INTRODUCTION:

Eye is one of the challenging organ for drug delivery because of its unique anatomy restricts drug absorption into deeper tissues. Poor bioavailability of drugs from conventional ocular dosage forms is mainly due to tear production, non-productive absorption, transient residence time, impermeability of corneal epithelium, binding by the lachrymal proteins,drainage of the instilled solution, tear turnoverand limited corneal area.¹

Several Novel drug delivery systems have been developed for ophthalmic use, not only to prolong the contact time of the vehicle on the ocular surface but also to slow down drug elimination. Successful results have been obtained with inserts and collagen shields.

However, these preparations have some disadvantages such as poor compliance, especially by elderly people and many patients sometimes lose the device without noticing it.

Nowadays, a major progress in development of ophthalmic formulations has been performed by the ophthalmic gel technology i.e in the development of “in situ gel” which consists of certain polymers undergoing sol–gel phase transition in response to environmental conditions such as pH, specific ions and temperature.²

In particular, a thermoresponsive in situ gel, an ophthalmic product vehicle responding to a shift in temperature, possesses liquid characteristic at room temperature and becomes gel when comes in contact with body temperature. One of well-known polymer types possessing thermoresponsive behavior is Pluronics, so called Poloxamers. They are a triblock copolymer poly(ethylene oxide)-b-poly(propylene oxide)- b-poly(ethylene oxide) (PEO–PPO–PEO) showing amphiphilic behavior due to hydrophilic ethylene oxide domains and hydrophobic propylene oxide domains. The gelation mechanism of Pluronics could be explained by the changes in micellar structure as a function of concentration and temperature.^3-5 However, a major disadvantage of Pluronics is their low mucoadhesive activity, therefore, some Pluronic-based ophthalmic formulations have been improved by adding polymers providing mucoadhesive property such as cellulose derivatives.⁴Levofloxacin hemihydrates is a fluoroquinolone derivative used to treat external infections of eye such as acute and subacute bacterial conjunctivitis, keratitis, keratoconjuctivitis and corneal ulcers.

The objective of present study was to develop and evaluate a temperature triggered in-situ ophthalmic gel system for levofloxacin hemihydrate. Poloxamer 407 was used as temperature activated gelling agent in combination with Hpmc k4m for formulation of levofloxacin eye drops (0.5% w/v), which undergo gelation in tear fluid and provide sustained release of drug. A 3² full factorial design has been applied with conc. of Poloxamer 407 and HPMC K4M as independent variables studied at three levels to study their influence on drug release and gelling capacity as dependent variables. The formulations were evaluated for clarity, pH measurement, gelling capacity, drug content estimation, rheological study, in vitro drug release and in-vivo studies in rabbit eye.

MATERIALS AND METHODS:

Levofloxacin hemihydrate was kindly supplied as a gift sample from H iran Argochem, Ankleshwar, Gujarat. Poloxamer 407 was obtained as a gift sample from signet chemicals Ltd. Hydroxyl propyl methyl cellulose (HPMC K4M) was gift sample from Colorcon Asia Pvt. Ltd. All chemicals were used of analytical grade.

Selection of vehicle

Solubility and stability of drug was tested in various buffers. Based on the observations citrophosphate buffer pH6.0 was selected as vehicle⁶.

Selection of excipients

Poloxamer 407 was selected as thermoreversible in-situ gelling agent in combination with HPMC K4M as viscofying agent. Sodium chloride and Benzalkonium chloride were selected as tonicity agent and antimicrobial preservative respectively.

Preparation of the formulations

The formulations of Levofloxacin hemihydrate in situ gel were prepared by the cold method⁷. Poloxamer 407 was completely dissolved in cold citrophosphate buffer pH 6.0. Then, levofloxacin hemihydrate, benzalkonium chlorideand sodium chloride were respectively added into the solutions of gelling agents and stirred continuously until homogeneous solutions were obtained. The batches for thermo-reversible ocular gels are shown in Table I.

Experimental design and Statistical analysis

A3² full factorial design Swas used in the study in which two factors were evaluated and experimental trials were performed with all 9 possible combinations. Concentration of Poloxomer 407 as (X1) and HPMC K4M as (X2) were selected as independent variables. % drug release (Y1) and gelling capacity (Y2) were selected as dependent variables. The experimental design is outlined in Table I. The fitted equations (full models) relating the responses i.e. Y1, Y2 to the transformed factor were shown Table II and III respectively. The polynomial equation can be used to draw conclusions after considering the magnitude of coefficient and the mathematical sign it carries, i.e. positive or negative⁸.

