Preparation Of Ocular Inserts Biology Essay

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The polymeric drug reservoir films were prepared by dissolving 1.0, 1.5 and 2.0% of HPMC-K4M in 15 mL of double distilled water. Along with this 26.95 mg of binary mixture containing Fluconazole b-CD was separately dissolved in dilute alkali hydroxide solution and then it was poured to the polymeric solution. The solution was stirred using magnetic stirrer at 100 rpm and PEG-400 (10% w/w), which was previously optimized for its concentration, was incorporated as a plasticizer as well as permeation enhancer to above solution under same stirring conditions.

After complete mixing the solution was casted in petri dish (previously lubricated with Glycerine) using a ring of 5.0 cm diameter and with a funnel inverted on the surface (for uniform evaporation of solvent). The cast solution was allowed to evaporate by placing it inside a hot air oven maintained at 37±20 C, 30±0.5 % of RH for 24 h. After drying the medicated films of 8 mm diameter each containing 0.69 mg of drug were cut using a stainless steel borer, which is previously sterilized.

II. Preparation of Rate controlling membrane (RCM)

A weighed quantity of cellulose acetate phthalate was dissolved in 10 ml of acetone to obtain 4, 5 and 6 % polymeric solutions. Stirring was continuously maintained until the clear solution was obtained. These solutions were poured in petri dish (previously lubricated with Glycerine) using a ring of 5.0 cm diameter. The solution was evaporated slowly by inverting a glass funnel on a petri dish at room temperature for 12 h. The dried films were cut into 9 mm diameter using a stainless steel borer.

III. Sealing

A medicated reservoir disc was sandwiched between two rate controlling membranes. Then this whole unit was placed for 4-5 minutes, over a wire mesh inside the desiccator. Desiccator was previously saturated with ethanol / acetone (60:40). This procedure resulted into successful sealing of the medicated reservoir film between two-rate controlling membranes. The sealed ocular inserts were stored in an airtight container under ambient conditions.

Plasticizer weight was based on weight of the polymer. All the above experimentation was carried out under laminar airflow to maintain the sterility conditions of ophthalmic products.

Compatibility studies

Interaction studies were conducted on the optimized formulation by comparing it with the pure drug on the basis of the ultraviolet, infrared and thin layer chromatography.

The solutions of pure drug and medicated formulations were prepared in pH 7.4 and filtered through Whatman filter paper No.42 and scanned for UV absorption between 200 to 400 nm.

The IR absorption spectra of the pure drug, optimized two medicated formulations were taken in the range of 400-4000 cm-1 by KBr disc method using IR spectrophotometer.

TLC analysis was conducted according to BP 2000. For visualization, plate was examined in ultraviolet light at 254 nm. Rf values were calculated for both standard and the sample using the formula:

Rf = Distance traveled by the solute from the origin line

Distance traveled by the solvent from the origin line

Stationary phase

Thin plates of Silica gel GF 254 having thickness of 0.25 cm. The plates were activated at 105oC for 30 minutes prior to use.

Mobile phase

Ammonia : methanol : methylene chloride in the ratio of 2: 18: 80

Separation technique



UV chamber at 254 nm

Reference solution

1 mg/ml of pure Fluconazole

Test solution

Drug-formulation mixture equivalent to 1 mg/ml of Fluconazole dissolved in solvents


10 µl of reference and test solutions were applied as spots on dry activated plate. The solvent front was developed up to 15 cm. The plate was dried in air and it was examined under UV chamber at 254 nm.

Evaluation of Polymeric Ocular Inserts

Thickness of Film

Films were evaluated for the thickness using a Dial Caliper (Mitutoyo, Japan). The average of 10 readings was taken at different points of film and the mean thickness was calculated. The standard deviations (SD) in thickness were computed from the mean value.

Uniformity in drug content

For drug content uniformity, the ocular inserts were placed in 5 ml of pH 7.4 phosphate buffer saline and were shaken in orbital shaker incubator at 50 rpm to extract the drug from inserts. After incubation for 24 hrs, the solution was filtered through a 0.45 mm filter and the filtrate was suitably diluted with buffer solution. The absorbance of the resulting solution was measured at 254 nm.

Uniformity of weight

The weight variation test was carried out using digital balance (………….), by weighing three patches from each formulation. The mean value was calculated and the standard deviations of weight variation were computed from the mean value.

Folding endurance

A small strip of film was cut evenly and separately folded at the same place till it broke. The number of times the film could be folded at the same place without breaking gave the folding endurance.

Percentage moisture absorption

The percentage moisture absorption test was carried out to check physical stability or integrity of ocular films. Ocular films were weighed and placed in a dessicator containing 100 ml of saturated solution of aluminium chloride and 79.5% humidity was maintained. After three days the ocular films were taken out and reweighed. The percentage moisture absorption was calculated using the following equation:

Percentage moisture absorption = Final weight - Initial weight x 100

Initial weight

Percentage Moisture Loss

The percentage moisture loss was carried out to check integrity of the film at dry condition. Ocular films were weighed and kept in a dessicator containing anhydrous calcium chloride. After 3 days, the ocuserts were taken out and reweighed; the percentage moisture loss was calculated using the following equation:

Percentage moisture loss = Initial weight - Final weight x 100

Initial weight

Determination of water uptake and swelling behavior

Water uptake was determined gravimetrically. Inserts were placed on a filter. The lower side of the filter was immersed in a beaker containing simulated lacrimal fluid (SLF) and incubated at 320C (eye surface temperature). The beaker was closed with soft paraffin to prevent evaporation during the experiment. The weight of each ocusert was determined with the digital balance at predetermined time points. The size changes of the inserts due to swelling were investigated macroscopically.

