Formulation and Evaluation of a Novel In Situ Gum Based Ophthalmic Drug Delivery System of Linezolid

A major problem in ocular therapeutics is the attainment of optimal drug concentration at the site of action, which is compromised mainly due to precorneal loss resulting in only a small fraction of the drug being ocularly absorbed [1]. The effective dose administered may be altered by increasing the retention time of medication into the eye by using in situ gel forming systems. The aim of the present investigation is to prepare and evaluate novel in situ gum based ophthalmic drug delivery system of linezolid. Hydroxypropyl guar (HPG) and xanthum (XG) were used as gum with the combination of hydroxyethyl cellulose (HEC), carbopol (CP), and sodium alginate as viscosity enhancing agents. Suitable concentrations of buffering agents were used to adjust the pH to 7.4. All the formulations were sterilized in an autoclave at 121°C for 15mins. The formulations were evaluated for clarity, pH measurement, gelling capacity, drug content estimation, rheological study, in vitro diffusion study, antibacterial activity, isotonicity testing, eye irritation testing. The developed formulations exhibited sustained release of drug from formulation over a period of 6hr thus increasing residence time of the drug. The optimized formulations were tested for eye irritation on albino rabbit (male) using 516 S. S. P. Hiremath et al.: the Draize test protocol with crossover studies. The formulations were found to be non-irritating with no ocular damage or abnormal clinical signs to the cornea, iris or conjunctiva observed. Thus these in situ gelling systems containing gums may be a valuable alternative to the conventional systems.


Introduction
Ophthalmic drug delivery is one of the most interesting and challenging factors facing the pharmaceutical research scientist [1,2].The anatomy, physiology, and biochemistry of the eye render this organ exquisitely impervious to foreign substances.The challenge to the formulator is to circumvent the protective barriers of the eye without causing permanent tissue damage [3].The development of newer, more sensitive diagnostic techniques and therapeutic agents render urgency to the development of more successful ocular delivery systems.The primitive ophthalmic solution, suspension, and ointment dosage forms are clearly no longer sufficient to combat these diseases, and current research and development efforts to design better therapeutic systems are the primary focus of this research work.The aim of the present investigation is to formulate an in situ gel using novel gum system.In situ gel solution increases the residence time and also sustain the release mechanism of the drug.

Results
In situ gel formulations were prepared using various polymers such as hydroxy ethyl cellulose, carbopol, sodium alginate, and gums such as hydroxypropyl guar, xanthum gum.Linezolid was used as model drug and cyclodextrin was used as Formulation and Evaluation of a Novel In Situ Gum Based Ophthalmic … 517 solubilising agent.All the formulations were prepared in the concentration range of 0.5gm/100ml of linezolid.
The prepared in situ gel formulations were evaluated for clarity, pH measurement, gelling capacity, drug content estimation, rheological study, in vitro diffusion study.The pH of in situ gel solution was found to be 7.4 for all the formulations.The formulation should have an optimum viscosity that will allow for easy instillation into the eye as a liquid (drops), which would undergo a rapid sol-togel transition (triggered by ion exchange) as shown in table II.

%CDR … % Cumulative drug release
The optimized formulations were subjected to isotonicity, antibacterial activity, in vivo ocular testing in rabbits and accelerated stability studies.One month accelerated stability study was carried out as per ICH guidelines, it was evident from the data that there was no change in clarity, pH, gelling capacity, rheological evaluation, and drug content and in vitro diffusion of the drug from the formulation.
Isotonicity testing of L 1 and L 8 formulation exhibited no change in the shape of blood cells (bulging or shrinkage), which reveals the isotonic nature of the formulation and compared with that of standard marketed ophthalmic of ciprofloxacin.Antibacterial sensitivity test-MIC was carried out using serial dilution method.