Validation of optimized model

Optimum formulations were selected to validate the chosen experimental design and polynomial equations⁹. The criterion for selection of optimum was primarily based on the highest possible values of % drug release and gelling capacity for in-situ gel formulations. The resultant experimental data of response properties were subsequently compared with predicted values as shown in Table IV.

Table I : Effect of Independent variable on dependent variable by 3² full factorial design

Formulation	Independent variable		Dependant variable
	X₁	X₂	Y₁	Y₂
F1	18	0.5	88.38	+++
F2	17	0.3	62.4	+
F3	18	0.4	82.1	+++
F4	17	0.5	71.9	++
F5	19	0.4	78.45	S
F6	19	0.3	76.67	++
F7	17	0.4	64.21	++
F8	19	0.5	68.79	S
F9	18	0.3	74.8	++

X1 – conc.of poloxamer 407(gm) , X2 - conc of hpmc k4m (gm)

Y1 = Rel_8hrs (%), Y2 = gelling capacity

Characterization of Thermo reversible ocular gels

Test for appearance/ clarity

All formulations were checked for general appearance i.e. color, odour, any suspended particulate matter etc. The clarity was checked using wooden board against black and white background. The vials were held horizontally and gently rotated immediately under the lamp and then inverted once or twice to detect foreign particles.

Determination of pH

The pH of each formulation was recorded using a calibrated digital pH meter immediately after preparation as well as after 24 hours of storage at room temperature.

Gelation Studies

The gelling capacity was determined by placing 2 drops of prepared system in a vial containing 2 ml of artificial tear fluid freshly prepared and equilibrated at 37^◦C. The gel formation was visually evaluated; time for gelation and the time taken for the gel to dissolve were noted. The lowest scores (+) were assigned to those products in which the phase transition occurred only after 60-90 sec. and the formed gels collapsed within 1-2 hrs. The highest scores (+++) were assigned to those products for which the phase transition commenced within 60-90 sec. And gels so formed were stable for about 7-8 hrs. The moderate scores (++) were assigned to the products, which could form the gel in 60-90 sec. but failed to maintain gel structure for more than 3hrs.¹⁰

Evaluation of ophthalmic in-situ gel was done by means of following tests

Drug content

The drug content was determined by using phosphate buffer pH 7.4. as medium. Levofloxacin concentration was determined at 288 nm by using UV-Visible spectrophotometer.

Rheological studies

The prepared solutions were allowed to gel in the simulated tear fluid and then the viscosity determination was carried out by using Brookefield viscometer with angular velocity run from 10 to 100 rpm.

In-vitro release study:

The in vitro release of levofloxacin from the formulations was studied through cellophane membrane using a modified franz diffusion cell. The dissolution medium used was artificial tear fluid freshly prepared (pH 7.4). Cellophane membrane, previously soaked overnight in the dissolution medium, was tied to one end of a specifically designed glass cylinder (open at both ends and of 5 cm diameter). A 1-ml volume of the formulation was accurately pipetted into this assembly. The cylinder was attached to the metallic driveshaft and suspended in 50 ml of dissolution medium maintained at 37⁰C so that the membrane just touches the receptor medium surface. The shaft was rotated at 100 rpm. Aliquots, each of 1-ml volume, were withdrawn at hourly intervals and replaced by an equal volume of the receptor medium. The aliquots were diluted with the receptor medium and analyzed by UV spectrophotometry at 288 nm.¹¹

In vivo drug release studies

In vivo release studies of levofloxacin hemihydrate from the prepared in situ gelling formulations were carried out using six male New Zealand albino rabbits each weighing 2.5–3.0 kg with no signs of ocular inflammation or gross abnormalities. All animals were maintained according to the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals.

Method:

50-μl of Levofloxacin hemihydrates optimized in situ gelling formulation (0.5%W/V) and marketed eye drop (0.5% w/v) were instilled in the lower cul-de-sac of each eye, and the upper and lower eyelids were gently held closed for 2 min to maximize drug-cornea contact. At 0.5, 1, 2, 4, 6, and 8 h of postdosing the eyes were anesthetized using 4% topical xylocaine solution and the aqueous humor was sampled from 6 eyes for each formulation using a 28-gauge needle. Aqueous humor samples (100 μl) were mixed with acetonitrile. The mixture was then centrifugeds at 3000 r/min for 15 min and 20 μl of the supernatant obtained was used to determine the Levofloxacin hemihydrate content by HPLC.

