FORMULATION AND EVALUATION OF STIMULI SENSITIVE pH TRIGGERED IN-SITU GELLING SYSTEM OF FLUCONAZOLE IN OCULAR DRUG DELIVERY

Received on 31 October, 2013; received in revised form, 12 December, 2013; accepted, 24 March, 2014; published 01 April, 2014

FORMULATION AND EVALUATION OF STIMULI SENSITIVE pH TRIGGERED IN-SITU GELLING SYSTEM OF FLUCONAZOLE IN OCULAR DRUG DELIVERY

S. Nagalakshmi*, Seshank, Radhika Ramaswamy and S. Shanmuganathan

Department of Pharmaceutics, Faculty of Pharmacy, Sri Ramachandra University, Porur, Chennai-116, Tamil Nadu, India

ABSTRACT: The present research finding focuses with the formulation and development of in situ gelling system of fluconazole using stimuli sensitive hydrogels. The quick elimination of the drug in the pre-corneal region thereby reducing the bioavailability of conventional ophthalmic solutions may overcome by means of in situ gel forming systems.  These are instilled as drops into the eye and undergo a sol-gel transformation in the cul-de-sac. Fluconazole is an effective antifungal agent against superficial fungal infections in the eye. Hence an effort was made to develop in situ gelling system that can be triggered by pH and for providing sustained release of fluconazole. The drug that has been loaded with polymeric carrier undergoes transition from solution to gel upon triggered by pH. The main purpose of the study was to formulate pH triggered in situ gelling system of fluconazole using carbopol and HPMC polymers in order to attain a better bioavailability, considerable increase in ocular residence time, and reduce frequent instillation, thereby improving patient compliance. Fluconazole in situ gelling system was formulated by using polymers of pH sensitive grade like polyacrylic acid (carbopol940) along with hydroxy propyl methyl cellulose (HPMC). Carbopol solutions are less viscous and transform into firm gels upon increase in pH of the eye as it is acidic in nature and when it combines with HPMC, a well-known ocular viscosity enhancing agent. The developed formulation provided sustained release over a period of 8 hours and it was proved that the formulation was stable and safe which can be considered an alternate to the conventional eye drops.

Fluconazole, Hydrogels, Carbopol, HPMC, pH triggered, In situ gel

INTRODUCTION:The most important and essential organs of our body are the eyes. Eye drops are one of the conventional ophthalmic drug delivery systems, ultimately lead to very less bioavailability in ocular area. This is attributed owing to its ocular anatomical and physiological constraints like relative impermeability of epithelial membrane of cornea, tear dynamics and drainage due to nasolacrimal fluid ¹.

The intention of pharmacotherapeutics is to seek the achievement of an adequate drug concentration at the specified site of action for an ample period of time to show a pharmacological response ². This has been a major challenge to develop a formulation   in ocular therapeutics. Recently, in situ gel formulations have extensively been studied to improve ocular bioavailability and period of drug therapy ³.

The reduced bioavailability and therapeutic response elicited by commercial ophthalmic solutions due to quick pre-corneal elimination of the drug can be overcome by the means of in situ gels ⁴. An increase in the pre-corneal residence time of drug and improved bioavailability could be achieved by the usage of ocular delivery systems based on the concept of in situ gel formation. These in situ gelling systems exhibit sol to gel transition in the cul de sac of the eye. The transition is due to alterations in the pH, temperature, and ionic strength and various physicochemical parameters ⁵.

The challenge of this research finding is to develop an in situ gelling system of fluconazole using carbopol940 and HPMC LV 50 that is activated by pH. To formulate fluconazole (0.3%w/v) eye drops, pH sensitive polymers were used as the vehicle. The challenge is to transform eye drops in to gel and to afford sustained release of the drug, in turn to improve the patient comfort by reducing the dose and frequency of administration.^4,5

MATERIALS AND METHODS:

Materials: Fluconazole, Carbopol940 (HIMEDIA LABORATORIES, LOBACHEMIE), Hydroxyl propyl methyl cellulose (LOBA CHEMIE), EDTA, Sodium Chloride, Benzalkonium chloride and pH 4 buffer. All chemicals either of analytical or pharmaceutical grade were used without further purification.

