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The main purpose of present study was to develop an ocucerts of Fluconazole -Î² CD (Beta cyclodextrin) complex and to evaluate both in vitro and in vivo. Fluconazole was made complex with Î² CD and the release rate was controlled by HPMC K4 M and Ethyl Cellulose polymers using Dimethyl sulfoxime as permeability enhancer. Drug- polymer interactions were studies by Fourier transform Infrared spectroscopic studies. The formulated ocuserts were tested for physicochemical parameters in vitro release and in vivo permeation in rabbits. The optimized formulations (F-4 and F-9) were subjected for stability studies. The formulated ocuserts were found to have good physical characters, thickness, diameter, uniformity in weight, folding endurance, less moisture absorption, controlled release of drug both in vitro and in vivo. The optimized formulations were retained their characteristics even after stabilities. The study clearly showed that this technique was an effective way of formulating ocuserts for retaining the drug concentration at the intended site of action for a sufficient period of time and to elicit the desired pharmacological response.
Keywords: Fluconazole, Ocuserts, Î² Cyclodextrin, In vitro, In vivo,
Eye drops and eye ointments are conventional ocular dosage forms. They have certain disadvantages like frequent administration, poor availability, massive and unpredictable doses, and drainage of medication by tear/ nasolacrimal fluid [1-3]. Ocuserts (Ophthalmic inserts) are sterile preparations, with a solid or a semi solid ingredients with suitable size & shape especially designed for ophthalmic purpose [4-6]. They mainly composed of a polymeric support with drug (s) incorporated as dispersion or a solution [7-9]. Fluconazole, a synthetic antifungal agent, is a triazole derivative. It is used in the treatment of a wide range of fungal infections  and it belongs to class II of Biopharmaceutical Classification System (BCS) having low water solubility . Cyclodextrins (CDs) are cyclic torus-shaped molecules with a hydrophilic outer surface and a lipophilic central cavity that can accommodate a variety of lipophilic drugs. CDs are available as Î±, Î² and Î³ forms. Among them Î²-CD is popularly included, which greatly modifies the physical and chemical properties of the drug molecule, mostly in terms of water solubility. Inclusion compounds of Cyclodextrin with hydrophobic molecules are able to penetrate in to body tissues; these can be used to release biologically active compounds under specific conditions . It was aimed to prepare ocular films containing Fluconazole Î²-CD complex.
2. MATERIALS AND METHODS
Fluconazole was a gift samples from Waksman Selman Pharmaceuticals, Anantapur, India. Î²-CD, Acetic acid, Propylene glycol were procured from Merck chemicals, Goa, India. All other reagents and solvents were of analytical grade.
Preparation of ocuserts
The preparation of ocuserts involved three different steps 
Preparation of Drug reservoir film
The polymeric drug reservoir films were prepared by dissolving 1.0, 1.5 and 2.0% of HPMC-K4M in 15 mL of double distilled water. Along with this 26.95 mg of binary mixture containing Fluconazole b-CD was separately dissolved in dilute alkali hydroxide solution and then it was poured to the polymeric solution. The solution was stirred using magnetic stirrer at 100 rpm and Di Butyl Pthalate (10% w/w), which was previously optimized for its concentration, was incorporated as a plasticizer as well as permeation enhancer to above solution under same stirring conditions.
After complete mixing the solution was casted in petri dish (previously lubricated with Glycerine) using a ring of 5.0 cm diameter and with a funnel inverted on the surface (for uniform evaporation of solvent). The cast solution was allowed to evaporate by placing it inside a hot air oven maintained at 37±2oC, 30±0.5 % of RH for 24 hours. After drying the medicated films of 8 mm diameter each containing 300 mg of drug were cut using a stainless steel borer, which is previously sterilized.
