INTRODUCTION:

The surface of the eye is rich in nutrients and consequently, supports a diverse range of microorganisms which constitutes the normal ocular flora. However acquisition of a virulent microorganism or uncontrolled growth of an existing organism due to lowered host resistance leads to infections of the external structures of the eye. Conjunctivitis and corneal ulcers are among the most common ocular infections and in more than 80% of cases, the infections are caused by Staphylococcus aureus, Streptococcus pneumoniae, or Pseudomonas aeruginosa.

Standard initial treatment consists of frequent instillation of eye drops with a broad-spectrum antibiotic. The drop application schedule requires strict discipline from the patient or care provider since a high and constant antibiotic concentration is intended at the site. However physiological constraints imposed by the protective mechanisms of the eye lead to low absorption of drugs, which results in a short duration of the therapeutic effect.

Ocular therapy in the bacterial infections would be significantly improved if the precorneal residence time of drugs could be increased.^1,2

The aim of the present work was to design polymeric ocular drug delivery system of ofloxacin 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. Flouroquinolones are one of the promising group of antibiotics currently being used topically to treat conjunctivitis and corneal ulcers. Ofloxacin has proved to possess superior antibacterial activity in-vivo and has better pharmacokinetic properties as compared with ciprofloxacin and norfloxacin. Ofloxacin is a broad-spectrum antibacterial agent with activities against gram-negative bacteria (E. coli, Klebsiela pneumoniae, Serratia species, Proteus species, Pseudomonas aerogenosa and H. influenzae) and gram-positive bacteria (Staphylococcus species, Streptococcus enterococci). It is used in the treatment of kerato-conjunctivitis, blepharo-conjunctivitis, corneal ulcer, preoperative prophylaxis and other ocular infections. It has a plasma half-life of 5.7±1 hours.^3,4

MATERIALS AND METHODS:

Materials:

Ofloxacin was gifted by Micro labs, Bangalore. Hydroxy propyl methyl cellulose and Ethyl cellulose were procured from Loba chemie Pvt. Ltd., Mumbai. Acrylic polymer Eudragit RS 100 was purchased from Rohm Pharma, Germany. All the other chemicals and solvents used were of either analytical or HPLC grade.

Preparation of Drug Reservoir film:

Required quantity of polymer (HPMC) was weighed and dissolved in Distilled water and stirred gently. Required amount of plasticizer (Glycerin- 30% w/w of polymer) was added followed by the drug (Previously dissolved in negligible quantity of 2% v/v glacial acetic acid) and stirring was continued to form a homogeneous clear solution. Homogeneous clear solution of 5 ml containing 72 mg of ofloxacin was casted on leveled glass moulds. After drying at room temperature for 24hr, circular films of 8mm diameter (an area of 0.5 cm²) each containing 1.5 mg drug were punched out with a sharp punch.⁵

Preparation of Rate Controlling Membrane:

The rate controlling membrane was prepared by solvent casting technique In this method polymer ethyl cellulose alone or in combination with polymer polymethacrylates (Eudragit RS 100) were dissolved in dichloromethane by continuous stirring and dibutyphthalate (30% w/w of polymer) was added as plasticizer and circular films of 10 mm diameter were punched out with a sharp punch.⁵

Preparation of Ocular Inserts:

The reservoir films containing the drug were sandwiched between the rate controlling membrane to control the release and it was fixed by applying chloroform on the edges of the controlling membrane. The ocular inserts were wrapped in a aluminium foil and stored in amber colored glass vials in a desiccator till further use.⁵

EVOLUTION OF OCULAR INSERTS

Interaction studies

The FT-IR spectrum of pure drug and Physical mixture of pure drug and polymers were analyzed to check the compatibility between the pure drug and polymers using Shimadzu Fourier Transform Spectrophotometer by KBr pellet method. The procedure consisted of dispersing a sample (drug alone or mixture of drug and polymers) in KBr and compressing into discs by applying a pressure of 5 tons for 5 min in a hydraulic press. The pellet was placed in the light path and the spectrum was obtained.