HPLC analysis:

Quantitative estimation of Levofloxacin in tear fluid was done by HPLC method. The Stationary phase used was HiQSil C18 column. A filtered and degassed mixture of phosphate buffer (0.02 M) pH 3.0 and acetonitrile (80:20) was used as the mobile phase. The mobile phase was delivered at a flow rate of 1.0 ml/min, the injection volume was 20 μl and detected at wavelength of 235 nm using gatifloxacin as internal standard.

Antimicrobial efficacy testing

Antimicrobial efficiency studies were carried out to ascertain the biological activity of sol-to-gel systems against microorganisms. This was determined in the agar diffusion medium employing “cup plate technique”. Marketed Levofloxacin hemihydrate eye drops were used as a standard. The standard solution and the developed formulations (test solution) were taken into separate cups bored into sterile Muller Hinton Agar (MHA) previously seeded with organisms Staphylococcus aureus (NCIM-2024) and Pseudomonas aeruginosa (NCIM-5029). After allowing diffusion of solutions for two hours, the plates were incubated for 24 h at 37⁰C. The zone of inhibition (ZOI) was compared with that of the standard.

Sterility testing:

Serility testing was done as per IP 1996 by employing membrane filtration technique. It was carried out by incubating formulations for not less than 14 days at 30 to 35⁰ C in the fluid thioglycolate medium to find the growth of anaerobic bacteria and at 20 to 25⁰ C in the soyabean-casein digest medium to find the growth of aerobic bacteria and fungi in the formulation.

RESULT AND DISCUSSION:

Selection of vehicle

Buffers as vehicles play a major role in formulating ophthalmic formulation. They contribute significantly to chemical stability and clinical response and also influence the comfort and safety of the product, hence it is important to select a suitable buffer which ensures product stability and desired drug solubility. The studies in various buffer solutions indicated that the drug was soluble in acetate buffer IP of pH 4.6, 4.8 and 5.0 and in citrophosphate buffer of pH 6.0 at the dosage level of 0.5%, w/v. The solutions were stable to elevated temperatures and autoclaving. However, their instability to light as evidenced by discoloration of the exposed solutions necessitated their packing in amber vials. It has been reported that the ocular penetration of Levofloxacin, is maximum at pH of about 6.5. Citrophosphate buffer, pH 6.0, was therefore selected as a vehicle for the formulated in-situ gel preparation.

Selection of excipients:

Poloxamer 407 were selected as thermoreversible gelling agent because of its property to transform into gel at temperature of the eye. Poloxamer 407 alone could not be used for ocular therapeutics due to its low mucoadhesive property and also to cause irritation to the eye if used in high concentration required to have effective gelling hence HPMC K4M was used along with Poloxamer 407 to reduce concentration of poloxamer in the final formulation and to impart mucoadhesive properties. Benzalkonium chloride is mostly used in ophthalmics as preservative and also it is stable over wide temperature and pH conditions. Sodium chloride is used as tonicity agent to maintain preparation isotonic.

Preliminary studies for optimum amount of Poloxamer 407 and HPMC K4M for insitu gellation

Preliminary studies were conducted using different concentrations of Poloxamer 407 and cellulose derivatives to decide their level in experimental design.

Experimental design and Statistical analysis

Factorial design enables all factors to be varied simultaneously, allowing quantification of the effects caused by independent variables and interactions between them. In this study, a 3² full factorial experimental design was used to optimize the formulation. The concentration of poloxamer 407 (17, 18, 19% w/v) and HPMC K4M ( 0.3, 0.4, 0.5%w/v) were selected as independent variables. Drug release and gelling capacity were selected as dependent variable.

Data Analysis

Various computations for the current optimization study using Response Surface Methodology (RSM) were carried out, employing the Design Expert Software (Version 8.0.7.1, Stat-Ease Inc., Minneapolis, MN). In this design there are 2 independent variables and 3 levels (low, medium, and high) of each variable has been studied.

