INTRODUCTION:

Eye drops that are conventional ophthalmic delivery systems often result in poor bioavailability and therapeutic response because high tear fluid turnover and dynamics cause rapid precorneal elimination of the drug. A high frequency of eye drop instillation is associated with patient non-compliance. Inclusion of excess drug in the formulation in an attempt to overcome bioavailability problem is potentially dangerous if the drug solution drained from the eye is systemically absorbed from the nasolacrimal duct. Various ophthalmic vehicles such as inserts, ointments, Suspensions, and aqueous gels, have been developed in order to lengthen the residence time of instilled dose and enhance the ophthalmic bioavailability. These ocular drug delivery systems, however, have not been used extensively because of some drawbacks such as blurred vision from ointments or low patient compliance from inserts^1,2.

One way of prolonging the availability of the drug in the precorneal area involves increasing the viscosity of the dosage form by use of in situ gel forming system prepared from water soluble polymers that are instilled as a drops into the eye it undergoes a sol-gel transition in cul-de-sac which upon exposure to physiological condition, thus increasing the precorneal residence of the delivery system and enhance ocular bioavailability. Three types of system are recognized as pH triggered system, temperature dependant system and ion activated system³.

Glaucoma is a group of disease of the eye characterized by damage to the ganglion cells and the optic nerve. If left untreated, these effects may lead to various degrees of loss of vision and blindness. Increased intraocular pressure (IOP) remains the most important risk factor for the development of glaucoma⁴. When monotherapy does not adequately lower the intraocular pressure, one or more agents are added. Clinical trials have demonstrated that the combination of dorzolamide hydrochloride and timolol maleate is safe, effective and generally well tolerated in lowering IOP in patients with open angle glaucoma or ocular hypertension, including individuals uncontrolled on beta-adrenoceptor antagonist or other monotherapy⁵.

MATERIALS AND METHODS:

Materials:

Dorzolamide Hydrochloride was gifted by Hetero Drugs Ltd. Hyderabad, Timolol Maleate was gifted by Micro Labs Karnataka and Pluronic F127 was purchased from Sigma Aldrich Bengaluru. All other reagents and solvents used for study were of analytical grades.

Selection of vehicle⁶:

The solubility of combination of dorzolamide hydrochloride and timolol maleate was tested in acetate buffer I.P (pH 5.0 and 5.5), citro-phospahte buffer I.P (pH 5.0 and 6.0) and phosphate buffer I.P (pH 5.0 and 5.5mixed). Solutions of dorzolamide hydrochloride (2.0%, w/v) and timolol maleate (0.5%, w/v) in the buffers in which they were soluble were prepared and these were tested for stability in light, temperature, autoclaving and analyzed by UV spectroscopy.

Method of preparation^{7, 8}:

Aqueous solutions of varying concentrations of Pluronic F127 and HPMC (formulation codes F1, F2, F3…..F8) were prepared and evaluated for gelling capacity in order to identify the compositions suitable for use as in situ gelling systems(Table 1). The gelling capacity was determined by placing 1 drop of the formulation in a vial containing 2ml of Artificial Tear Fluid (freshly prepared and equilibrated at 37°C) and visually assessing time for gelation and the time taken for the gel redissolve. The composition of artificial tear fluid used was NaCl 0.670 g, sodium bicarbonate 0.200 g, calcium chloride·2H₂O 0.008 g, in 100.0 g. of purified water. The viscosity was measured using a Brookfield viscometer (DV- I+ model) using a small volume adapter. Depending on the gelling capacity of Pluronic F 127 and HPMC, formulation F3, F4, F6 and F7 was used for further study.

The composition of the in situ gel-forming system is as shown in Table 2. The buffer salts were dissolved in 75ml of distilled water; different concentrations of HPMC was added and allowed to hydrate. Pluronic F127 was sprinkled over these solutions and allowed to hydrate overnight. The solution was stirred with an overhead stirrer. A solution of dorzolamide hydrochloride and timolol maleate in distilled water was separately prepared. Benzalkonium chloride (BKC) solution was added to this solution and filtered through whatman filter paper. This solution was added to the solution of Pluronic F127 and HPMC under constant stirring until a uniform solution was obtained. Sufficient distilled water added to make up the final volume, pH of solution was adjusted with 0.5M sodium hydroxide solution. Finally packed in an amber colored vial and sterilized by autoclave at 121°C and 15 p.s.i for 20 minutes.

Evaluation of Formulation:

Visual appearance and clarity⁹: Visual appearance and clarity was done under fluorescent light against a white and black back ground for presence of any particulate matter.