The IR absorption spectra of the pure drug and physical mixture were taken in the range of 400-4000 cm^-1.⁶

Physico-chemical evaluations:

Determination of Film Thickness :

Thickness of the film is an important factor while considering its drug release from ocular delivery systems. If thickness varies from one film to another, the drug release from the film also will vary. So it is must to keep the thickness of the film uniform to get reproducible results. In the present study, the thickness of the formulated films was determined by using a Digimatic caliper. The thickness was measured at five different places and the mean value was calculated.⁵

Fig. No.1 In vitro release of ofloxacin from formulations F1-F6

Determination of Weight Variation:

As weight variation between the formulated films can lead to difference in drug content and in vitro behaviour, a study was carried out by weighing 5 films in an electronic balance. The average weight of a film and its standard deviations were calculated.⁵

Drug Content:

Drug content was estimated by placing an ocular insert in 25 ml of phosphate buffer pH 7.4 with occasional stirring for about 24h at 37°C. 1 ml of the above solution was withdrawn and measured by UV-visible spectrophotometer at 294 nm. Averages of three films were taken and it was determined from the standard curve.⁷

Fig. No.2 In vitro release of ofloxacin from formulations F7-F12

Table No.-1: Composition of various polymers and plasticizer in different formulations of extended release ofloxacin ocular films

Formulation Code	Drug Reservoir			Rate controlling membrane
	Drug (mg)	Polymer	Plasticizer	Polymers		Plasticizer
		HPMC(%)	Glycerin (%w/w of polymer)	EC (%)	ERS (%)	DBP (%w/w of polymer)
F1	72	3.0	30	3.5	-	15
F2	72	3.5	30	3.5	-	15
F3	72	4.0	30	3.5	-	15
F4	72	3.0	30	3.0	0.5	15
F5	72	3.5	30	3.0	0.5	15
F6	72	4.0	30	3.0	0.5	15
F7	72	3.0	30	2.5	1.0	15
F8	72	3.5	30	2.5	1.0	15
F9	72	4.0	30	2.5	1.0	15
F10	72	3.0	30	2.0	1.5	15
F11	72	3.5	30	2.0	1.5	15
F12	72	4.0	30	2.0	1.5	15

Percentage Moisture Absorption:

The percentage moisture absorption test was carried out to check physical stability or integrity of the ocular inserts. Ocular inserts were weighed and placed in a desiccator containing 100 ml of saturated solution of Aluminium chloride. After three days the ocular inserts were taken out and reweighed, the percentage moisture absorption was calculated.⁵

Percentage Moisture Loss :

The percentage moisture loss was carried out to check integrity of the film at dry condition. Ocular inserts were weighed and kept in a desiccator containing anhydrous calcium chloride. After three days the ocular inserts were taken out and reweighed, the percentage moisture loss was calculated.⁵

In-vitro release study by using Commercial Semi-permeable Membrane :

The in vitro release studies were carried out using bichambered donar-receptor compartment model using commercial semipermeable membrane. Membrane was tied at one end of the open - end cylinder which is acted as the donar compartment containing 0.7ml of pH 7.4 isotonic phosphate buffer. This membrane was used to simulate ocular in vivo conditions like corneal epithelial barrier. The surface of the membrane is in contact with receptor compartment, which contain 25ml of pH 7.4 isotonic phosphate buffer and stirred continuously using a magnetic stirrer. Samples were withdrawn from the receptor compartment at periodic intervals and replaced with equal volume of pH 7.4 isotonic phosphate buffer. The drug content was analysed in UV Spectrophotometer at 294 nm against reference standard using phosphate buffer pH 7.4 as blank.^5,8

Transcorneal permeation studiesby using excised goat cornea:

