Formulation Development and Characterisation of Gemi loxacin Mesylate and Loteprednol Etabonate Ophthalmic Ocuserts

Gemi loxacinMesylate is a luoroquinolone antibacterial drug preferably used in the treatment of bacterial conjunctivitis. The addition of Loteprednol Etabonate enhances the anti-in lammatory activity of the developed formulation. The objective of the present work was to develop ocular inserts of Gemi loxacin Mesylate with Loteprednol Etabonate and thereby evaluate its potential as a sustained ocular delivery system. Poor bioavailability and poor therapeutic responses are associated with conventional ophthalmic solutions due tomany pre-corneal constraints. These constrain trigger the researcher’s mind to formulate a controlled and sustained drug delivery system. Ocular inserts based on the solvent cast technique were formulated and characterized by in vitro drug release studies using a low-through apparatus that simulated the eye conditions. Compatibility of Gemi loxacin Mesylate, Loteprednol Etabonate, polymer, and excipients was checked based on preformulation studies. Different combinations of Gemi loxacin Mesylate, Loteprednol Etabonate, Carbopol 974, 98 981, PEG 400, and glycerine were formulated by the solvent cast method and evaluated. Clarity, smoothness, surface pH, drug content, and in-vitro drug release study were the various parameters evaluated on the formulated ocusert. Formula GLE 74 ful illed the needs of all organoleptic parameters and also the in-vitro release study. Based on in vitro correlation stability studies, it was concluded that this ocular inserts formulation could be a promising controlled release formulation.


INTRODUCTION
The eye serves as a portal for ocular drug delivery generally for local therapy to avoid the risk of eye damage from high blood concentrations of the drug. Constant challenges presented to the formulators were in understanding the anatomy, physiology and biochemistry of the eye, which is impervious to foreign substances. Poor bioavailability and poor therapeutic responses are associated with eye drop due to rapid precorneal elimination of the drug, which often results in patient compliance problems Barbu et al. (2005); Sreenivas et al. (2006). Design, construction and technology of ocular insert in the ield of the controlled and sustained ocular delivery systems are gaining rapid improvement to over-come conventional ocular dosage constraints (Attia et al., 1988;Bharath and Hiremath, 1999). Thus the aim of the present work was to formulate ocusert with a de inite concentration of Gemi loxacin and Loteprednol Etabonate for the treatment of ocular conjunctivitis and compared for the sustained release of the active. The formulation was formulated with the objective of increasing the residence time of the drug, reducing the dosing frequency by combining with Carbopol 974, 980, 981, PEG 400, polyvinyl alcohol, and glycerine.

Materials
Gemi loxacin Mesylate was obtained as a gift sample from Glenmark Pharmaceuticals, Solan, Himachal Pradesh and Loteprednol Etabonate was from Ajanta Pharma, Pvt, Ltd. Carbopol 974, 980 and 981 were gifted by Lubrizol Pvt, Ltd, Mumbai. PVA, PEG 400, Beta cyclodextrins used were procured from Hi-Media. Analytical grade chemicals were used for analytical purposes.

Preformulation Studies
Preformulation studies were performed on the procured drug samples and excipients with respect to the description, melting point, solubility, IR spectra, ultraviolet (UV) spectroscopic studies, and Differential Scanning Calorimetry (DSC) (Keny and Shah, 2020;Kumar et al., 2013b).

Determination of wavelength of maximum absorption
Pure Gemi loxacin Mesylate and Loteprednol Etabonate were weighed separately and diluted in distilled water. The prepared solutions were scanned in the wavelength region of 200 -400 nm. UV-visible spectrophotometer (UV-Shimadzu make) was used for scanning purposes.

Determination of Linearity and Range
25mg of Gemi loxacin Mesylateand 25 mg Loteprednol Etabonate were weighed separately and transferred to two different 25 ml volumetric lask, dissolved and diluted up to mark with water and methanol to give a stock solution having the strength of 1mg/ml and further diluted to get 0.1mg/ml. Aliquots of 0.25ml, 0.5ml, 0.75ml, 1.0ml, 1.25ml, 1.5ml, 1.75ml, 2.0ml, 2.25ml and 2.5ml of working standard solution of individual drugs were transferred to a series of 10 ml standard volumetric lask and diluted with Phosphate buffer pH 6.8 to get 2.5µg /ml, 5µg/ml, 7.5µg/ml, 10 µg/ml, 12.5µg/ml, 15µg/ml, 17.5µg/ml, 20 µg/ml, 22.5µg/mland25µg/ml of Gemi loxacin Mesylateand Loteprednol Etabonate individually. The resulting solution was estimated in a UV-spectrophotometer at 263.8nm and 245.8nm, respectively, for Gemi loxacinm Mesylateand Loteprednol Etabonate against the blank solution prepared using methanol and phosphate buffer pH 6.8. A graph of concentration against absorbance was plotted, and Beer's Lambert law was veri ied (Keny and Shah, 2020;Azharuddin et al., 2011).