Table 1: Various batches using different polymers concentration

Sr. No.	Batch Code	Polymer Conc. (% w/v)		Gelling Capacity
Sr. No.	Batch Code	Pluronic F127	HPMC	Gelling Capacity
1.	F₁	15	-	-
2.	F₂	15	0.5	+
3.	F₃	15	1.0	++
4.	F₄	15	1.5	++
5.	F₅	20	-	+
6.	F₆	20	0.5	++
7.	F₇	20	1.0	++
8.	F₈	20	1.5	+++

Where, -, no gelation

+, gels after few minutes, dissolves rapidly
++, gellation immediate, remains for few hours
+++, gellation immediate but for extended periods

Table 2: Composition of optimized formulations

Ingredients (%w/v)	F3	F4	F6	F7
Dorzolamide Hydrochloride	2.0	2.0	2.0	2.0
Timolol Maleate	0.5	0.5	0.5	0.5
Pluronic F 127	15.0	15.0	20.0	20.0
HPMC	1.0	1.5	0.5	1.0
Sodium Chloride	0.51	0.51	0.51	0.51
Glacial Acetic acid (ml)	0.6	0.6	0.6	0.6
Sodium acetate	1.36	1.36	1.36	1.36
Benzalkonium Chloride	0.01	0.01	0.01	0.01
Dist. Water q.s (ml)	100	100	100	100

pH⁹: pH of the in-situ gels after addition of all ingredients was measured using digital pH meter.

Rheological study¹⁰: Using Brookfield viscometer model (DV- I+), the developed formulation (pH 5.0) was poured into the small sample adaptor and the angular velocity increased gradually from 0.5 to 100 rpm. The hierarchy of the angular velocity was reversed and the average of two readings was used to calculate viscosity. The formulation was then poured into an ointment jar and the pH raised to 7.4 by adding 0.5 M NaOH. The rheology of the resultant gel was studied using the T bar F.

Drug content: Drug content was determined by suitably diluting formulation with ATF and analyzed by UV spectroscopy at 254 nm for dorzolamide hydrochloride and at 294 nm for timolol maleate.

In vitro release study^9,11: Drug released study from prepared formulation was studies using Franz- diffusion cell. Cellophane membrane and artificial tear fluid (ATF) pH 7.4 was used as a diffusion membrane and medium respectively. The cellophane membrane (previously soaked overnight in the receptor medium) was tied at one end of the glass diffusion cell. Accurately weighed 1ml of gel was spread uniformly on a cellophane membrane, which was in contact with receptor medium. The receptor medium was stirred continuously at 20rpm to simulate blinking action of eyelids. The whole assembly was adjusted on magnetic stirrer and maintained at 34±1°C. At specify intervals (0.5 hour, 1 hour, 2 hours…8 hours) 1 ml of sample was withdrawn from receptor compartment, replace with 1ml of freshly ATF and analyzed by UV spectroscopy.

Determination of isotonicity¹⁰: Isotonicity is important characteristic of the ophthalmic formulations. Isotonicity has to be maintained to prevent tissue damage or irritation of eye. Formulations were mixed with few drops of blood and observed under microscope at 40X magnification and compared with standard marketed ophthalmic formulation (eye drop) containing dorzolamide hydrochloride and timolol maleate. The shape of blood cell (bulging or shrinkage) was compared with standard marketed ophthalmic formulation containing dorzolamide hydrochloride and timolol maleate.

Sterility study¹²: It was performed for aerobic, anaerobic and fungi microorganisms using fluid thioglycollate and soyabean-casein digest medium as per IP 2007. Formulation took into laminar flow and passed through a membrane filter of 0.45µm with the help of vaccum pump. After filtration the filter paper was removed and cut into two halves. One half was dropped in fluid thioglycollate and other in soyabean-casein digest. Both the media kept for incubation at 37°C for 7 days, and observed for any microbial growth.

Stability study: Selected sterilized formulations store at 4±1°C, room temp. (27±1°C), 45±1°C for a period of 2 months. The formulation was evaluated at periodic intervals for drug content, clarity, ph, sol-gel transition, rheology and in vitro drug release.

RESULTS AND DISCUSSION:

Selection of vehicle:

Buffers play a pivotal role in formulating ophthalmic drops. They contribute significantly to chemical stability and clinical response and also influence the comfort and safety of the product. Hence the importance of selecting a suitable buffer which ensures product stability and desired drug stability. The studies in various buffer solutions indicated the drugs were soluble in acetate buffer pH 5.0 and 5.5 and in citro-phosphate buffer pH 5.5, mixed at the desired dosage level. The solutions were stable to elevated temperatures and autoclaving. However, their instability to light as evidenced by discoloration of the exposed solutions necessitated their packaging in amber vials. Acetate buffer pH 5.0 was selected as a vehicle for the formulation of in situ gelling system at the dosage level desired; it is easily neutralized by the buffering action of the tear fluid.