The in vitro transcorneal permeation study of the best formulation was carried out using bichambered donar-receptor compartment model using excised goat cornea. Whole eye ball of goat was transported from the local butcher shop to the laboratory in cold (4⁰C) normal saline within 1 hour of slaughtering of the animal. The cornea was carefully excised along with 2-4 mm of surrounding scleral tissue and was washed with cold normal saline till the washing was free from proteins. Isolated cornea was mounted by sandwiching surrounding scleral tissue between clamped donor and receptor compartments in such a way that its epithelial surface faced the donor compartment. The donor compartment was filled with 0.7ml of pH 7.4 isotonic phosphate buffer. The test formulation was placed on the cornea in the donor compartment and opening of the donor compartment was sealed with a glass cover slip. The surface of the cornea is in contact with receptor compartment, which contain 25ml of pH 7.4 isotonic phosphate buffer and stirred continuously using a magnetic stirrer. Samples were withdrawn from the receptor compartment at periodic intervals and replaced with equal volume of pH 7.4 isotonic phosphate buffer. The drug content was analysed in UV Spectrophotometer at 294 nm against reference standard using phosphate buffer pH 7.4 as blank.^9,10

Fig No.3 In vitro correlation between commercial semipermeable membrane and excised goat cornea for the release of ofloxacin from formulation F10

Fig No.4 In vitro-in vivo correlation for the release of ofloxacin from formulation F10

Ocular Irritation Test :

The potential ocular irritation and/ or damaging effects of the ocular inserts under test were evaluated by observing them for any redness, inflammation (or) increased tear production. Formulation was tested on six rabbits by placing the inserts in the cul-de-sac of the left eye. Both eyes of the rabbits under test were examined for any signs of irritation before treatment and observed upto 24 hours.¹¹

Estimation of minimum inhibitory concentration (MIC) of ofloxacin:

MIC is the lowest concentration of drug that prevents growth of a particular pathogen. Determination of antimicrobial effectiveness against specific pathogen is essential to proper therapy. Test can show which agents are most effective against a pathogen and give an estimate of the proper therapeutic dose. The antibiotic ofloxacin in various concentration range 1-10 µg/ml was added in a series of nutrient broth tubes inoculated with standard test organism (Staphylococcus aureus- Gram positive bacteria). The lowest concentration of the antibiotic resulting in no growth (indicated by no turbidity) after incubation for 24 hr is called as the MIC of ofloxacin for Staphylococcus aureus.

In Vivo Drug Release Study:

Selection of animals:

Male rabbits (Orytolagus cuniculus), 10-12 weeks old, weighing 2.5-3 kg were used in the present study. They were housed individually with husk bedding and fed with standard laboratory diet and water as much as required. Light and dark cycle of 12 hr was maintained throughout the study. The temperature was maintained at 28±2°C.

The study protocol was approved by Institutional Animal Ethical Committee for the use of animals in the research.

Estimation of drug release in the rabbit’s eye from the ocular insert:

A group containing six healthy rabbits was treated as control. Similarly another set containing same number of rabbits was used as study group. All of them were kept in hygienic conditions to avoid vulnerability to any disease including ophthalmic type. The sterilized ocular inserts of formulation F₁₀ (Drug reservoir with 3% HPMC and 2% EC + 1.5% ERS 100 as rate controlling membrane) were placed in the lower eyelid of rabbits of the test group.

At specific time intervals, the films were removed carefully and analyzed for the remaining drug content by UV spectrophotometrically. Drug free films were placed in the lower eyelid of rabbits of the control group considered as blank.

The drug content obtained was subtracted from the initial drug content in the ocusert. The value obtained denotes the amount of drug released in the rabbit’s eye.^5,7

Estimation of ofloxacin concentration in the tear fluid at 12^th and 24^th hours:

Two healthy rabbits were used for the study. For the measurement of ofloxacin in the tear fluid, the extended release ofloxacin insert was applied in the lower cul-de-sac of one eye of each rabbit. The upper eyelids were gently held closed for 10 seconds to maximize the corneal contact. At the 12^thand 24^thhr, tear samples of 2.0 µl were collected by using glass microcapillaries avoiding contact with the cornea and then gently blown into 98 µl of acetonitrile and centrifuged at 13000 rpm for 15 minutes and 20 µl of the supernatant was analysed for the presence of ofloxacin by HPLC- U.V detector, by comparing with the HPLC peak of standard solution of ofloxacin (100 µg/ml). There exists the possibility of drug binding with the tear proteins resulting in a reduction in the free drug concentration available for estimation. Therefore the tear Samples underwent treatment with Acetonitrile prior to injection to precipitate/eliminate tear proteins.^12-15