Fig. IV. Blood cells with ciprofloxacin as standard
In L 1 formulation, pure drug sample & L 8 formulation the MIC conc.was found to be 4mcg/ml.Hence it was concordant with that of the standard linezolid and no reduction in the efficacy of the formulation was observed.Stability data recorded over a 1-month period under accelerated temperature conditions indicated the stability of the formulation.
The developed formulation is a viable alternative conventional eye drops by virtue of its ability to enhance bioavailability through its longer precorneal residence time and ability to sustain drug release.

Conclusion
The present work was carried out to develop a novel in situ gum based ophthalmic drug delivery system of linezolid.The methodology adopted for preparation of in-situ gel solution was very simple and cost effective.It is newer approach to improve easy eye instillation, residence time and bioavailability and prolong drug release.From the study conducted, the following conclusions were drawn, by varying the concentration of polymers with two different gums ratio, it is to obtain the increased residence time and sustained drug release.Among the novel gum systems used such as hydroxypropyl guar and xanthan gum.Xanthan was found to be best gum and viscosity enhancer in combination with polymers with respect to increased duration of action and drug release.The study revealed that an appropriate ratio of hydroxypropyl guar, xanthan to polymers is an important factor in achieving increased duration of action and also release from the dosage form to achieve sustained effect.The gel formed in situ afforded sustained drug release over 6 hrs periods.The formulations exhibited therapeutic efficacy.The developed formulation is a viable alternative conventional eye solution by virtue of its ability to enhance bioavailability through its longer precorneal residence time and ability to sustain drug release.

Preparation of in situ gel
The polymeric dispersion was prepared by dispersing required quantity of gums and polymers in water using a magnetic stirrer and allowing it to swell overnight.In the aqueous solution of cyclodextrin linezolid was added with agitation until it is fully dissolved [4].Buffering and osmolality adjusting agents were added.

Physical parameters
The formulated in situ gel solution was tested for clarity, pH, gelling capacity, and drug content estimation.The results are as shown in table II.

Gelling capacity
The gelling capacity of the prepared formulation was determined by placing a drop of the formulation in a vial containing 2ml of freshly prepared simulated tear fluid and visually observed.The time taken for its gelling was noted [5,6].The results are as shown in table II.

Rheological studies
The viscosity measurements were done using Brookfield viscometer DV-2 model.The in situ gel formulations were placed in the sampler tube.From the literature it was evident that, the formulation before gelling should have a viscosity

In vitro drug release studies
In vitro release study of in situ gel solution was carried out by using Franz diffusion cell.The formulation containing 5 mg/ml concentration of linezolid was placed in donor compartment and freshly prepared simulated tear fluid in receptor compartment.Between donor and receptor compartment dialysis membrane is placed (0.22µm pore size).The whole assembly was placed on the thermostatically controlled magnetic stirrer.The temperature of the medium was maintained at 37°C ± 0.5°C.1ml of sample was withdrawn at predetermined time interval of 1hr for 6 hrs and same volume of fresh medium was replaced [3][4][5].The withdrawn samples were diluted to 10ml in a volumetric flask with acetonitrile:methanol:water (4:4:2) and analyzed by UV spectrophotometer at 254nm using reagent blank.The drug content was calculated using the equation generated from standard calibration curve (y= 0.0505 x+0.0291).The % cumulative drug release (%CDR) was calculated.The data obtained was further subjected to PCP DISSO software for curve fitting for drug release data [11].The best fit model was found to be Krosmeyers peppas with the regression in the range of 0.9204-0.9959and the formulation exhibited fickinian diffusion mechanism with a value of 0.1095-0.1609.
Formulation Samples were analyzed every month for clarity, pH, gelling capacity, drug content, rheological evaluation, and in vitro dissolution.
The L 1 and L 8 formulation were subjected to accelerated studies and were analyzed for isotonicity evaluation, antibacterial activity and eye irritation testing.