Method: The novel formulation of insitu gel was developed as per the method reported by Kumar and Himmestein, et al.⁶Different concentrations of pH sensitive polymers were used to develop eye drops of fluconazole and the composition is shown in table 1. Combination of polymers of required quantity was allowed to hydrate in pH 4 buffer. Calculated quantities of EDTA, sodium chloride, benzalkonium chloride were mixed to the buffered polymeric solution. The resulting solution was mixed with the drug solution and was stirred continuously to get a uniform solution. The final volume was adjusted by using pH 4 buffer. The developed formulations were stored in aseptic conditions. Sterilization procedure was carried out by autoclaving (121ºC and 15 psi) for 20 minutes and characterization studies were performed further.

TABLE 1: COMPOSITION OF VARIOUS INSITU GELS

EVALUATION:

Visual Appearance, Clarity and pH: The prepared formulations were checked for clarity, color and transparency visually. The pH was measured by using a pH meter ^{4, 5, 6}.

 Gelling capacity: The developed in situ gel was assessed for its gelling capacity. The dilution of formulation was done by mixing with simulated tear fluid (25:7). The time taken for gelation and dissolution by the developed gel was noted ^{5, 7}.

Drug content estimation: The content of drug in the in situ gel was calculated by taking a sample (2ml) of the in situ gel and mixed with simulated tear fluid of pH 7.4 in 100 ml flask to get the concentration of 10µg/ml in 100 ml.

The absorbance values were read by using UV spectrophotometer at 260 nm in order to calculate the percentage of drug content ⁸.

In vitro Release Studies: Dialysis method was used to study the release of drug. Open ended glass cylinders of dimensions of about 10 cm height, 3.7 cm outer diameter were used as a permeation cell.

A cellophane membrane (0.8µm pore size previously soaked in phosphate buffer of pH 7.4) was attached to one end of the cylinder by adhesive tape. 2 ml of the prepared formulation (eye drops) solution was placed inside the cell and the cell is submerged to a depth of 1 cm in 100 ml phosphate buffer pH 7.4. The cell was maintained at 37±1˚C throughout the experiment.

Aliquots were withdrawn from the receptor compartment respectively (0.5, 1, 1.5, 2, 2.5hrs). After each withdrawal, the volume of liquid in the receptor compartment was replaced by fresh phosphate buffer of pH 7.4.The  drug concentration was determined spectrophotometrically at 260nm.The release pattern of the in situ gel preparation was calculated by plotting the drug release with time. The in-vitro drug release for all the developed formulation was calculated and amongst them the formulation showing good release profile was compared with the commercially available eye drops ^{8, 9}.

In vitro Release Studies using goat’s cornea: Goat’s cornea was used in the permeation study of the drug across the corneal membrane. Eye ball of the goat was procured from the slaughter house and was transferred while in cold condition submerged in normal saline at 4^oC to the laboratory. Cornea was removed along with 5-6mm of the surrounding scleral tissue and was washed with cold saline. The drug release from the prepared formulation was studied by using dialysis method using goat’s cornea as semi permeable membrane ¹⁰.

Sterility testing: IP method (1996) was followed for the sterility testing of the eye drops. Sterility testing was performed by incubating the formulations for not less than 14 days at 30-35˚C in fluid thioglycollate medium to check the growth of bacteria and at 20-25^oC in the soya bean casein digest medium to check the growth of fungi in the formulations ^{1, 11}.

Antifungal assay: The antifungal activity of the selected formulation was carried out on Candida albicans species by antifungal susceptibility test.

The nutrient agar medium was prepared by dissolving potato dextrose in hot distilled water and media was autoclaved at 121˚C for 15 minutes. By using diffusion method, test organisms were previously seeded in the nutrient agar medium. The aliquot test samples were poured in to petri plates containing nutrient agar medium using a micropipette.

The plates were left undisturbed for 20min and then incubated at 25˚C for 24hr.The diameters of zone of inhibition for Candida albicans were measured up to 48-72hrs respectively ¹².

FTIR studies: The FTIR study reveals the possibility of interaction between drug and the polymer. Potassium bromated (KBr) pellets were used to record the FTIR graph for pure drug and the polymer ¹³.

Accelerated stability testing: The developed formulations were stored in amber colored vials which were then subjected to accelerated stability studies by storing at 40±2˚C and 75±5% RH as per ICH guidelines. During the stability studies, samples were periodically analyzed for any change in their appearance, pH, gelling capacity and drug content ¹⁴.