Table-2 Comparison of pharmacokinetic factors between rabbit and human

0PHARMACOKINETIC FACTORS	RABBIT	HUMAN
Tear volume (µl)	5-10	7-30
Tear turn over rate (µl/min)	0.5-0.8	0.5-2.2
Aqueous humor volume(µl)	250-300	100-250
Aqueous humor turnover rate (µl/min)	3-4.7	2-3

Estimation of ofloxacin concentration in the aqueous humor at 12^th and 24^th hours:

Two healthy rabbits were used for the study. For the measurement of ofloxacin transcorneal penetration into the aqueous humor, the extended release ofloxacin insert was applied in the lower cul-de-sac of one eye of each rabbit.. The upper eyelids were gently held closed for 10 seconds to maximize the corneal contact. At the 12^thand 24^thhour after ocular insert application, eyes were anesthetized using 4% Xylocaine solutions topically and 50 µl aqueous humor was sampled from eyes by introducing a 26 gauge needle between the junction of sclera and cornea. After extraction the eyes were treated with ciprofloxacin eye drops for the prevention of infection. The aqueous humor samples were immediately frozen and stored at -18 °C. For analysis, each sample was mixed with an equal volume of acetonitrile, then it was centrifuged at 13000 rpm for 15 minutes and 20 µl of the supernatant was analyzed for the presence of ofloxacin by HPLC- U.V detector, by comparing with the HPLC peak of standard solution of ofloxacin (100 µg/ml). There exists the possibility of drug binding with the aqueous humor proteins resulting in a reduction in the free drug concentration available for estimation. Therefore the aqueous humor Samples underwent treatment with Acetonitrile prior to injection to precipitate/eliminate tear proteins.^16,17

Chromatographic Details

Mobile Phase : methanol: acetonitrile:: citric acid 0.4 M (3:1:10)

Flow Rate : 1.0 ml/min

Column : C₁₈ universal column

Detector : UV-detector at 294 nm

Retention time : 6.8

Preparation of standard solution of ofloxacin for HPLC study:

100 µg/ml solution of ofloxacin was prepared in mobile phase. Inject 20 µl of the above solution for analysis and sample concentration determination by external standard method.

20 µl of the solution has 2 µg of ofloxacin.

Fig No. 5 The effective concentration of ofloxacin above

MIC showing no growth (no turbidity)

Sample Concentration¹⁸:

Peak area in sample × Concentration of standard

Peak area in standard

Kinetic modeling:

Several kinetic models have been proposed to describe the release characteristics of a drug from polymer matrix. The following 3 equations are commonly used, because of their simplicity and applicability. Equation 1, the zero-order model equation (Plotted as cumulative percentage of drug released vs time); Equation 2, Higuchi’s square-root equation (Plotted as cumulative percentage of drug released vs square root of time); and Equation 3, the Korsmeyer-Peppa’s power law equation (Plotted as Log cumulative percentage of drug released vs Log time).

M_t/M_∞ = K₀t (1)

M_t/M_∞ = K_Ht^1/2 (2)

M_t/M_∞ = Ktⁿ (3)

Where, M_t/M_∞ is the fraction of drug released at any time t; and K_o, K_H, and K are release rate constants for Equations 1, 2, and 3, respectively. In Equation 3, n is the diffusional exponent indicative of mechanism of drug release.

Equations 1 and 2 fail to explain the drug release mechanism from polymeric matrices that undergo swelling and/or erosion during dissolution. In such cases, based on the value of n obtained by fitting the data into Equation 3, we can describe the mechanism of drug release from the formulation

Fig No.6 The concentration of ofloxacin below MIC

showing growth indicated by turbidity

When a graph of the cumulative percentage of the drug released from the matrix against time is plotted, zero order release is linear in such a plot, indicating that the release rate is independent of concentration.

