DESIGN AND CHARACTERIZATION OF SUSTAINED RELEASE LEVOFLOXACIN OCULAR INSERTS

Levofloxacin is a fluoroquinolone antibacterial drug effective in the treatment of bacterial conjunctivitis. The objective of the present work was to develop ocular inserts of levofloxacin and evaluate their potential for sustained ocular delivery. Conventional ophthalmic solution shows the poor bioavailability and therapeutic response due t many pre-corneal constraints. These constrains necessitates the controlled and sustained drug delivery to become standard one in modern pharmaceutical era Matrix type ocular inserts were prepared by the film casting technique in Teflon coated Petri dishes and characterized in vitro by drug release studies using a flow through apparatus that simulated the eye conditions. Nine formulations were developed, which differed in the ratio of polymers chitoson, polyvinyl alcohol (PVA). All the formulations were subjected to evaluation of thickness, weight variation, folding endurance, drug content uniformity, in vitro release study, Surface pH, Swelling Studies (Swelling Index), % Moisture absorption, Release Kinetics, and Ocular Irritation Studies. On the basis of in vitro drug release studies, the formulation L9 was found to be better than the other formulations and it was selected as an optimized formulation.


Introduction
Continuous delivery of drugs to the eye offers major advantages over conventional therapies that involve administration of drug solutions or suspensions as eye drops. Eye drop administration often results in poor bioavailability and therapeutic response due to rapid precorneal elimination of the drug and is also associated with patient compliance problems 1-2 . A basic concept in ophthalmic research and development is that the therapeutic efficacy of an ophthalmic drug can be greatl improved by prolonging its contact with the corneal surface. Ophthalmic inserts offer many advantages over conventional dosages forms, like increased ocular residence, possibility of releasing drug at a slow and constant rate, accurate dosing, exclusion of preservatives and increased shelf life. Design, construction and technology of ocular insert in a controlled and sustained ocular delivery device are gaining rapid improvement to overcome these constraints [3][4] . Levofloxacin is a broad spectrum antibacterial with a half-life of 6 to 8 hrs frequently used in ocular infections, and is sparingly soluble in water 5 . 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 and naso-lacrimal fluid 6-8. These factors require formulating a controlled release ocular drug delivery system which maintains a steady state drug release. The literature survey reveals good scope of work in this direction. In the present study, an attempt was made to formulate ocuserts of levofloxacin using polymers like chitoson and PVA IJBAR (2011) 02 (12) www.ssjournals.com formulation with the combination of these two polymers. The chitoson was natural polymer and pva is synthetic polymer. Levofloxacin is a pale yellow or bright yellow, crystal-line powder. Levofloxacin is antibacterial and is the most commonly used fluroquinolone. It inhibits the enzyme bacterial DNA gyrase, which nicks double stranded DNA, introduces negative supercoils and then reseals the nicked ends. This is necessary to prevent excessive positive supercoiling of the strands when they separate to permit replication or transcription. The bactericidal action probably results from digestion of DNA by exonucleases whose production is signaled by the dam-aged DNA 9 . Levofloxacin has a broad antimicrobial spectrum against gram-positive and gramnegative microorganisms. This drug is routinely used in the many ocular conditions like infections, inflammations, conjunctivitis, blepharitis, iritis, corneal ulcer etc 10,11 . In the present study, it was aimed to prepare ocular films containing Levofloxacin along with hydrophilic polymers with better solubility and longer duration of action delivering the drug in zero order kinetics. The aim of the present work was to design polymeric ocular drug delivery system of Levofloxacin to overcome the disadvantages associated with conventional ophthalmic dosage forms (eye drops and suspensions), to achieve long duration of action and to improve ocular bioavailability. Quantity of drug instilled in one day (6-12 drops/ day) = 1500-3000 µg