Visual appearance and clarity: Formulations were found to be transparent and clear.

pH: The pH of formulations was found to be 4.87 – 5.01 as shown in (Table 3).

Table 3: Physio-chemical properties of optimized formulations

Parameters	F3	F4	F6	F7
Visual appearance	Transparent	Transparent	Transparent	Transparent
Clarity	Clear	Clear	Clear	Clear
pH	4.96	5.01	4.87	4.89
Gelling capacity	++	++	++	++

Rheological study: Results of rheological studies revealed that, the viscosity of all formulations at non-physiological (pH 5.0) and physiological (pH 7.4) conditions were decreased, as a shear rate increased as shown in (Fig. 1 and 2). Hence, the formulations may possess the characteristics of pseudoplastic fluid.

Fig. 1: Rheological profile of in-situ gelling systems at pH 5.0

Fig. 2: Rheological profile of in-situ gelling systems at pH 7.4

Drug content: Drug content in optimized formulations were found to be 98.97 - 94.49 % for dorzolamide hydrochloride and 99.18 – 96.28 % for timolol maleate as shown in (Table 4).

Table 4: Drug content of optimized formulations

Sr. No.	Batch Code	Drug Content(%) ± S.D (n=3)
Sr. No.	Batch Code	DH	TM
1.	F₃	98.97 ± 0.41	99.18 ± 0.20
2.	F₄	94.49 ± 0.65	97.87 ± 0.45
3.	F₆	98.21 ± 0.60	98.57 ± 0.13
4.	F₇	97.82 ± 0.31	96.28 ± 0.16

In vitro release study: In-vitro drug release studies of optimized dorzolamide hydrochloride and timolol maleate in-situ gel formulations are by plotting cumulative % drug release Vs time. Results of this study revealed that, formulation F3 containing Pluronic F127 (15%) and HPMC K15M (1.0 %) released 92.72 % of dorzolamide hydrochloride and 91.71 % of timolol maleate respectively, when compared to other formulations over a period of 480 minutes (Fig. 3). In case of marketed eye drop, almost all the both drugs were released within 90 minutes (Fig. 4). The prolonged period of drug release may be due to slow diffusion of drug from combined effect of polymers. The prolonged release may be probably due to the formation of hydrogen bonds between drug and polymers, which have helped in rate control release of drug.

Fig. 3: In-vitro release profile of F3 formulations

Fig. 4: In-vitro release profile of marketed eye drop

Isotonicity study: The optimized formulation (F3) was subjected to isotonicity study and exhibited no change in the shape of blood cells (bulging or shrinkage), which reveals the isotonic nature of the formulation and compared with that of standard marketed ophthalmic eye drop of dorzolamide hydrochloride and timolol maleate. Fig. 5, 6 and 7 showed photograph of RBC during isotonicity study.

Fig. 5: Blood cells with standard marketed eye drop

Fig. 6: Blood cells with isotonic formulation F₃

Fig. 7: Blood cells without maintained isotonicity of formulation F₃

Sterility study: There was no microbial growth in optimized formulation (F3) after 7 days of incubation period, showing that the method used for sterilization was reliable.

Stability study: Stability studies was carried out on formulation F3 for a period of 2 months and was found to be clear with no change in pH (about 5.0), drug content (98.80- 99.11 %), viscosity, in vitro release, gelling capacity.

CONCLUSION:

Dorzolamide hydrochloride and timolol maleate (anti-glaucoma agent) in combination were successfully formulated as temperature dependent in situ gel-forming eye drop. The developed formulations are viable alternative to conventional eye drops by virtue of its stability to enhance bioavailability through its longer precorneal residence time and ability to sustain drug release. Also important is the ease of administration afforded and decreased frequency of administration resulting in better patient acceptance. Therefore, based on present study, it can be concluded that, the formulations F3 of polymeric in-situ gels containing Pluronic F 127 (15%) and HPMC 1.0 % have considerable influence on their physiochemical characteristics, permeability properties and decreased frequency of administration with appreciable strength and safety.

ACKNOWLEDGEMENTS:

The authors thank College of Pharmacy, IPS Academy, for providing required facilities to carry out this research work. They also thank Hetero Drugs Ltd., Hyderabad for providing gift sample of Dorzolamide Hydrochloride and Micro Labs, Bengaluru for providing gift sample of Timolol Maleate.

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Received on 29.08.2011 Accepted on 26.10.2011

Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 93-97