Preparation of Rate controlling membrane (RCM)
A weighed quantity of Ethyl Cellulose was dissolved in 10 mL of acetone to obtain 4, 5 and 6 % polymeric solutions. Stirring was continuously maintained until the clear solution was obtained. These solutions were poured in petri dish (previously lubricated with Glycerine) using a ring of 5.0 cm diameter. The solution was evaporated slowly by inverting a glass funnel on a petri dish at room temperature for 12 hours. The dried films were cut into 9 mm diameter using a stainless steel borer.
A medicated reservoir disc was sandwiched between two rate controlling membranes. Then this whole unit was placed for 4-5 min, over a wire mesh inside the desiccator. Desiccator was previously saturated with ethanol / acetone (60:40). This procedure resulted into successful sealing of the medicated reservoir film between two-rate controlling membranes. The sealed ocuserts were stored in an airtight container under ambient conditions.
Plasticizer weight was based on weight of the polymer. All the above experimentation was carried out under laminar airflow to maintain the sterility conditions of ophthalmic products.
Evaluation of Polymeric Ocuserts
The compatibility of drug with the excipient used was studied by Fourier Transform Infrared (FTIR) spectroscopy. The FTIR spectrums of Cetirizine Hydrochloride and Formulation (F-5) blend were studied by using FTIR spectrophotometer (Perkin Elmer, spectrum-100, Japan) using the KBr disk method (5.2510 mg sample in 300.2502 mg KBr). The scanning range was 500 to 4000 cm-1 and the resolution was 1 cm-1. This spectral analysis was employed to check the compatibility of drugs with the polymers used.
The ocucets were evaluated for their physical characters such as shape, colour, texture, appearance etc.
Thickness of Film
Films were evaluated for the thickness using a vernier caliper (For-bro Engineers, Mumbai, India). The average of 5 readings was taken at different points of film and the mean thickness was calculated. The standard deviations (SD) in thickness were computed from the mean value .
Uniformity in drug content
For drug content uniformity, the ocuserts were placed in 5 mL of pH 7.4 phosphate buffer saline and were shaken in orbital shaker incubator at 50 rpm to extract the drug from ocuserts. After incubation for 24 h, the solution was filtered through a 0.45 mm filter and the filtrate was suitably diluted with buffer solution [15, 16]
The absorbance of the resulting solution was measured at 254 nm.
Uniformity of weight
The weight variation test was carried out using electronic balance (Sartorius GmbH, Gottingen, Germany), by weighing three patches from each formulation. The mean value was calculated and the standard deviations of weight variation were computed from the mean value.
A small strip of film was cut evenly and separately folded at the same place till it breaks. The number of times the film could be folded at the same place without breaking gave the folding endurance .
Percentage moisture absorption
The percentage moisture absorption test was carried out to check physical stability or integrity of ocular films. Ocular films were weighed and placed in a dessicator containing 100 mL of saturated solution of aluminium chloride and 79.5% humidity was maintained. After three days the ocular films were taken out and reweighed. The percentage moisture absorption was calculated using the following equation .
Percentage moisture absorption = Final weight - Initial weight x 100
Percentage Moisture Loss
The percentage moisture loss was carried out to check integrity of the film at dry condition. Ocular films were weighed and kept in a dessicator containing anhydrous calcium chloride. After 3 days, the ocuserts were taken out and reweighed; the percentage moisture loss was calculated using the following equation [19, 20]
Percentage moisture loss = Initial weight - Final weight x 100
Determination of the Swelling Index and the Surface pH of the Fluconazole Films in Distilled Water
The ocuserts were coated on the lower side with ethyl cellulose (to avoid sticking to the dish) then weighed (W1) and placed separately in petri dishes containing 25 ml of distilled water. The dishes were stored at room temperature. After 5, 10, 15, 20, 30, 45 and 60 minutes, the films were removed and the excess water on their surface was carefully removed using filter paper. The swollen discs were weighed (W2) and the percentage of swelling was calculated by the following formula [19- 21]
Swelling index = W2 - W1 / W1 X100
The films used for determination of swelling index were used for determination of their surface pH using universal pH paper 
In vitro drug release studies
The ocuserts from each batch were taken and placed in a 15 mL vials containing 10 mL of pH 7.4 phosphate buffered saline. The vials were placed in an oscillating water bath at 32 ± 10C with 25 oscillations per minute. 1mL of the drug releasing media was withdrawn at various time intervals of 1, 2, 4, 8, 12, 16 and 20 h and replaced by the same volume of phosphate buffer saline pH 7.4. These samples were filtered through 0.45 mm membrane filter. The filtrate was diluted suitably with the buffer [23, 24]. The drug was estimated in each batch by double beam UV-Vis spectrophotometer (Elico SL 210, Mumbai, India) at 254 nm. The obtained data was treated with mathematical kinetic modeling.
