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��ࡱ�>��	��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������[�	��,�bjbj����	4�ΐΐ.z+w�����������___����sss8���s+E�k����&1<1H1~C�C�C�C�C�C�C$�F�kIN�CA_P1�0^&1P1P1�C����1�D�>�>�>P1v�l�_�~C�>P1~C�>�>3,�>�����еj0ߔ�s�7&�>jC�D0+E�>�I�9��I�>�>n�I_�>tP1P1�>P1P1P1P1P1�C�C�>P1P1P1+EP1P1P1P1���������������������������������������������������������������������IP1P1P1P1P1P1P1P1P1�	�:	 Development of Controlled release bioerodable anti infective ophthalmic ocuserts
 R. Thakur1, Mahfoozur Rahman2 ,  Yogesh Kumar2 , K. Kanwar1, M. S. Rathore1 and A.K. Sharma1 , G. Swami1,*
1Department of pharmaceutics, CT Institute of Pharmaceutical Sciences, Jalandhar, Punjab, India
2Assistant professor and researcher at Dreamz college of Pharmacy, H.P. India 175036

*Corresponding author: 
Dr. G. Swami
Department of Pharmaceutics,
CT Institute of Pharmaceutical Sciences, Jalandhar, Punjab, India
Contact No: 91-8146046196
Email: gauravswam@gmail.com

Abstract 
 An attempt has been made to formulate the bioerodable ocusert for prolong release and better ocular availability of azithromycin in ophthalmic infections of eye. A modified solvent casting method was used for preparation of azithromycin ocuserts, in which hydroxyl propyl methyl cellulose (HPMC), Eudragit RL100 were used as a drug reservoir and rate controlling membrane. Further formulation were evaluated for uniformity of thickness, uniformity of weight, surface pH, folding endurance, Percentage moisture loss, percentage moisture absorption, drug content, in-vitro release, kinetics studies(zero order, first order, Higuchi and peppas) and stability studies.  The Formulation H8 (amongst the range of H1-H10) comprise of 1.5% HPMC, 3% Eudragit RL100, was found as optimized formulation on the basis of uniformity of thickness (0.26�0.004 mm), uniformity of weight (24.9�0.27 mg), surface pH (7.1�0.063), folding endurance (18.3�0.81), Percentage moisture loss (7.49�0.30%), percentage moisture absorption (5.7%), drug content (1.98mg ), in-vitro release (99%) and stability studies (Shelf life- 346 days). The formulation H8 showed a steady, controlled release up to 12 hours with non-Fickian diffusion release mechanism, against the normal release of drug up to 2-3 hours. These ocuserts showed a promising results which can be used to treat wide range of ocular infection with potential scope of market and socio benefit by the industries.