The general form of the model is represented as in equation,

Y = b0 + b1X1 + b2X2 + b11X11 + b22X22 + b12X1X2

Where Y is the dependent variable, b0 is the mean response of the 9 runs, and b1 is the estimated coefficient for factor X1. The main effects (X1 and X2) represent the average result of changing a factor at a time from its low to high value. The interaction term (X12) shows how the response changes when the factors are simultaneously changed. Polynomial terms (X11 and X22) are included to investigate nonlinearity. A, B are the coded levels of the independent variables and they represent result of changing one factor at a time from its low to high value. AB is interaction term that shows how response changes when factors are simultaneously changed. The coefficients of the polynomial equations were generated using multiple linear regression analysis (MLRA) for % drug release and gelling capacity. All the data of Summary output of regression analysis for effect of A & B on Y1 and Y2 respectively are enlisted in Table 3,4 respectively. 3-D plots are generated to show graphically, effect of independent variable on dependent variable.

Concerning Y1, the equation of multiple linear regression analysis is as follows:

Y₁=82.49+4.28A+2.58B-4.42AB-11.39A²-1.13B²

The Y1 for all batches A1- A9 shows good correlation co-efficient of 0.8980. The equation suggests that both factor A and B has positive effect on % drug release, upto particular concentration. After particular concentration factor A and B have negative effect on % drug release as indicated by negative sign of co-efficient of A²and B². Poloxamer 407 is a non-ionic surfactant; it releases the drug slowly by forming PP0 block dehydration in combination with HPMC K4M as mucoadhesive agent.

Table II : Summary output of regression analysis for effect of X1 and X2 on Y1

Regression statistics for Y1
F value	12.33
Predicted R square	-0.0368
R square	0.8980
Adjusted R square	0.8252
Standard error	1.38
Observations	9
Coefficients
Coefficient	Coefficient value
A	4.28
B	2.58
AB	-4.42
A²	-11.39
B²	s-1.13
Equation
Y₁=82.49+4.28A+2.58B-4.42AB-11.39A²1.13B²

Table III : Summary output of regression analysis for effect of A and B on Y2

Regression statistics for Y2
F value	15.71
sPredicted R square	0.2543
R square	0.9182
Adjusted R square	0.8597
Standard error	1.42
Observations	9
Coefficients
Coefficient	Coefficient value
A	0.50
B	0.33
AB	-0.75
A²	-1.64
B²	-0.14
Equation
Y2 = 2.90+0.50A+0.33B-0.75AB-1.64A²-0.14B²

Thus % drug release increases with increase in the poloxamer concentration upto particular concentration. After that the gel becomes stiff as proportion of both Poloxamer and HPMC increases and % drug release decreases. The 3-D plots also confirm the above observation as shown in fig.I.

Concerning Y2, the equation of multiple linear regression analysis is as per below:

Y2 = 2.90+0.50A+0.33B-0.75AB-1.64A²-0.14B²

The Y2 for all batches A1- A9 shows good correlation co-efficient of 0.9182. From table 4, The equation suggests that both factor A and B has positive effect on gelling capacity. As level of A and B increase, gelling capacity also increases. Above the particular concentration of both A and B gel becomes too stiff and drug release gets affected. The 3-D plots shows that as the concentration of Poloxomer 407 increase the gelling capacity increases significantly and concentration of HPMC K4M increases the gelling capacity also increases as shown in Figure II.

Validation of optimum formulations

For all 4 checkpoint formulations, the results were found to be within limits. Table IV lists the checkpoints, the predicted and experimental values of all the response variables, and the residual error in prognosis. Thus, the low magnitudes of error as well as the significant values of R² in the current study indicate a high prognostic ability of in-situ gel formulations.

EVALUATION OF FORMULATION:

Appearance, clarity, pH and drug content:

The appearances of all formulations were light yellow in colour and were clear. Terminal sterilization by autoclaving had no effect on the formulations. The haziness observed during autoclaving due to precipitation of HPMC at elevated temperature was found to disappear and the clarity was regained after overnight standing. The pH of all the formulations was found to be within the range of 6.0 to 6.5, which is desirable for absorption of levofloxacin and patient compliance. The drug content of all formulations was within the range of 99.35% to100.13%, showed the uniform distribution of drug in the ophthalmic formulations.

Gelling capacity:

The viscosity and gelling capacity plays important role for in situ gelling system. The formulation should have an optimum viscosity for easy instillation into the eye as a liquid which undergo sol-to-gel transition. As per Table I, formulations F1 and F3 showed better gelling capacity. The other formulations were not having desirable gelling capacity.