In vivo drug release study
Out of 5 batches of formulations F-4 and F-9 were taken for in vivo study on the basis of in vitro drug release studies. The ocuserts were sterilized by using UV radiation before in vivo study. The ocusert and other materials were exposed to UV radiation for 1 h. After sterilization, ocuserts were transferred into polyethylene bag with the help of forceps inside the sterilization chamber itself. The pure Fluconazole that was sterilized along with ocuserts was analyzed for potency by UV spectrophotometer at 254 nm after suitable dilution with pH 7.4 phosphate buffer [25, 26].
The albino rabbits of either sex (New-Zealand strain), weighing between 2.5-3.0 kg were used for the experiment. The animals were housed on individual cages and customized to laboratory conditions for one day (received free access to food and water).
The ocuserts containing Fluconazole were taken for in vivo study, which were previously sterilized on the day of the experiment and were placed into the lower conjunctival cul-de-sac. The ocuserts were inserted into each of the 7 rabbits at same time the other eye of seven rabbits served as control.
Ocuserts were removed carefully at 1, 2, 4, 8, 12, 16 and 20 h and analyzed for drug content as dilution mentioned in drug content uniformity. The drug remaining was subtracted from the initial drug content of ocuserts that will give the amount of drug released in the rabbit eye. Observation for any fall out of the ocuserts was also recorded throughout the experiment. After one week of washed period the experiment was repeated for two times as before.
The potential ocular irritation and/or damaging effects of the ocusert under test were evaluated by observing them for any redness, inflammation (or) increased tear production. Formulation was tested on five rabbits by placing the inserts in the cul-de-sac of the left eye. Both eyes of the rabbits under test were examined for any signs of irritation before treatment and were observed up to 12 h 
Stability testing has become integral part of formulation development. It generates information on which, proposed for shelf life of drug or dosage forms and their recommended storage conditions are based.
In the present study, the formulation F-4 was selected for the study and ocuserts were packed in amber-colored bottles tightly plugged with cotton and capped. They were exposed to various temperatures (60 o, 40o, 20 o, 10o and 0o C) for 30 days. At regular intervals, the ocuserts were taken in 10 mL of pH 7.4 buffer and were shaken for 12 h in an orbital shaker. The resultant solutions were filtered, properly diluted and estimated spectrophotometrically by keeping pH 7.4 buffers as blank . The logarithmic percent of un-decomposed drug was plotted against time and decomposition rate constants (K) were obtained at each temperature. The logarithm of decomposition rate constants were plotted against reciprocal of absolute temperature and the resulting line was extrapolated to K at 25o C.
Shelf life can be obtained by using formula,
T90 = 0.104/K at 25o C.
Results and Discussion
The Thickness of formulated ocuserts were uniform and ranged from 0.16±0.001 to 0.17±0.005 mm. The little variation was observed with formulation F-5 might be due to the more concentration of rate controlling membrane. The values of uniformity of weight found to vary from 15.89±0.028 to 18.48±0.153 mg. All formulations (F-1 to F-9) showed good uniformity in weight. After the moisture loss the ocuserts showed no change in integrity and it was ranged from 6.29±0.109 to 9.68±0.045% and the moisture absorption was ranged from 4.78±0.222 to 9.84±0.148%. The highest moisture absorption was marked from formulations F-6 (9.84±0.148%), this may be due to the presence of larger concentration of hydrophilic polymer HPMC-K4M. The Folding endurance was ranged from 74±6.681 to 98±5.621 and no cracks were observed. Formulations F-9, F-3 and F-8 showed maximum folding endurance.