DSC
The thermal property of drug and excipients alone and in combination was studied using DSC (DSC-60 Shimadzu, TA-60 WS collection software). Endothermic and exothermic parameters of the drug and polymer were subsequently obtained.

IR
The FT-IR spectrums of the obtained sample were compared with the reference standard FT-IR spectrum of Gemi loxacin Mesylateand Loteprednol Etabonate by potassium bromide method (Kumar et al., 2013a).

Preparation of Beta cyclodextrin and Loteprednol Etabonate complex
Loteprednol Etabonate being a poorly water-soluble drug, needs to be complexed with β-cyclodextrin to enhance its solubility. Six different molar ratios were prepared and evaluated. The solubility pro ile of the drug was checked, and the ratio to be used (Drug: β cyclodextrins) was inalized based on % cumulative drug release (Fialho and da Silva-Cunha, 2004).

Preparation of Ocusert of Gemi loxacin Mesylate and Loteprednol Etabonate
Petri dish with a 9 cm diameter was chosen and the area of the same was calculated. Drug quantity was calculated depending on the area of the petri dish. The proportion of 1: 9 (carbopol: PVA) was kept for overnight soaking in 20ml of distilled water, followed by incorporation of drug and PEG 400 and glycerin with stirring on a magnetic stirrer for 6 hours as represented in Table 1. On completion of 6 hours, the preparation was poured in the mentioned Petri dishes and was dried at 50 o C in a hot air oven for 4 hours.1cm 2 x 1cm 2 areas of the prepared ilms were used for the evaluation purpose (Rajalakshmi et al., 2013).

Surface pH
The ocular insert should be non-irritating to the eye and should be compatible with lachrymal luid. The prepared ilms were allowed to swell in 0.1ml of double distilled water at room temperature for 30 minutes. The swollen ilms were placed under a digital pH meter, and the surface pH was determined (Parmar and Tank, 2013).

Drug Content
1cm x 1 cm ilm was cut and dissolved in 10 ml phosphate buffer pH 6.8. Further, 1ml is diluted to 10ml to be analyzed using a UV-visible spectrophotometer at the wavelength of 263.8 nm and 245.8 nm, respectively (Pawar et al., 2012).

In-Vitro drug release study
Franz Diffusion Cell was employed to determine the in vitro drug release. The set up was placed on the magnetic stirrer with a minimum stirring rpm closely relating to eye blinking movement and the room temperature was maintained. Semi-permeable membrane (dialysis membrane 50, HIMEDIA) was used as a drug release study. 1 ml sample receptor compartment was withdrawn at periodic intervals and subsequently replaced with 1ml Phosphate buffer. The withdrawn sample was analyzed and drug release was calculated (Chowdhury et al., 2017).

Antimicrobial activity
The cup-plate technique with an agar diffusion medium was used. The cup was bored at the center of the plate. The developed ilm and standard solution of the pure drug were taken separately into the soyabean casein digest medium earlier seeded with Staphylococcus Aureus organism. On placing the ilm and standard solution on the plate, they were incubated for a day at 37 o C. Compared with the standard, the zone of inhibition (ZOI) was calculated (Mishra and Gilhotra, 2008).

Sterility Testing
The standard procedure from Indian Pharmacopoeia 1996 was employed to perform this test. Fluid thioglycolate and soyabean casein digest media were used. The formulated ilms were cut into two equal halves under laminar air low and dropped in the two test tubes simultaneously. Both the media were checked for microbial growth by incubating at 37 o C for seven days. The results were compared with positive and negative control samples (Pharmacopoeia, 1996).

Isotonicity evaluation
Tissue damages if the tonicity of the ilm is not maintained, hence the isotonicity of the ilm is a mandatory parameter. Sodium chloride solutions of three different concentrations namely hypertonic (HT -3%w/v), hypotonic (HP -0.2%w/v) and isotonic (IS -0.9%w/v) concentrations were prepared. Four clean slides were taken and labeled as HT (hypertonic), HP (hypotonic), IS(isotonic) and Test. A drop of blood with heparin (1% w/v) was taken to prevent coagulation, further placed on all slides. An optimized ilm drop was placed on a test slide and all four sides were covered with a coverslip and checked using a 45x magni ication microscope. Morphology of RBC's was studied (Rathore, 2011).