INTRODUCTION
Designing of stable & effective ocular drug delivery is a challenging task due to several restricted barrier associated with drug penetration via corneal or non-corneal routes, such as transportation to anterior or posterior chambers of eye. The attainment of an optimal concentration of drugs at the site of action, during the ocular infections is also a major problem in ocular therapeutics. Poor bioavailability of  drugs  from  conventional  ocular  dosage  forms  is mainly due to the pre-corneal loss factors,  which include tear dynamics, insufficient  residence  time, non-productive  absorption and impermeability of corneal epithelium. The bioavailability problem pertaining to ocular route arise due to drugs which are hydrophobic, unstable at pH i.e. comfortable to an eye, and other associated problems which cannot be solved with conventional eye formulation i.e. eye drop.
Various novel ocular drug delivery systems are proved to control the drug release & enhance the residence time i.e. attain better ocular bioavailability example: minitablets, disposable lenses, collegen shields and biodegradable polymeric systems. Ophthalmic inserts offer many advantages over conventional dosage forms, like increased ocular residence, possibility of releasing drugs at a slow and constant rate, accurate dosing, exclusion of preservatives, and increased shelf life with better patient compliance due to lower frequency of administration and lower incidence of side effects1- 3.
 Presently, Azithromycin is available in the 1.0% eye drop which needs to be administrated 1-2 drops in 6-8 hrs for the treatment of meibomian gland dysfunction and Posterior blepharitis4, 5. It inhibits the RNA dependent protein synthesis in bacteria due to bind with 50S subunit of 70S bacterial ribosome. Once at the site of infection, large concentrations of azithromycin are slowly released by exposure of the phagocyte to the cell membrane of the bacteria where ribosomal binding occurs and suppression of NF-�B decreases the levels of proinflammatory cytokine IL-6 and IL-8 which are pertinent to ophthalmology for their anti-inflammatory and immunomodulatory effects6, 7. Treatment with oral delivery of azithromycin has effective8, 9 but systemic side effects of the oral formulation may limit its�use. The high tissue concentration, long duration of action, and simple dosing regimen, makes topical azithromycin�an ideal treatment option for trachoma. Trachoma, the leading cause of infectious blindness (chronic keratoconjuntivitis) caused by Chlamydiae trachomatis.  Several studies have researched the safety and efficacy of topical azithromycin�1.5% for the treatment of trachoma. 
Various attempts have been made for ocular delivery of azithromycin using different approaches. However, many of these approaches are associated with some or other limitations. The nanoparticle consisting of PLGA can damage corneal epithelium by disrupting the cell membrane10. The crystal growth was observed in the suspension & ointment formulations which interfere with the dose uniformity, clear vision& bioavailability and clearly no longer sufficient to combat some present virulent microorganisms. Major topical formulation problem arise of the fact that the azithromycin is hydrophobic and sparingly soluble in water and at neutral pH-711. 
Keeping above in view the objective of present study, is to develop azithromycin ocusert to improve the efficiency of the therapy and increasing patient compliance. Ocular inserts are polymeric systems that contain the hydrophilic or hydrophobic drugs, which is molded as solid or semisolid sterile preparation of appropriate size and shape, designed to be inserted behind the eyelid or held on the eye and to deliver drugs for topical or systemic effect. There has been a growing interest in using bio-adhesive films, in the ocular cul-de-sac for enhanced or prolonged localized drug delivery.
In this study one hydrophilic (HPMC) and one hydrophobic polymer eudragit RL100 were used as a drug reservoir and rate controlling membrane. The given below Table No. 1 and II showing various formulation of ocular inserts.
MATERIALS AND METHODS 
Material
Azithromycin was received as a gift sample from Overseas Pharma Limited, Punjab.  Dialysis membrane was procured from Himedia.  Other chemicals and solvents used in the study were of analytical grade.
Preparation of ocular inserts
Solvent casting method used for preparation of azithromycin ocuserts15. HPMC (1%, 1.5%, 2%, 2.5% and 3%) and Eudragit RL100 (1%, 2%, 3%, 4% and 5%) were used as drug reservoir (DR) & rate controlling membrane (RCM) respectively for preparation of ocuserts. In addition, Glycerin and dibutylphthalate were added as plasticizers. For DR, azithromycin (108 mg) was dissolved in the HPMC  and Eudragit polymeric solutions containing plasticizer, these polymeric solutions were stirredat 100 rpm by using magnetic stirrer. The polymeric solutions (for DR and RCM) were spared on mercury surface in Petri dish (Area: 3849.5mm2). The cast solution was allowed to evaporate by placing it inside stability chamber maintained at 37 � 2p C, 30 � 0.5% of RH for 24 hours. After drying, DR & RCM membranes were cut in 78.5 mm2area (2 mg azithromycin in the each DR) by using sterile stainless steel borer.
Medicated reservoir disc was sandwiched between two rate controlling membranes, respectively. Chloroform was used a sealing agent and this whole unit was placed for 4-5 min in desiccator previously saturated with ethanol/acetone (60: 40) for successful sealing of the medicated reservoir �lms between two rate controlling membranes. The sealed ocuserts were stored in an airtight container under ambient conditions. 
All the above experimentation was carried out under laminar air�ow to maintain the sterility conditions of ophthalmic products. The composition of ocusert formulations (H1-H10) were represented in Table I.
Drug reservoir was selected on the basis of drug content and physiochemical parameters. Different concentration of drug reservoir polymer was used with drug and then analyzed at 243.0 nm using phosphate buffer pH 7.4 as blank on UV/Visible spectrophotometer (1800, Shimadzu Inc., Japan) (Table II.).