RESULTS:

Visual Appearance, Clarity and pH: The nature and the color of prepared eye drops were seem to be light yellow in color, except F2 and F6. All the formulations were found to be clear without any haziness. The developed ophthalmic formulations were found to have pH within the range of 6-6.4 (refer table 2).

Table 2: EVALUATION OF INSITU GELS

Gelling capacity: Formulations F1 and F6 gels slowly and dissolves. F2 and F5 showed immediate gelation and remained for few hours. Formulations F3 and F4 also showed immediate gelation but remained for an extended period (refer table 2).

Drug content estimation: The drug content was within the range of 98.35% to 99.88% and the results are shown in table 2.

In vitro Release Studies: The best formulation was found to be F4 and the release pattern was compared with the marketed formulation. The release pattern of all the gels are shown in figure 1 and the comparative release is shown in figure 2.

FIGURE 1 FIGURE 2

Sterility testing: The formulation F4 passed the test for sterility testing.

Antifungal assay: Test sample (formulated product) representeda maximum zone of inhibition and the results for antifungal assay are shown in table 3 and refer figure 3.

TABLE 3: EVALUATION ANTI FUNGAL ASSAY

No of organism: 1No (Candida albicans); Amphotericin B (Conc. 100 mg/ml -100µl for 100 ml media)

FIGURE 3: ZONE OF INHIBITIONS FOR THE TEST FORMULATION, SAMPLE CONTROL, MARKETED PREPARATION AND AMPHOTERICIN B

FTIR studies: The FTIR graph representing pure drug and mixture of drug and polymers were studied. From the observed peaks for pure drug and mixture of drug and polymers, it was confirmed that there were no significant interaction between the drug and polymers.

Accelerated stability testing: There were no significant variations in the drug content, consistency, pH and physical appearance of the formulations after storing at the prescribed temperatures as mentioned earlier.

DISCUSSIONS:

Visual Appearance, Clarity and pH: Sterilization had no specific effect on the formulations. The lack of clarity which was noted during autoclaving procedure mainly occurred by the precipitation of HPMC at elevated temperatures. The haziness disappeared and the solution attained clarity on overnight standing. The pH was found to be suitable for eye drops.

Gelling capacity: The rheology and gelling capacity are significant parameters for the development of insitu gel formulation. The formulations should provide favorable viscosity for easy instillation into the eye as a drop which then transforms in to gel form.

Drug content estimation: The percentage drug content tells that the drug has been uniformly distributed in all the developed formulations.

In-vitro Release Studies: It was evident from in-vitro dissolution data that F4 showed better sustaining effect amongst all formulations. The in-vitro release profile of F4 was then compared with marketed formulation of fluconazole and was found that the drug release was about 52% at the end of 8hrs study.

Sterility testing: The formulation F4 passed the sterility tests, since no turbidity appeared in the formulation and hence there was no proof of microbial growth when incubated in not less than 14 days at 30-35˚C in case of fluid thioglycollate medium and at 20-25˚C in the case of soyabean casein digest medium.

Antifungal assay: The test sample showed a maximum zone of inhibition when compared to the sample control, marketed product and amphotericin, which proves that the test formulation has better efficacy and can provide an excellent antifungal property.

FTIR studies and Accelerated stability testing: No interaction with the drug and polymers were to be found which states that the formulation was stable. And the results from accelerated stability tests confirmed that the formulated in situ gel did not show any variations and could withstand the various temperatures and humidity.

CONCLUSION: The novel pH triggered ocular insitu gelling drug delivery was successfully developed and evaluated for its appearance, drug content, clarity, pH, gelling capacity, in vitro release and stability studies etc. The formulation was in the form of a liquid at pH 6 and undergoes immediate gelation when the pH was raised to 7.4. The gel exhibited release over a period of 8 hours in a sustained manner. Hence this developed formulation is a viable alternate choice over the commercial eye drops by the virtue of its improved bioavailability through its increased corneal residence time and ability to sustain drug release. Thus patient compliance could be achieved by decreasing the frequency of drug administration.

ABBREVIATIONS:

EDTA-Ethylene diamine tetra acetic acid

IP- Indian Pharmacopoeia

FTIR- Fourier Transform Infrared Spectroscopy

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