Factorial Design:

A 3² factorial design was adopted to optimize the variables and nine experiments were conducted. Experimental values of all responses were compared with the predicted values and Software was validated.

Characterization of formulation:

Visual appearance, Clarity and pH:

The formulations were examined for visual appearance and clarity by observing the formulated in situ gels against a white and black background to check the presence of any particulate matter⁵. The developed formulations were evaluated for pH by using Equiptronic digital pH meter⁶.

In vitro gelling capacity:

Gelling systems of various concentrations of Poloxamer 407 and 188 were prepared and evaluated for gelling capacity to decide the suitable composition for use as an in-situ gelling system⁷.

Measurement of the Sol–Gel Transition Temperature:

The sol-to-gel phase transition temperature was measured for prepared thermos-sensitive Besifloxacin HCl In-situ gel⁸.

Rheological Studies:

Rheological properties were evaluated for both sol and gel phase of the formulation using Brookfield viscometer (RVDV+PRO model)⁹.

In vitro drug release study:

In vitro drug release study was carried out by using Franz diffusion cell in triplicate¹⁰.

Drug content:

1ml of the Besifloxacin HCl In-situ gel was diluted to 100ml with ATF (pH 7.4), of which 1mL was withdrawn and diluted to 10mL. Drug content was determined by using UV-visible spectrophotometer (UV-630, Jasco, Japan) at 296nm.

Ex-vivo drug permeation studies:

An ex vivo corneal permeation study was carried out using freshly excised goat cornea. Whole eyeballs of goat were procured from a slaughter house and transported to laboratory in normal saline (0.9% NaCl solution) maintained at 4ºC. The corneas were carefully removed using forceps and scissors along with a 5–6 mm of surrounding scleral tissue and washed with cold saline. The washed corneas were kept in freshly prepared solution of ATF of pH 7.4. Franz diffusion cell was used for the study and the tissue was carefully placed between the donor and receptor compartments with the endothelial side facing the receptor compartment. The same steps carried out as in the in vitro release study were followed in this study. The percent drug released was plotted against time to get dissolution rate curves¹¹.

Antimicrobial efficacy studies:

Antimicrobial efficacy studies were carried out using Staphylococcus aureus and Escherichia coli strains. Inhibitory effect of the Besifloxacin HCl in situ gel was evaluated by cup plate method using nutrient agar media. Microbial inoculum was spread evenly with the help of sterile spreader. Each 1 mL of prepared standard solution (0.6 % w/v Besifloxacin HCl in water) and test solution (Besifloxacin HCl in situ gel) was transferred into wells. Plates were allowed for diffusion of solution for 2 h. and incubated at 37°C for 24 h. An average of 3 readings were taken as zone of inhibition¹².

Histopathology study:

In this study goat cornea were checked for change in the structure of cornea. The excised cornea was kept in contact with formulation for the period of 12h. and immediately placed in formalin solution (8% w/w). It was dehydrated using alcohol gradient and solidified using melted paraffin. Cross sections of cornea were taken and examined microscopically, eosin and haematoxylin were used as staining agent⁹.

Results and discussion:

Evaluation of ophthalmic in situ gel:

Visual Appearance and clarity:

All batches of Besifloxacin HCl in situ gel were clear and slightly yellow in colour (Table 5). The pH of the formulations was found to be satisfactory and was in the range of 6.7-7.3 which is acceptable for ocular administration.

In vitro Gelling capacity:

The gelling capacity of prepared formulations shown in Table 2, represent that the formulations F1, F2 and F4 showed immediate gelation and rapid dissolution. Formulations F3 and F5 showed rapid gelation but was stable for few hours. On the other hand, formulations F6 – F9 showed gel transitions within few seconds and remains stable for longer period. This indicates that as the concentration of polymer increase, Sol-Gel transition time was decreased and the dissolution time of formed gel was increased.

Table 2: Evaluation of 9 batches of Besifloxacin HCl in situ gel

Batch code	Appearance	Clarity	pH	Gelling Capacity
F1	Slightly Yellow	Clear	6.8	++
F2	Slightly Yellow	Clear	7	++
F3	Slightly Yellow	Clear	7.3	+++
F4	Slightly Yellow	Clear	6.6	++
F5	Slightly Yellow	Clear	7.2	+++
F6	Slightly Yellow	Clear	6.7	++++
F7	Slightly Yellow	Clear	6.9	++++
F8	Slightly Yellow	Clear	7.1	++++
F9	Slightly Yellow	Clear	7	++++

++ (Phase transition within 60 sec and gel structure stable for 6 h.),

+++ (Phase transition within 40 sec and gel structure stable for more than 6 h.),

++++ (Phase transition within 30 sec and gel structure remain for long time).