Antibacterial Activity
The serial dilution method was employed to carry out the microbiological assay. The test organism employed was Staphylococcus aureus. Two samples for testing were coded as A ( ilm) and B (pure sample) for minimum inhibitory concentration (MIC). The concentration of pure drug taken was 5mg/ml. 51µl of this drug solution contains 256µg of the drug.
Series of 14 test tubes were taken and numbered as 1 -14. To 1 st test tube, 2000µl of broth was added. And 1000µl broth was added from test tube 2 nd -14 th . 51µl of broth from test tube 1 st was withdrawn and discarded and replaced with drug solution corresponds to 256µg of the drug. 1000µl of the content from the 1 st test tube was transferred to 2 nd and so on.
This procedure is repeated until the second last test tube contains an amount of drug corresponding to 128µg, 64µg, 32µg, 16µg, 8µg, 4µg, 2µg, 1µg, 0.5µg, and 0.25µg. The last test tube serves as a negative control. 10µl of S.aureus broth is added in each tube except negative and kept for incubation at 37 0 C for 24 hours. Further, MIC was calculated, and results were tabulated (Prakash et al., 2009).

Short term Stability Studies
Short term stability study was carried out for a period of 3 months on the formulated products at room temperature. Samples were withdrawn for each month till three months and analyzed for visual appearance, pH and drug content (Gevariya et al., 2009).

RESULTS AND DISCUSSION
Results of preformulation studies performed on drug and excipients are represented in Table 2 and IR spectra of pure drugs and excipients were plotted and compared with standard samples. Drugs and excipients were found to be compatible with each other, as represented in Table 3.

Determination of Linearity and range
The linear calibration curve was obtained for Gemiloxacin Mesylate and Loteprednol Etabonate in the concentration range of 2.5-20 µg/ml at λ max of 263.8nm and 245.8 nm, respectively.
It followed Beer's Lamberts Law with a regression coef icient (R 2 ) value of 0.999 for both Gemi loxacin Mesylate and Loteprednol Etabonate as represented in Table 4 and Figure 4 and Figure 5.

Preparation of Beta cyclodextrin and Loteprednol Etabonate complex
Loteprednol Etabonate was complexed with βcyclodextrins in six different molar ratios before incorporating in the occusert development. The solubility pro ile of the drug was checked and the ratio of 1:0.5 (Drug: β cyclodextrins) was inalized based on % cumulative drug release, as shown in Figure 6 and Table 5.

Drug Content
The Drug content was determined based on the UVsimultaneous estimation method developed for the combined dosage form. Other evaluated parameters of the prepared occuserts with respect to surface pH, tensile strength, thickness are recorded in Table 6.

In-vitro Release Study
It was performed using Franz Diffusion Cell and it was found that Formulation GLE 74 gave the best results compared to the other two formulations. The values are shown in Table 7 and graphical representation in Figure 7 and Figure 8.

Measurement of ZOI by cup plate method
The zone of inhibitions of the formulated ilms was compared with that of the pure drug against positive and negative control. Readings of this study were tabulated, and images on ZOI's were depicted in Table 8 and Figure 9.

Sterility Testing
When the formulations were incubated for prescribed time and temperature, no turbidity was observed. This indicates it passes the test for sterility, as shown in Figure 10.

Isotonicity evaluation
Isotonicity test proved that the optimized ilm produces no change in the blood cells, neither bulging nor shrinking, as shown in Figure 11. This proves the formulated ilm was isotonic in nature.

Antibacterial Activity
Below the concentration of 0.5 µg/ml for the ilm and 8µg/ml for the pure drug, the turbidity was present, which indicates the growth of the organism.The MIC concentration was found to be 0.5 µg/ml for the ilm and 8µg/ml for the pure drug, as shown in Table 9.

Short term Stability Studies:
Stability studies proved that the formulations GLE 74, GLE 80 and GLE 81 showed no signi icant variations. There was a slight decrease in drug content but were not signi icant. The variation in drug content can be attributed to moisture content. Thus from the results, we can interpret that ilms can be stored at room temperature for the short term.

CONCLUSION
The formulated ocular ilms prove to be a novel drug delivery system with a promising approach in achieving greater drug absorption in comparison to the conventional ocular drops. The results concluded that ilm GLE 74 was the best amongst the three formulations in terms of drug content, in vitro drug release and anti-microbial activity. The optimized ilm showed no interactions between drugs, excipients and also beta-cyclodextrin complex when characterized by IR and DSC studies. Hence a combination of Gemi loxacin and Loteprednol Etabonate as an ocular ilm serves as a boost for the researchers and boon to the patients in the future.