Isotonicity evaluation
Isotonicity is important characteristic of the ophthalmic [13].Isotonicity has to be maintained to prevent tissue damage or irritation of eye.L 1 and L 8 were subjected to isotonicity testing, since they exhibited good release characteristics and gelling capacity and the required viscosity.Formulations were mixed with few drops of blood and observed under microscope at 45X magnification and compared with standard marketed ophthalmic formulation containing ciprofloxacin.The shape of blood cell was compared with standard marketed ophthalmic formulation containing ciprofloxacin.
with observation of the various criteria made at a designed required time interval of 1hr, 24hrs, 48 hrs, 72hrs, and 1week after administration [18,19].Three rabbits (male) weighing 1.5 to 2kg were used for the present study.The sterile formulation was instilled twice a day for a period of 7 days, and a cross-over study was carried out (a 3 day washing period with saline was carried out before the cross-over study).Rabbits were observed periodically for redness, swelling, watering of the eye.The results are as shown in table VI, VII, & VIII.

Fig. III .
Fig. III.Comparative in vitro diffusion profile of L 1 & L 8 formulations

of 5
to 1000 m Pa s.And after ion gel-activation by the eye, will have a viscosity of from about 50-50,000 m Pa s.The samples were analyzed both at room temperature at 25°C and thermostated at 37°C ± 0.5°C by a circulating bath connected to the viscometer adaptor prior to each measurement.[7-10] The angular velocity of the spindle was increased 20, 30, 50, 60, 100, 200 and the viscosity of the formulation was measured.All the formulations exhibited Newtonian and pseudoplastic flow characteristics before and after gelling in the simulated tear fluid respectively.The formulations L 1 and L 8 exhibited the required viscosity range, hence this formulation were taken for further study.Results are as shown in table III & figure I & II.
Figure IV, V & VI, depicts the isotonic nature of Standard ciprofloxacin, L 1 and L 8 respectively.
Rheological studies of formulation L 1 & L 8 +: Gels after few minutes, dissolves rapidly.++:Gelation immediate remains for few hours.+++:Gelation immediate, remains for extended period.Rheological evaluation of all the formulation exhibited Newtonian flow before gelling (as shown in figure I) and exhibited pseudoplastic flow after gelling (as shown in figure II and table III) in the eye.There was 5-fold increase in the viscosity after gelling.Additionally, the gel formed in situ should maintain its integrity without dissolving or eroding for a prolonged period.Tab.III. 1 : 0.25% HPG, 0.3% HEC, 0.5% SA, L 8 : 0.2%XG, 0.5% SA, 0.4% CP From the in vitro results it was observed that the L 8 formulation contains xanthum gum, sodium alginate and carbopol (57.08%) has shown highest % cumulative drug release (% CDR) at 6 th hrs where as the L 1 formulation contains HPG with the varying concentration of Hydroxyethyl cellulose and Sodium alginate has shown 47.20 % CDR (as shown in figure III).Hence L 1 and L 8 formulation were taken for further study.
Formulation and Evaluation of a Novel In Situ Gum Based Ophthalmic …523In vivo eye irritation testing was carried out using rabbits and as per Draize test protocol.Optimized formulations L 1 & L 8 were used for this test.The formulations were found to be non-irritating with no ocular damage or abnormal clinical signs to the cornea, iris or conjunctivae observed.Hence the formulation was suitable for the eye instillation.
DiscussionBy varying the concentration of polymers with two different gums ratio, it is to obtain the increased residence time and sustained drug release.Both the gums, HPG and xanthan are suitable candidates for ophthalmic in situ gel system.The combination of and appropriate ratio of hydroxypropyl guar, xanthan:polymers is an important factor in achieving increased duration of action and also release from the 1 N NaoH/ 0.1N HCL.The ingredients of the formulations are depicted in table I.
L 1 and L 8 exhibited required release characteristics with the regression value of 0.9855, 0.9959 respectively as show in table IV & figure III.The L 1 and L 8 formulation were further subjected isotonicity evaluation, antibacterial activity, eye irritation testing and accelerated stability studies.