Peppas equation could adequately describe the release of solutes from slabs, spheres, cylinders and discs, regardless of the release mechanism. The value of ‘n’ gives an indication of the release mechanism. When n = 1, the release rate is independent of time (typical zero order release / case II transport); n = 0.5 for Fickian release (diffusion/ case I transport); and when 0.5 < n < 1, anamalous (non-Fickian or coupled diffusion/ relaxation) are implicated. Lastly, when n > 1.0 super case II transport is apparent. ‘n’ is the slope value of log M_t/M_∞ versus log time curve.

In the case of the Fickian release mechanism, the rate of drug release is much less than that of polymer relaxation (erosion). So the drug release is chiefly dependent on the diffusion through the matrix. In the non-Fickian (anomalous) case, the rate of drug release is due to the combined effect of drug diffusion and polymer relaxation. Case II release generally refers to the polymer relaxation. Nature of release of the drug from the designed ocular inserts was inferred based on the correlation coefficients obtained from the plots of the 3 kinetic models.^19-21

RESULT AND DISCUSSION:

Interaction studies:

The FT-IR spectra of the physical mixture exhibited absorption peaks similar to those of the pure drug sample. The results of FT-IR analysis indicated that there was no chemical interaction between the drug and the excipients in the ocular insert.

Fig No.7 Comparison between the above samples (Fig No.5 and 6)

Physico-chemical evaluations:

Thickness :

The extended release ofloxacin ocular inserts were flexible and elastic. As the delivery system was designed to release the drug in controlled manner and minimize the irritation, the batches were formulated to have minimum thickness and the patches were found to have thickness in the range of 0.259±0.01 to 0.301±0.04 mm (Table No.3- Formulations F1-F3) and the thickness was found to be in the range of 0.236 ± 0.01 to 0.291±0.01 mm (Table No.3 - Formulations F4-F12). This indicated that as the concentration of the polymers increased, there was increase in the thickness of the ocular inserts.

Weight variation:

The weight of Extended release ofloxacin ocular inserts varies between 0.280±0.35 to 0.380±0.25 mg (Table No.3- Formulations F1-F3) and the weight was found to be in the range of 0.252±0.21 to 0.375±0.23 mm (Table No.3- Formulations F1-F3). The minimum standard deviation values revealed that the process is reproducible in its capability of giving films of uniform magnitude.

The minimum intra batch variance revealed the suitability of the process used in the study for preparing ocular controlled therapeutic systems.

Drug content :

Reliability of the process in the purview of getting uniform drug loading was confirmed by drug content analysis data and it was between 1.321±0.03 to 1.388±0.01mg (Table No.3 ). The drug content uniformity values owed the fact that the process used in the study is capable of giving films with uniform drug content, with unsubstantial differences in targeted drug loading.

Percentage moisture absorption:

In the percent moisture absorption studies the batches F1 to F3 had shown increase in percent moisture absorption with increase in concentration of HPMC, which is due to high degree of hydrophilicity of polymer HPMC where as the batches F4 to F6, F7 to F9 and F10 to F12 had shown increase in percent moisture absorption with increase in concentration of ERS and decrease in the concentration of EC (Table No.3). This is probably because the EC is hydrophobic and offers resistance in moisture absorption and ERS reduces the resistance offered by the EC film alone, and by increasing pores and/or their diameter the moisture diffuses with less resistance.

Percentage Moisture Loss:

In the percent moisture loss studies the batches F1 to F3 had shown increase in percent moisture loss with increase in concentration of HPMC, which is due to high degree of hydrophilicity of polymer HPMC whereas the batches F4 to F6, F7 to F9 and F10 to F12 had shown increase in percent moisture loss with increase in concentration of ERS and decrease in the concentration of EC (Table No.3). This is probably because the EC is hydrophobic and offers resistance in moisture loss and ERS reduces the resistance offered by the EC film alone, and by increasing pores and/or their diameter the moisture diffuses with less resistance.

