freshly prepared eye drops. The drug contents were with in I.P limits The PHof the eye drop was closes the expected value of 6.5 and 7.2. The solutions were found to be clear and free from particulate matter. The eye drop complied with the test for sterility.

Biological evaluation:

A. Eye irritation study:

This study is of importance because the infected /inflamed eye already caused irritation and pain to the patient and if the eye drop too causes irritation, it will result in grater discomfort. Move over, an irritating solution when instilled in to the eye results in profuse reflex tearing and hence the medication would be quickly washed out from the eye by nasolacrimal drainage, thus resulting in decreased effectiveness it is therefore imperative that the eye drop be non irritating to the eye.

The Draize system of scoring is based on the reaction of individual components of the eye to the instillation of the solution under test. Reflex tearing, conjunctival redness etc one mild reaction and their severity scores are therefore multiplied by a factor of 2, however damage to the cornea or the iris are more severe reactions and their severity scores are multiplied by a factor of 5.In our study the only parameter that could be scored were conjunctival, redness appearing immediately after instillation and reflex tearing occurring on instillation. The sum of the scores of all tissues response for a marginally irritating substance is rated as 49. In our cases, the sum of the score was zero for the formulation. Which indicated that the solution were none irritating to the eye.

B. Efficacy study:

Evaluation of the performance of formulation for use in the eye can be difficult since the most common animal, the rabbit, seems to correlate poorly with the human performance. The gels are much better retained in the human eyes and that the rabbit don’t respond well to variation in the osmolarity of the preparation. Thus rabbit eye is more efficient in eliminating gels than the human eye. Anti inflammatory activity showed that the percentage protection by the in-situ gel was more compared to the control and the eye returned to its normal level within 24 hours compared to control eye.

IN VITRO RELEASE STUDY:

With membrane:

Diclofenac potassium release is retarded and it also indicates that there is no interaction of the drug with the polymer.

Without membrane:

All diclofenac potassium is released from the reference solution with in first hours. On the other hand, the gelrite sample gels as soon as it comes in contact with the dissolution medium, preventing any escape of the bulk and entrapping the drug. The release is faster than with the dialysis membrane. A higher release rate should be expected in vivo, as the leaching action of the tears will tend to speed up the release process.

RHEOLOGICAL STUDY:

The viscosity curve obtained for the gelrite sample in simulated lacrimal fluid is typical of pseudoplastic behavior and it is expected to hinder drainage from the cul-de-sac of the eye. Gelrite polymer is a linear polymer with a tetrasaccharide repeating unit of l-rhamnose, D-glucose and D-gluconate. The formulation exhibits a non-Newtonian fluid characteristic.

GELATION OF GELLAN GUM:

The gelation study indicates that the gelrite will gel when it comes in contact with the divalent cations of the eye, even if the concentration of the ions in the eye is reduced to as much as 10% of the original value i.e. 10% of 0.008gm is 0.8mg which is equivalent to 800mg and also if the concentration is reduced to 10% of 0.8mg is 0.08mg which is equivalent to 80µg.

CONCLUSION:

A combination of diclofenac potassium and gelrite was developed for the first time, in the form of eye drops, as a clear solution. The formulation was simple and prepare with relatively ease. The eye drops were found to be non-irritating to rabbit eyes and were found to be effective in faster recovery from the inflammation. The gelrite also showed a good gelling property, hence it can be concluded that the in-situ gel can be successfully be used to prolong the duration of action of diclofenac potassium as compared to conventional solution.

ACKNOWLEDGEMENTS:

The author likes to thanks Jagdale scientific research foundation, Bangalore for help in carrying out the work at its research centre.

FIGURE: 3

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