Physicochemical Evaluation Azithromycin ocusert
Uniformity of thickness 
Each ocusert was evaluated for the thickness (n=5) using a screw gauze (HC memorial scientific corporation, Ambala, Haryana, India). The average of five readings of randomly selected ocuserts was taken at di�erent points of �lm, and the mean thickness was calculated. The standard deviations (SD) in thickness were computed from the mean value16.
Uniformity of Weight
To check the uniformity of weight, five ocuserts from each batch was selected randomly and weighed individually using digital balance (Deluxe Sigma, India). The average weight as well as the  standard  deviations (SD)  were  calculated  from  individual  weight  of  the inserts17.



Drug content uniformity 
For drug content uniformity, the ocuserts were placed in the chloroform and agitated for 5 min in the glass shaker at 37�2�C. After that filter the resulting solution from 0.45 �m filter and amount of Azithromycin in each insert was calculated by using following equation.
				

where As is the absorbance of sample solution, Ca is the concentration of the azithromycin in the standard  solution,  and  Aa is  the  absorbance  of  standard solution of azithromycin. The same procedure adopted for all of the batches and drug content was determined18.
Surface pH
The inserts were allowed to swell in closed Petri plate at room by keeping them in contact with 5 ml of distilled water for 1 hr.The pH was noted by bringing the digital pH meter (Hanna Instruments, USA) near the surface of the formulation (ocular inserts) and allowing it to equilibrate for 1 min19.
Folding Endurance
The evaluation was important to check the ability of the sample to withstand folding which also gives an indication of brittleness. The ocusert was folded at the same place, by placing in between figure and thumb and then opened till it breaks. The number of times the ocusert could be folded at the same place without breaking gave the folding endurance20.
Percentage Moisture Absorption
This percentage moisture absorption test was performed to check physical stability or integrity of ocular �lms. The Ocuserts were weighed accurately and then placed in a desiccator containing 100 ml of saturated solution of aluminum chloride, and humidity was maintained at 79.5%. The ocular �lms were taken out after three days and then reweighed. The percentage moisture absorption was calculated using the following equation21:

.
Where, Fw= Final weight and IW=Initial weight 
Percentage Moisture Loss
The percentage moisture loss was performed to check integrity of the �lm at dry condition. Ocuserts were weighed and kept in a desiccator containing anhydrous calcium chloride. The ocuserts were taken out after three days and reweighed; the percentage moisture loss was calculated using the following equation22:
			

Where, Iw=Initial weight and Fw= Final weight 

In Vitro Drug Release Studies
The bi-chambered donor receiver compartment model, designed using commercial semi-permeable membrane of transparent and regenerated cellulose type (dialysis membrane), was used to carry out the in vitro drug release studies23. The ocusert was placed in the donor compartment, and 5�l of phosphate buffer with pH 7.4, Kinetic modeling of release data was maintained at the same level throughout the study in the donor compartment to simulate tear volume. The entire surface of the membrane was in contact with the reservoir compartment that contained 60 ml of phosphate buffer with pH 7.4, which stirred continuously using a magnetic stirrer at 20 rpm to simulate blinking action. Drug release was determined by withdrawing a de�ned quantity of sample from the sampling port at periodic intervals, which was replaced with equal volume of phosphate buffer pH 7.4. Drug content was analyzed at 243.0 nm using phosphate buffer pH 7.4 as blank on UV/Visible spectrophotometer (1800, Shimadzu Inc., Japan). The drug release kinetic was obtained by treating the drug release data as per Zero order, First order, Higuchi release kinetics and Peppas kinetics. Regression coef�cients were compared to get the release kinetic.
Kinetic Analysis 
The in vitro and in vivo data were analyzed by a zero order, first order, peppas and Higuchi kinetics equation to understand the release profile and release mechanism24 and graph of the cumulative  percentage  of  the  drug released  from  the  ocusert against  time  is  plotted and release kinetic  is calculated indicating  that  the release profile and release mechanism.  
Ocular irritation
To check the potential ocular irritation or damaging effects of the ocusert, the ocular irritation test was carry out by placing the formulation of ocuserts in the cul-de-sac of the left eye on five rabbits and then observing  them for any redness, inflammation (or) increased tear production25. Both eyes of the rabbits under test were examined for any signs of irritation during treatment for up to 12 hours.
In Vivo Drug Release Study
To carryout In-vivo drug release studies two groups containing eight and ten healthy rabbits were used to study the In-vivo drug release from formulation H8 and H9, respectively, which showed the desired In-vitro drug release26. Good hygienic conditions were provided for each rabbit in order to avoid vulnerability to any disease including ophthalmic type. Then pre-sterilized ocuserts using � -radiation were used for In-vivo drug release studies which were placed in the cul-de-sac of each rabbit while the other eye served as a control. At periodic intervals, the inserts were taken out carefully from the cul-de-sac of each rabbit and analyzed spectrophotometrically for the remaining drug content.
Stability studies
The formulation H8 was selected as the optimized formulation for accelerated stability studies. The ocuserts were packed in amber colour container with induction sealing. The processing temperature and relative humidity were selected according to ICH guidelines. At the regular time intervals, ocuserts were analysed proceeded for drug content determination and undecomposed rate constant (k) was determined. Further k value was used for estimation of shelf life at different conditions.