In-vitro drug release study:

From the in-vitro release it was found that as the concentration of HPMC was increased and keeping the concentration of EC constant, there was a decrease in the release rate (fig no.1). The release rate of the drug from the ocular inserts decreased with an increase in polymer (HPMC) proportion because of an increase in the gel strength with a longer diffusional path. This could have caused a decrease in the effective diffusional coefficient of the drug and therefore a reduction in the drug release rate. As the polymer ERS 100 was added in the rate controlling membrane and concentration of EC was decreased keeping the total concentration of polymers in the rate controlling membrane constant, there was a increase in the release rate in comparison to formulations F1,F2,and F3 in which the concentration of EC was more. The two polymers used in the rate controlling membrane were actually compensates each other on their release rates and on property of deforming ocular inserts. This is probably because the ERS 100 reduces the resistance offered by the EC film alone, and by increasing pores and/or their diameter the drug diffuses with less resistance. The inherent problems encountered with ERS 100 are the rapid penetration of the lachrymal fluid into the device, the blurred vision caused by the solubilization of ocular insert where as Ethyl cellulose, a hydrophobic polymer, can be used to decrease the deformation of the insert and thus to prevent blurred vision.

From the release study by commercial semi permeable membrane (CSM) it was found that the formulation F10 is the best formulation because it showed a maximum

Table No.3: Physico-chemical evaluations of extended release ofloxacin ocular insert

Formulation Code	Thickness (mm)	Weight Variation (mg)	Drug content (mg)	Percentage Moisture absorption	Percentage Moisture Loss
F1	0.259±0.01	0.280±0.35	1.388±0.01	6.81±0.22	10.51±0.75
F2	0.280±0.01	0.322±0.21	1.353±0.02	7.12±0.25	11.62±0.41
F3	0.301±0.04	0.380±0.25	1.325±0.02	7.65±0.32	12.32±0.35
F4	0.251±0.02	0.270±0.41	1.341±0.03	6.88±0.08	10.55±0.51
F5	0.273±0.02	0.311±0.45	1.379±0.01	7.22±0.12	11.67±0.32
F6	0.291±0.01	0.375±0.23	1.364±0.01	7.71±0.13	12.37±0.32
F7	0.242±0.01	0.261±0.36	1.321±0.03	6.98±0.21	11.61±0.22
F8	0.265±0.03	0.292±0.31	1.365±0.02	7.34±0.18	12.23±0.44
F9	0.284±0.02	0.361±0.37	1.358±0.02	7.85±0.20	13.12±0.51
F10	0.236±0.01	0.252±0.21	1.332±0.01	7.11±0.25	12.11±0.35
F11	0.258±0.02	0.285±0.28	1.348±0.03	7.45±0.21	12.91±0.55
F12	0.276±0.02	0.355±0.25	1.343±0.03	7.98±0.15	13.88±0.61

*Average of 5 determinants

cumulative percentage drug release of 97.533% after 24 hours (fig no.2).

The formulation F10 is flexible and smooth and its thickness was also uniform. Hence from the above study it was found that the formulation F10 was the best due to the appropriate balance between drug release rate and uniformity of thickness as well as flexibility.

Transcorneal permeation studies by using excised goat cornea:

From the release study of the best formulation F10 by excised goat cornea it was found that Cumulative % drug release is 88.865 % after 24 hours. Probably the reason for less release rate in case of Excised Goat Cornea in comparison to Commercial Semi-permeable Membrane is that the cornea carries charged groups and therby becomes less permeable to charged species or ions. Hence there is decrease in the percentage of drug Permeation. In vitro release study of the formulation F10 through excised goat cornea has confirmed the fact that the formulation is capable of releasing the drug for extended period of 24 hours in the same concentration independent manner as for in vitro study through semi- permeable membrane. Despite the fact the cumulative percent drug release was 88.865 % at the end of 24 hours; the in vitro correlation between the two was extremely good. This was confirmed by the regression analysis of data. The correlation value was found to be 0.9978.

Therefore for in vitro and in vivo correlation study we can use the data of release study through excised goat cornea because it better relates with the actual release rate and permeation through rabbit cornea.

Sterility testing:

Ultra-Violet radiation was used to sterilize the ocular inserts and sterility testing was carried out under aseptic conditions. It was found visually that the Alternate thioglycolate, Soyabean casein digest media, Fluid thioglygolate media containing sterilized ocular inserts were free from turbidity. This confirmed the absence of aerobic organism, anaerobic organism and fungi. From this it confirms the sterility of ocular inserts; therefore, the sterilized inserts were considered suitable for in vivo studies.