The surface pH values of all films were in the range 4.5-6.5.
The formulated ocuserts were found to have uniformity in drug content, formulation F-4 showed least dug content (85.65±9.657%) and formulation F-9 showed highest dug content (97.26±2.255%). Water uptake studies were performed for optimized formulations (F-4 and F-9). The water uptake was gradually increasing with time indicating the good wetting nature of the ocuserts. Based on the highest regression value (r), which is nearing to unity, the formulations F-1, F-2, F-4, F-6, F-8 and F-9 followed Higuchi-Matrix kinetics. This suggests that the drug release by swellable polymer matrix through the diffusion of tear fluids. The 'n' values of formulations F-1 to F-9 were 0.5652, 0.5329, 0.7126, 0.5984, 0.7687, 0.6295, 0.6985, 0.5987 and 0.7748 respectively. This indicates that the release by non-Fickian-diffusion mechanism. Cumulative percent drug release for F-4 and F-9 were found to be 92.31 and 93.03% respectively at 20th h. For in vivo drug release, formulations F-4 and F-9 were selected based on their uniform drug content and highest in vitro drug release. The cumulative percent drug release from F-4 and F-9 were found to be 90.24% and 87.45% after 20th h respectively, which is found to be less when compared to in vitro drug release studies. The ocuserts were retained in the rabbit eye during the entire study.
Stability data indicates the formulations were stable and no major degradation was found and a shelf life of 1.499 years was assigned to the ocuserts (F-9).
In the present study an attempt was made to develop ocuserts of Fluconazole with improved bioavailability, avoidance of repeated administration and dose reduction. From the experimental finding, it can be concluded that Hydorxy Propyl methyl cellulose is a good film forming hydrophilic polymer and is a promising agent for ocular delivery. Ethyl Cellulose was a satisfactory polymeric ingredient to fabricate the rate-controlling membrane of the ocusert system. Incorporation of Propylene glycol enhances the permeability of Fluconazole and thus therapeutic levels of the drug could be achieved. Complexation of Fluconazole with b-cyclodextrin suggested, enhancing the solubility profile of poorly soluble drug Fluconazole and also permeability of the drug through cornea. The kinetic treatment of in vitro dissolution data indicated that the ocusert followed non-Fickian diffusion kinetics. In vivo release profile indicated that drug release was less compared to in vitro and there was complete absence of eye-irritation, redness of the rabbit eye. The drug remained intact and stable in the ocuserts on storage and shelf life of 1.499 years. Further future work will be progressed to establish the therapeutic utility of these systems by pharmacokinetic and pharmacodynamic studies in human beings.
Table 1: Composition of various polymers in different formulations per ring
12 mL of the cast solution was poured into petri dish to prepare circular cast film. * Based on polymer weight
Table 2: Physicochemical Evaluation of different formulations
*All values in mean ±SD and number of trials (n=5)
Table 3: Water uptake and swelling behavior
Water uptake (mg)
All values in mean ±SD and number of trials (n=5)
Table 4: Kinetic values obtained from Zero order release profile
Table 5: Kinetic Values Obtained From First Order Release Profile
Table 6: Kinetic Values Obtained From Higuchi-Matrix Release Profile
Table 7: Kinetic values obtained from Korsmeyer Peppa's release profile
Table 8: Kinetic values obtained from Hixson Crowell release profile
Table 9: Data Obtained From Stability Studies
Ab. Temp (T)
Temp = Temperature; Ab. Temp = Absolute Temperature:
Rec. T = Reciprocal of absolute temperature;
D.R.C. (K) = Decomposition rate constant (Day-1);
Log. K = Logarithm of decomposition rate constant.