Table 3: Optimized composition for in-situ gel

Ingredients	Quantity
Besifloxacin HCl	0.6 gm
Poloxamer 407	24.80 gm
Poloxamer 188	14.53 gm
BKC	0.02%
Distilled water	Upto 100 mL

Evaluation of optimized batch:

In Vitro Drug release:

Cumulative %drug release of Besifloxacin HCl ophthalmic in situ gel after 8 hours was found to be 96.47%.

Drug content uniformity:

Drug content uniformity was performed by randomized sampling of optimized batch. Drug content was found to be within the range of 98.83–99.05%, which indicates uniform distribution of drug in the prepared formulation (Table 4).

Table 4: Content uniformity

Sr. No.		Drug Content
	1	98.83
	2	99.16
	3	99.05

Ex-vivo drug permeation studies:

Ex vivo transcorneal permeation study was conducted on the optimized formulation through goat cornea. The formulation showed 95.19% release after 8 h. (Fig.3).

Antibacterial Activity:

The study indicated Besifloxacin HCl retained its antibacterial activity when formulated in-situ gel against selected microorganism, Staphylococcus aureus and Escherichia coli. (Table 5). The results of antibacterial activity are as shown in the Fig.4 and Fig. 5.

Table 5: ZI against Staphylococcus aureus and Escherichia coli

Species	Zone of inhibition
Species	Standard (mm)	Test (mm)
Staphylococcus aureus	25	22
Escherichia coli	34	30

Histopathology study:

Histopathological studies revealed no change in histopathology of the tissues for the optimized formulation. Normal histomorphological features of cornea with intact cellular layer of anterior epithelium (Stratified squamous epithelium) was observed. There was absence of any inflammatory changes and any ulceration or erosion of epithelial tissue layer. The nucleus of epithelial tissue was intact and normal (Fig.6).

Accelerated stability study as per ICH Guidelines: (Table 6).

Table 6: Stability study data as per ICH guidelines

Time (month)	Temperature/ %RH	Parameters
Time (month)	Temperature/ %RH	pH	Appearance	Drug Content
1	25±1°C/ 60 ± 5	6.8	+	98.89
	30±1°C/ 65 ± 5	6.9	+	99.02
	40 ± 2^oC/ 75 ± 5	6.9	+	98.95
2	25±1°C/ 60 ± 5	7.0	+	98.86
	30±1°C/ 65 ± 5	7.1	+	98.74
	40 ± 2^oC/ 75 ± 5	7.0	+	98.93
3	25±1°C/ 60 ± 5	6.9	+	99.05
	30±1°C/ 65 ± 5	6.8	+	99.14
	40 ± 2^oC/ 75 ± 5	6.7	+	98.98

+ sign indicates no change in formulation.

CONCLUSION:

Besifloxacin HCl, a broad-spectrum antibacterial agent used in the treatment of bacterial conjunctivitis, was successfully formulated as thermo-sensitive ophthalmic in-situ gel using poloxamer 407 and poloxamer 188 by cold method. Upon administration into the eye as eye drop, the formulation undergoes gelation in the cul-de-sac. The formed gel prolonged the in vitro drug release over 8h period. The optimized batch represents a promising alternative to the conventional eye drops due to its ability to prolonged drug release and longer pre-corneal residence time. In addition, ease of administration and expected reduced frequency of administration will result in better patient compliance.

Fig.1 In-vitro drug release

Fig.2- In vitro drug release of optimized batch

Fig.3 Ex-vivo drug release of optimized batch

Fig.4 Antibacterial activity against E. Coli a) Standard b)Test formulation

Fig.5 Antibacterial activity against S. Aureus a) Standard b) Test formulation

Fig.6 Histopathological sections of goat cornea (a) negative control (untreated cornea) (b) test sample (treated with Besifloxacin HCl in situ gel)

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Received on 05.07.2023 Modified on 14.12.2023

Research J. Pharm. and Tech 2024; 17(5):2035-2039.

DOI: 10.52711/0974-360X.2024.00322

Ingredients	F1	F2	F3	F4	F5	F6	F7	F8	F9
Besifloxacin HCl	0.6	0.6	0.6	0.6	0.6	0.6	0.6	0.6	0.6
P407	1.5	2.0	2.5	1.5	2.0	2.5	1.5	2.0	2.5
P188	0.5	1.0	1.5	0.5	1.0	1.5	0.5	1.0	1.5
BKC	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
Distilled water	q. s	q. s	q. s	q. s	q. s	q. s	q. s	q. s	q. s