RESULTS 
The drug reservoir was selected on the base drug content and optimum thickness. The higher drug content (1.98+0.016 mg) and optimum thickness (0.11+0.009 mm) were found in the drug reservoir (H2) as compared to other drug reservoir (H1, H3, H4, &H5), so H2 drug reservoir was selected as the optimized drug reservoir, shown in Table II.
	The average thickness of ocular inserts (H6 to H10) was found to be in range 0.23�0.007 mm to 0.28�0.007 mm as shown in Table II. The low standard deviation of the measured thickness of all the ten formulations ensured uniformity in thickness. The weighed ocuserts (H6 to H10) was found to be in the range 22�0.57 to 28.21�0.23 mg (Table III). The Formulation H8 and H9 showed the good uniformity of weight uniformity24�0.27mg and 26.2�0.52mg respectively. The formulations (H6-H10) were showed the drug content in range of 1.96+0.019mg to 1.99+0.012mg (Table III). The formulated H8 and H9 had highest drug content (1.99+0.012 and 1.97�0.011) with little variations as compared to other formulation batches. The surface pH of prepared inserts (H6 to H10), was found be in range of 6.95�0.054 to 7.35�0.05 (Table III). The formulation H8 showed the surface pH 7.1�0.063, which is occur in the optimum range (7.0 to 7.4).
	Folding endurance indicate the breaking strength and endurance nature of film. The observed folding endurance for all batches (H6 to H10), were found between 25.2�0.75 to 15.5�1.04 (Table III). A  positive  linear  correlation  was  found  between the  moisture  absorption  capacity  and  the  concentration  of  the  hydrophilic  polymers  in  the  formulations (H6 to H10) (Table III). The formulations H6 to H10 were subjected to In-vitro drug release study. The cumulative percentage drug release for formulation (H6 toH10) was 99.9, 99.83, 99.33, 95.66 & 90.66%, respectively. The drug release kinetic studies was performed for all formulations (H6 to H10) by using four drug release kinetics models, such as zero order, first order, higuchi & Korsmeyer�s peppas. Ocular irritation test was carried out with formulation (H8) sterilized using � -radiation before undertaking the In-vivo release study. Results of the Ocular irritation test revealed that all occular inserts prepared using HPMC as drug reservoir and Eudragit RL 100 as rate controlling membrane, at different time intervals were nontoxic and non-irritating to the eye.
	The In-vivo release studies were performed using rabbits with the selected formulations H8 and H9.  Ocular inserts were removed carefully at periodic intervals and analyzed for residual drug content. The drug remaining was subtracted from the initial drug content of the insert, which gave the amount of drug released in the rabbit eye. In pharmacokinetic studies, the area under curve (AUC), tmax and Cmax is calculated. It was found that AUC for Invitro and In-vivo release is 38828.33 & 39783.33 �g.min/ml respectively which represent the drug release accumulation in the body, and is found higher than the pure drug (1190 �g.min/ml). The Cmax and tmax also observed for In-vitro and In-vivo release, 402 �g/ml, 397�g/ml and 50min, 40min, respectively. Stability studies at different climatic conditions (as per ICH guidelines) revealed no significant change in physical appearance, weight, thickness and folding endurance. It was found that at 25 � 10C, room temperature and 40 � 10C along with 75 % relative humidity, the shelf life of ocusert  is 312.3 days, 622.7 days, 370.6 days. However, storage condition like 5 � 10C temperature and 40 � 10C both temperatures reduced the stability due to excessive heat and moisture. So the prepared ocusert stored on temperature not exceeding 400C temperature and moisture proof packing material.  