Rheological studies:

Viscosity of the instilled formulation is an important factor in determining residence time of drug in the eye. The formulations were shear thinning and an increase in shear stress was observed with increase in angular velocity. The administration of ophthalmic preparations should influence as little as possible the pseudoplastic character of the precorneal tear film. Since the ocular shear rate is very large ranging from 0.03 s^-1 during interblinking periods to 4250–28,500 s^-1 during blinking, viscoelastic fluids with a viscosity that is high under conditions of low shear rate and low under conditions of high shear are preferred. At pH 6.0, the formulations were in a liquid state and exhibited low viscosity. An increase in pH to 7.4 (the pH of the tear fluid) caused the solutions to transform into gels with high viscosity.

In-vitro release:

Figure III shows the percentage of levofloxacin hemihydrate released as a function of time for optimization batches. The in vitro drug release conditions may be very different from those likely to be encountered in the eye. However, the results clearly showed that the gels have the ability to retain levofloxacin hemihydrate for a prolonged period of time (8 h) and premature drug release does not occur. From the results it is concluded that the high viscosity plays important role in controlling the release of drug from the formulations. When the polymer concentration increases, drug release increases upto certain point and later decreased. Comparison of in-vitro release of optimized batch with marketed eye drop is shown in Figure IV.

Figure III :In-vitro release kinetics of optimization batches

Figure IV : Comparison of in-vitro release of marketed formulation(MF) with optimized formulation(F)

In-vivo release:

The in vitro release studies were completely free from any complications due to variability in precorneal factors, such as blinking, lachrymation, tear turnover, and drug washout. The in vitro studies provided the relative permeation characteristics of levofloxacin from different formulations, but they could not simulate real in vivo conditions. It is, therefore, necessary to study the in vivo ocular absorption of the drug from the formulations. The results of the in vivo studies are shown in Figure V .The relative bioavailability of optimized temperature triggered in-situ gel was calculated by comparing with marketed eye drop. Cmax, Tmax and AUC_0-8of optimized formulation was found to be 2.42 μg/ml, 3 hr and 18.87 μg.h/ml respectively. The relative bioavailability of optimized in-situ gel formulation was found to be 1.487 fold over marketed eye drop.

Figure V : Tear fluid concentration profile of marketed eye drop & Temperature activated in-situ gel

Antimicrobial efficacy studies:

The study indicated that the levofloxacin retained its antimicrobial efficacy even after formulated as an in situ gelling system in infected condition of eye i.e at STF Ph 6.0.

Sterility study:

The optimized formulation passed the test for sterility as there was no appearance of turbidity and hence no evidence of microbial growth when incubated for not less than 14 days a 30-35°C in case of fluid thioglycolate medium and at 20-25°C in the case of soyabean casein digest medium.

CONCLUSION:

Levofloxacin hemihydrate which is a broad spectrum Anti-bacterial agent used in the treatment of various ocular infections was successfully formulated as Temperature triggered in situ gel using Poloxomer 407 as polymer. The formulated systems provided sustained release of the drug for 8 hr period. The developed formulation is a viable alternative to conventional eye drop due to its ability to enhance bioavailability through its longer precorneal residence time and ability to sustain release of the drug. Also important is its ease of administration and decreased frequency of administration resulting in better patient compliance.

ACKNOWLEDGEMENTS:

The authors wish to express their gratitude to Maxim Pharmaceuticals, Pune for providing gift sample of Levofloxacin hemihydrate. The authors are also thankful to Dr. A. R. Madgulkar, Principal, AISSMS College of Pharmacy for providing necessary facilities to carry out the research work.

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Received on 18.06.2012 Accepted on 26.06.2012

Asian J. Pharm. Res. 2(3): July-Sept. 2012; Page 100-106

Batch code	Pluronic 127	HPMC K4m	response	Predicted value	Experimental value	Residual error
O1	18.11	0.44	Drug release	83.39	84.34	0.95
O1	18.11	0.44	Gelling capacity	2.99	3.00	0.01
O2	18	0.43	Drug release	83.28	84	0.72
O2	18	0.43	Gelling capacity	3.00	3.00	0.00
O3	17.96	0.44	Drug release	83.25	83.89	0.64
O3	17.96	0.44	Gelling capacity	3.00	3.00	0.00
O4	18.12	0.43	Drug release	83.50	83.99	0.49
O4	18.12	0.43	Gelling capacity	2.99	3.00	0.01