Ocular irritation test:

The rabbits subjected to ocular irritation test did not show any signs of irritation, inflammation or abnormal discharge. Polymeric ocular inserts appeared to be devoid of any irritant effect on cornea, iris, and conjunctiva up to 24 h after application, which probably suggest its suitability for ophthalmic drug delivery.

However, the animal behavior was slightly agitated from the normal animals but the intake of food and water was normal. There was no drag out of ocular inserts at the time of experiment, which suggest that the particular dimension (10 mm) was suitable as ocular films.

In vitro-in vivo correlation:

The best Formulation F10 was subjected to in vivo study in the rabbit’s eye. The drug release at the end of the 24 hrs was found to be 80.666 % An attempt was made to correlate in vitro and in vivo release (fig no.4). In vivo release study of the formulation F10 has confirmed the fact that the formulation is capable of releasing the drug for extended period of 24 hours in the same concentration independent manner as for in vitro study. Despite the fact the in vivo percent drug release was only 80.666 % at the end of 24 hours; the in vivo- in vitro correlation was extremely good. This was confirmed by the regression analysis of data. The correlation value was found to be 0.999076, which affirmed the adaptability of the delivery system to the biological environment where it can release the drug in ideal zero order pattern.

Estimation of minimum inhibitory concentration (MIC) of ofloxacin ocular insert:

The minimum inhibitory concentration of ofloxacin was found to be 4.375µg/ml. This is the lowest concentration at which there was no growth of bacteria S.aureus as indicated by no turbidity. But the concentration below 4.375µg/ml there was growth of the bacteria as indicated by turbidity (Fig No. 5,6 and 7).

Estimation of ofloxacin concentration in the tear fluid after 12^th and 24^th hours

Calculations-

After 12 hours

Sample concentration = 15692×2 / 124317

= 0.25245 µg/ 20µl

20 µl of the solution contains = 0.25245 µg of ofloxacin

7.5 µl of Tear fluid (volume of Tear fluid in the eye of rabbit) contains = 0.25245 × 7.5/20

= 0.09466 µg

The concentration of ofloxacin present after 12 hours

= 12.622 µg/ml (greater

than MIC of ofloxacin i.e.4.375 µg/ml as calculated before.)

After 24 hours

Sample concentration = 13582×2 / 124317

= 0.2185 µg/ 20µl

20 µl of the solution contains = 0.2185 µg of ofloxacin

7.5 µl of Tear fluid (volume of Tear fluid in the eye of rabbit) contains = 0.2185×7.5/20

= 0.08193 µg

The concentration of ofloxacin present after 24 hours

= 10.9252 µg/ml (greater than MIC of ofloxacin i.e. 4.375 µg/ml as calculated before.)

Estimation of ofloxacin concentration in the aqueous humour after 12^th and 24^th hours

Calculations-

After 12 hours

Sample concentration = 214601 ×2/124317

= 3.4524 µg / 20 µl

20 µl of the solution contains = 3.4524 µg of ofloxacin

275 µl of aqueous humor (volume of aqueous humor in the anterior chamber) contains = 3.4524 ×275/20

= 47.4716 µg

After 24 hours

Sample concentration = 467541 ×2/124317

= 7.52175 µg / 20 µl

20 µl of the solution contains = 7.52175 µg of ofloxacin

275µl of aqueous humor (volume of aqueous humor in the anterior chamber) contains = 7.52175 ×275/20

= 103.4239 µg

Kinetic modeling:

The different models, viz.-zero-order, Higuchi’s equation and Korsmeyer-Peppas equation were used to study the in vitro release of the ocular insert. The zero order plots of formulations were found to be fairly linear as indicated by their high regression values (Table No.4). Therefore, it was ascertained that the drug release from ocular inserts followed either near zero or zero order kinetics. The zero order curves alone are not sufficient to predict zero order since each curve, albeit straight, has a different slope. Hence to confirm the exact mechanism of drug release from the films, the datas were computed and graphed according to Higuchi’s equation and Korsemeyer’s Peppa’s equation. Regression values of Higuchi’s plot revealed that the mechanism of drug release was diffusion (Table No.4). The in-vitro kinetic data is subjected to log time-log cumulative % drug release transformation plot (Korsmeyer-Peppas plot),all the slope values ranges from 1.16893 to 1.396967 (n>1, Table No. 4) revealed the fact that the drug release follows super case II transport diffusion , possibly owing to chain distanglement and swelling of hydrophilic polymer.