DISCUSSION
The bioerodable azithromycin ocuserts prepared by solvent casting method. Among the all formulation (H1 to H10) H8 was selected as optimized formulation on the basis of different evaluation parameters. The H8 formulation comprises 1.5 % HPMC as drug reservoir membrane and 3% Eudragit RL100 as RCM. The formulation H8 was contain the 1.98�0.016 mg azithromycin which is the approximately 99% present in the drug reservoir membrane and delivery of drug through RCM is up to 12 hour. The formulation H8 and H9 had showed the good thickness along with the 98 to 99 % of drug content  and thickness of formulation were kept optimum (0.26� 0.004mm) to prevent any irritations which may cause while  placing it  inside the cul de sac.
The average uniformity of weight were observed in formulation H8 and H9 as 24�0.27 & 26.2�0.52mg respectively, which indicates that good distribution of drug, polymer and plasticizer. The surface is pH also an important parameter ,in case of ocusert and from the results, formulation H8 porses the surface pH 7.1�0.063, which indicates that the prepared inserts would not alter the pH of the Lacrimal fluid  and eye comfort. All ocusert formulation were also evaluated for their mechanical strength by calculating the folding endurance. The mean folding endurance was found 20.35�0.895, which showed the good strength of ocusert under handling shocks .The cccuserts would not break and maintain their consistency and compatibility with general skin folding, when used. Presence of moisture also affect the physical properties of ocusert, it was also observed for formulations (H6 to H10) and formulation H10 has shown the highest moisture absorption due to presence of highest concentration of HPMC & H7 showed the highest moisture loss due to presence of higher concentration of Eudragit RL100.The formulation H6, H1 & H8 showed the more then 99% drug release and r2 value of H8 & H9 showed the good linearity to control the release of drug. The In-vitro drug release data was analyzed by zero order kinetics (cumulative % drug released v/s time) to understand the release profile and release mechanism (Fig1). The formulation H6 containing 1% of polymer concentration in the rate controlling membrane showed the fast release of the azithromycin. As the concentration of the polymer was increased, the release of the drug was more sustained and uniform but at higher concentrations (> 4%) there was an incomplete release of the azithromycin (H2)28, 29.The Ocusert H8  containing HPMC  (1.5%) and Eudragit RL100 (3%) while H9 containing HPMC  (1.5%) and Eudragit RL100 (4%)  showed a release of 99.30% and 98% respectively, at the end of 12 hours which indicated that, the polymer combination with same quantities can be used for the formulation of ocular film for therapeutic drug management in the  systemic  circulation.
It was found that formulation H8 showed the good, controlled linear drug release on the basis of r2 value and according to higuchi model. The release of azithromycin from formulation H8 through non-Fickian diffusion mechanism and it was found that drug release control by the dissolution fluid permeating through the membrane until an internal pressure is attained that drive the drug release from the drug reservoir. On the basis of cumulative release, r2 value and kinetic models H8 formulation found to be optimized formulation, in which 3% of eudragit RL100 produces almost constant and controlled drug release.
The developed ocusert formulation H8 was non toxic and non irritating to eye and the inserts were not expelled out of the cul-de-sac of the rabbits, it concluded that the insert�s dimensions were appropriate to use. The indirect In-vivo studies was performed for optimized formulation (H8). The In-vivo drug release study from formulation H8 and H9 was found to be in accordance with that of then vitro drug release study (Fig-3). Hence, we tried to correlate In-vivo results with the In-vitro percentage drug release of H8. The In-vitro /in vivo correlation for formulation H8 was found to be strong and productive. The strong In vitro and In-vivo correlation (r2= 0.9953) revealed the efficacy of the formulation (Fig.-4).When compare the Cmax, Tmax and AUC of In-vitro, In-vivo and pure drug. It was found that formulation H8 increase the Cmax & AUC  with comparison to pure drug. The stability studies was performe according to ICH guidelines and It was found that ocusert formulations were more stable at room temperature and with the shelf life was 622.7 days.