Materials and
The quantity of levofloxacin in the ocular insert can be between 1.5-3.0 mg for one day treatment using eye drops. Thus, 2.25 mg of levofloxacin is taken as the minimum required quantity for one day treatment. It was presumed that there would be a negligible loss of drug through ocular inserts when the drug is released at a constant rate over a period of 24hrs. Assumption was also made that the ocular inserts will produce a better clinical response than the eye drops. The amount of levofloxacin to be incorporated for each ring was calculated in the following manner: 13 Area of circle = r 2 Diameter of the ring = 5.5 cm Radius = 5.5 / 2 = 2.75 cm 2 Diameter of the ocuserts = 0.8 cm Area of the single ocular film = 0.502 cm 2 IJBAR (2011) 02 (12) www.ssjournals.com Number of films casted on the ring theoretically = 48 films Amount of drug loaded on each ring = No. of single film x Dose (mg) = 48 x 2.25 = 108 mg 2.

Preparation of Ocular Inserts
The inserts were prepared by solvent casting method 14 . The polymers solution were prepared by dissolving CHITOSON and PVA in distilled water (10 ml) and PEG 400 (w/v of polymer) was added as plasticizer to this solution under stirring condition. The weighed amount of levofloxacin (108 mg) was added to above dispersion. After proper mixing the casting solution (5 ml) was poured in clean glass petridish (an area of 63.59 cm 2 ). The petridish was dried at room temperature for 24 h. The dried film thus obtained were cut by cork borer into circular pieces of definite size 8 mm diameter (an area of 0.502 cm 2 ) contain 2.25 mg of drug. 15,16 . The ophthalmic inserts were stored in an airtight container (desiccator) under ambient condition as shown in the Table 1: To check the uniformity of the drug in the circular film, three inserts were taken out from each film. Each inserts were then placed in volumetric flask containing 100 ml of phosphate buffer pH 7.4 and shaken to extract the drug from film. One ml of above resulting solution was withdrawn, after suitable dilution with phosphate buffer pH 7.4 and analyzed by spectrophotometrically. The absorbance of the solution was measured by U.V. visiblespectrophotometer at 290 nm and against blank 17 .

Thickness:
Ocular insert films prepared by solvent casting technique were evaluated for the thickness uniformity. The thicknesses of the prepared films were measured by using digital micrometer (Mitutoyo co., Japan) with an accuracy of 1µ measurement. Digital micrometer contain two screws one is fixed and another is a movable screw. Initially before measuring the thickness of the film, movable screw was rotated tightly (to touch the fixed screw) to get the zero adjustment in the display screen of the instrument, then slowly the movable screw was released and the film was placed in between the rotating and fixed screws, then the rotating screw was slowly tightened similarly, the same procedure was repeated at different points on the surface of the film. Thickness measurements were carried out for a minimum of three films of each IJBAR (2011) 02 (12) www.ssjournals.com formulation and average thickness calculated 18,19 .

Uniformity of weight:
For uniformity of weight, 3 films Mfrom each batch were taken randomly and their weights were determined using electronic balance (Asco, India) 20 .

Folding endurance:
Three film of each formulation of size (2 x 2 cm) were cut by using sharp blade. Folding endurance was determined by repeatedly folding a small strip of the film at the same place till it broke. The number of times the film could be folded at the same place without breaking gave the value of folding endurance 21

Swelling Studies (Swelling Index)
Three films were weighed and placed separately in beakers containing 4 ml of distilled water. At regular intervals of time (every 5 min), the films were removed and the excess water on their surface was removed using a filter paper and then weighed again. The procedure was continued till there was no increase in the weight. The swelling index was then calculated by dividing the increase in weight by original weight and was expressed as percentage 18 .