SUMMARY AND CONCLUSION:

In the Present study, efforts have been made to prepare extended release ocular inserts of ofloxacin for “Once A Day Therapy” using different polymers such as HPMC, EC and ERS100. The drug delivery system was designed as a reservoir and the release was controlled by using polymeric rate controlling membrane.

When Commercial Semi-permeable Membrane was used, the formulation F10 containing 3.0 % HPMC as monolithic drug reservoir of ofloxacin and sandwiched with rate-controlling membranes of 2.0 % Ethyl cellulose and 1.5 % Polymethacrylate (ERS 100) has shown best release with 97.144 % at the end of 24 hours in concentration independent manner and when the same formulation was used to study release rate through excised goat cornea has shown release of 88.511 % at the end of 24 hours in concentration independent manner. Excellent correlation was observed between the two (0.997877)

In vitro release studies revealed that the ocular inserts followed zero-order release kinetics. Higuchi’s plot and Peppa’s plot revealed that the mechanism of drug release involved in all the formulations was super case II transport diffusion, possibly owing to chain distanglement and swelling of hydrophilic polymer.

On administration of formulation F10, The concentration of ofloxacin present in the tear fluid after 12^th and 24^th hours was 12.622µg/ml and 10.9252 µg/ml (greater than MIC of ofloxacin i.e. 4.375 µg/ml) It shows that the formulation would be able to inhibit the bacteria S.aureus effectively during the period of application of ocular insert.

The amount of ofloxacin present in the aqueous humor in the anterior chamber of the rabbit eye after 12 and 24 hours was found to be 47.4716 µg and 103.4239 µg respectively. Acoording to literatures, in case of eye drops and ophthalmic ointments the drug content in the aqueous humor was not detected after 4^th hours. Hence, extended release ofloxacin ocular insert was found to overcome the disadvantage of the see-saw pattern of conventional formulations by maintaining almost constant amount of drug for longer period of time.

There was a good correlation between in vitro and in vivo release data indicated correctness of the in-vitro method followed and adoptability of the delivery system to the biological system, where it released the drug in concentration independent manner.

The in-vitro and in-vivo studies revealed that the formulation was capable of releasing the drug in concentration independent manner for the extended period of 24 hours. As it fulfilled many perquisites of Novel “Once A Day” delivery system and it may improve patient compliance so it may be considered as formulation of choice for further studies to establish the therapeutic utility of these systems by Pharmacokinetic and Pharmacodynamic studies in human beings.

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Received on 01.09.2008 Modified on 12.09.2008

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008; Page 460-468

¹Nandha College of Pharmacy, Erode-638052, Tamil Nadu, India.

²Department of Pharmacy, Faculty of Science and Technology, Annamalai University, Chitambaram, India

*Corresponding Author E-mail:karthii69@yahoo.co.in

Preparation of Ocular Inserts:

Physico-chemical evaluations:

In-vitro release study by using Commercial Semi-permeable Membrane :

Transcorneal permeation studies by using excised goat cornea:

After 12 hours

1Nandha College of Pharmacy, Erode-638052, Tamil Nadu, India.

2Department of Pharmacy, Faculty of Science and Technology, Annamalai University, Chitambaram, India

*Corresponding Author E-mail: karthii69@yahoo.co.in

Preparation of Ocular Inserts:

Physico-chemical evaluations:

In-vitro release study by using Commercial Semi-permeable Membrane :

Transcorneal permeation studies by using excised goat cornea:

After 12 hours

¹Nandha College of Pharmacy, Erode-638052, Tamil Nadu, India.

²Department of Pharmacy, Faculty of Science and Technology, Annamalai University, Chitambaram, India

*Corresponding Author E-mail:karthii69@yahoo.co.in