CONCLUSION
In present study an attempt was made to develop ocusert of azithromycin with prolong availability at the target site, avoidance of repetition of administration. On the bases of invitro & invivo studies , formulation H8 comprising of 1.5 %, HPMC & 3 % eudragit RL100, as drug reservoir membrane and rate controlling membrane respectively, was found be optimized formulation. The Ocular pharmacokinetic parameters of azithromycin ocusert data indicated the ocuserts follow the diffusion mechanism. The invivo studies in rabbit were in conformance to the results found in In-vitro studies. Stability studies revealed that developed formulation was stable for at least 622 days. However, their potential to improveocular bioavailability in humans needs to be investigated further.


ACKNOWLEDGMENT
The authors are grateful to management of CT group of institutions, Jalandhar for providing the research facilities.

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Q�of liposomal formulations of tropicamide for ocular delivery. International Journal of Pharmaceutics. 1999; 190(1):63�71.
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Table I. Composition of various polymers and plasticizers in different formulations
Formulation 
CodeDrug reservoir 
(HPMC)[ %w/w]Rate Controlling membrane
Eudragit RL 100
[% w/w]Plasticizers 
Glycerin dibutyl phthalate [%w/w]H11-               1H21.5-1H32-1H42.5-1H53-1H61.511H71.521H81.531H91.541H101.551 
Table II.Optimization of Azithromycin ocuserts.
Formulation codeThickness* (mm)Weight*
(mg)Surface pHFolding
Endurance*%Moisture
loss *% Moisture
absorption*Drug content* (mg)H10.09+0.0089.85+0.296.98+0.1224.3+2.163.48+0.235.1+0.161.88+0.011H20.11+0.00910.4+0.427.2+0.0832.3+2.733.83+0.135.23+0.261.98+0.016H30.14+0.0110.7+0.57.15+0.0728.8+3.72.43+0.45.5+0.21.91+0.01H40.15+0.0210.78+0.46.98+0.1227.2+3.42.45+0.25.58+ 0.31.88+0.019H50.18+0.0211.3+0.36.98+0.1923.6+2.81.6+0.206.47+0.361.85+0.03


Table II.Optimization of azithromycin ocuserts.
Formulation codeThickness* (mm)Weight*
(mg)Surface pHFolding
Endurance*%Moisture
loss *% Moisture
absorption*Drug content* (mg)H60.23�0.00722�0.577.1�0.0525.2�0.757.5�0.24.5�0.321.98�0.013H70.25�0.00723.8�0.207.23�0.05421�1.038.41�0.295.34�0.211.96�0.019H80.26�0.00424.9�0.277.1�0.06318.3�0.817.27�0.067.49�0.301.99+0.012H90.27�0.00726.2�0.527.21�0.07515.8�0.756.37�0.297.48�0.161.97+0.011H100.28�0.00728.2�0.237.35�0.0515.5�1.045.27�0.089.76�0.121.98+0.021* indicates average of five readings, Values are given as mean + SD (n=5)

Table IV Kinetic analysis of in-vitro drug release studies (r2).
Formulation Coder2n-valueZero orderFirst orderPeppasHiguchi-MatrixPeppasHiguchi-MatrixH60.76990.89620.89410.88880.14540.0942H70.78810.90670.91210.89650.15420.0932H80.80160.91480.92410.90130.17090.0853H90.81440.90270.90920.90630.19180.0810H100.85000.93040.90250.93140.20840.0756

Table V Comparison of pharmacokinetics parameters on release as invitro, in vivo release and for pure drug.
Pharmacokinetics parametersIn-vivoIn-vitroStdCmax (�g/ml)397402 36.4Tmax (min)405050AUC (�g.min/ml)39783.3338828.331190



Fig. 1 In vitro diffusion of Azithromycin ocuserts from H6 to H10 formulations.



Fig. 2 Area under curve (AUC) for In-vitro and In-vivo release of azithromycin.


Fig. 3 In-vivo release profile of azithromycin from ocusert (H8 and H9).

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Fig. 4 In-vitro and In-vivo correlation.









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