Percentage Moisture loss:
The percentage moisture loss was carried out to check the integrity of the film at dry condition. The films were weighted and kept in dessicator (8x 6 inch, polylab, India) containing anhydrous calcium chloride. After three days, the films were taken out and reweighed 23 . The percentage moisture loss was calculated using the formula: %Moisture loss = Initial weight -Final weight × 100 Initial weight

Percentage Moisture absorption
The percentage moisture absorption test was carried out to check physical stability or integrity of the film at humid condition.
The study was carried out as per procedure reported earlier (Dhanaraju et al., 2002) The films were weighed and placed in desiccators (8x 6 inch, polylab, India) containing 100 ml of saturated solution of aluminium chloride and 80% humidity was maintained. After three days, the films were taken out and reweighed 20  The lower one is fixed and upper one is movable. The test film of specific size (4*1 cm 2 ) was fixed between these cell grips and force was gradually applied till the film breaks. The tensile strength of the film was taken directly from the dial reading in kilograms 31,32 . Tensile strength (kg/cm2) = break force (kg)/cross-sectional area of the sample (cm2).

Ageing study:
The optimized inserts (L9) were stored in amber colored glass bottles at 3 different temperatures 4 0 C, Room temperature (R.T.) and 37 0 C for a period of 3 months. The samples were withdrawn after 30, 60 and 90 days and analyzed for physical appearance, drug content and sterility 33 .  Table 2). The minimum intra batch variations revealed the suitability of the process used to prepare the ocuserts.

Folding Endurance:
The folding endurance for all formulations was good.
The maximum folding endurance of formulation L3 was 96.3±4.5 foldings and formulation L1 showed minimum folding endurance of 61±4.5 foldings ( Table 2). This showed that as the concentration of polymer increased in the formulation, folding endurance was decreased.   (12) www.ssjournals.com

Surface pH:
The surface pH of the prepared inserts varied between 6.5 to 7.5, indicating that the inserts did not have an irritation potential as the pH is within the accepted ocular range (Table 4).

Swelling Studies (Swelling Index):
The Polyvinyl alcohol (PVA) and chitoson are hydrophilic polymer and are soluble in water. Due to its hydrophilic nature the polymers can be expected to absorb water. So to verify this fact, a swelling index test was carried out. The result showed that there was no significant variation in the water absorption properties of formulations. The formulation L1 showed maximum swelling index of 2.141 and formulation L9 showed minimum swelling index of 1.141. The % moisture absorption was calculated for all 9 formulations in triplicate ( Table  7). According to the results obtained, the moisture absorption is more in the formulations in which hydrophilic polymers are present. Formulation L9 has shown maximum % moisture absorption, 12.13 ± 0.48%, which may be due to the presence of PVA which is relatively more hydrophilic in nature. The formulation L1 has shown the minimum % moisture absorption, 5.48 ± 0.50%, as it contains polymer of less hydrophilic nature.          The release profile of the formulations is depicted in the figure 1 to 9. The formulation with chitoson and Polyvinyl alcohol (PVA) showed complete release in 11 to 12 hr. The release of the drug form the formulation L1 and L2 were found to be 90.52 % and 91.09 % at the end of 12 hr respectively. The release of the drug from the formulation L3 and L4 were found to be 92.44 % and 93.79 % at the end of 12 hr respectively. The release of the drug from the formulation L5 and L6 were found to be 94.12 % and 95.48 % at the end of 12 hr respectively. The release of drug from formulation L7 and L8 were found to be 96.57 % and 98.10 % at the end of 12 hr. The releases of drug from formulation L9 were found to be 98.99782 % at the end of 12 hr. IJBAR (2011) 02 (12) www.ssjournals.com

In vivo Drug release study:
The results of in vivo release study of the ocusert L9 is shown in table 9 and figure  10. The ocusert showed 98.01% of drug release after 12 hours which was comparable to in vitro drug release (Table  9). Thus there was good in vitro -in vivo correlation for the ocusert L9 ( Figure 10) indicating the effectiveness of the formulation to be used in vivo.

Ocular irritation studies
The ocular irritation studies were performed on rabbit eye. Five male rabbits were selected for the observation of any redness or inflammation after instillation of formulation. The formulation L9 were instilled on the left eye of the rabbit and the right eye was kept as control as described in the methodology. There was no redness or inflammation observed in the left eye for a duration of 12 hrs. The observation was done periodically after every 30 min. The control eye also showed no redness and inflammation for 12 hrs.

stability studies
Stability studies as per ICH guidelines performed showed that the optimized formulation was stable.