In vitro drug release studies

The inserts from each batch were taken and placed in a 15 mL vials containing 10 mL of pH 7.4 phosphate buffered saline. The vials were placed in an oscillating water bath at 32 ± 10C with 25 oscillations per minute. 1ml of the drug releasing media was withdrawn at various time intervals of 1, 2, 4, 8, 12, 16 and 20 hrs and replaced by the same volume of phosphate buffer saline pH 7.4. These samples were filtered through 0.45 mm membrane filter. The filtrate was diluted suitably with the buffer. The drug was estimated in each batch by double beam UV-Vis spectrophotometer (Elico Sl 210, Mumai, India) at 254 nm. The obtained data was treated with mathematical kinetic modeling.

In vivo drug release study

Out of 5 batches of formulations F-4 and F-5 were taken for in vivo study on the basis of in vitro drug release studies. The inserts were sterilized by using UV radiation before in vivo study. Inserts were taken in a petri dish along with 100 mg of pure drug (Fluconazole), which was spread to a thin layer. This petri dish along with polyethylene bags and forceps were placed in UV sterilization chamber (hood).

The inserts and other materials were exposed to UV radiation for one hour. After sterilization, inserts were transferred into polyethylene bag with the help of forceps inside the sterilization chamber itself. The pure Fluconazole that was sterilized along with inserts was analyzed for potency by UV spectrophotometer at 254 nm after suitable dilution with pH 7.4 phosphate buffer.

The albino rabbits of either sex (New-Zealand strain), weighing between 2.5-3.0 kg were used for the experiment. The animals were housed on individual cages and customized to laboratory conditions for one day (received free access to food and water).

The ocular inserts containing Fluconazole were taken for in vivo study, which were previously sterilized on the day of the experiment and were placed into the lower conjunctival cul-de-sac. The inserts were inserted into each of the 7 rabbits at same time the other eye of seven rabbits served as control.

Ocular inserts were removed carefully at 1, 2, 4, 8, 12, 16 and 20 hours and analyzed for drug content as dilution mentioned in drug content uniformity. The drug remaining was subtracted from the initial drug content of inserts that will give the amount of drug released in the rabbit eye. Observation for any fall out of the inserts was also recorded throughout the experiment. After one week of washed period the experiment was repeated for two times as before.

Stability Studies

Stability testing has become integral part of formulation development. It generates information on which, proposed for shelf life of drug or dosage forms and their recommended storage conditions are based.

In the present study, the formulation F-4 was selected for the study and inserts were packed in amber-colored bottles tightly plugged with cotton and capped. They were exposed to various temperatures (600, 400, 200, 100 and 00 C) for 30 days. At regular intervals, the inserts were taken in 10 ml of pH 7.4 buffer and were shaken for 12 hrs in an orbital shaker. The resultant solutions were filtered, properly diluted and estimated spectrophotometrically by keeping pH 7.4 buffers as blank. The logarithmic percent of undecomposed drug was plotted against time and decomposition rate constants (K) were obtained at each temperature. The logarithm of decomposition rate constants were plotted against reciprocal of absolute temperature and the resulting line was extrapolated to K at 250 C.

Shelf life can be obtained by using formula,

T90 = 0.104/K at 250 C.


The purity of drug was confirmed by comparison of its I.R spectrums (Fig. 1 and 2) with the spectrum of the standard drug Fluconazole. The spectrum showed the following functional groups at their respective frequencies [at 3441cm-1 showed -NH stretch (amine), at 3077 cm-1 showed -OH stretch (hydroxyl), 1507 cm-1 showed -C=O stretch (ketonic) and 1095 cm-1 showed -O- stretch (ether).

The pure drug was found to be slightly soluble in water, phosphate buffer saline pH 7.4, freely soluble in dimethyl sulfoxide, very slightly soluble in alcohol. It dissolved in dilute solutions of mineral acids and alkali hydroxides.

The study was carried out by open capillary method and the melting point of pure drug was found to be 256oC - 257oC, which complied with standard specifications.

Compatibility evaluations were studied by extracting the drugs from the inserts and TLC was performed on silica gel plates using ammonia / methanol / methylene chloride in the ratio of 2:18:20. The spots were examined in UV light at 254 nm.

The Rf values of Fluconazole, F-4 and F-5 were 0.81, 0.84 and 0.80 respectively.

From the I.R spectral analysis it was found that I.R spectrum of pure drug Fluconazole (Fig. 1) and combination of pure drug with polymers like b-cylcodextrin, HPMC-K4M, cellulose acetate phthalate showed the all characteristic peaks of Fluconazole confirming the compatibility of the pure drug and polymer. The spectral representation of combination of all polymers with Fluconazole was shown in Fig. 2. An I.R spectrum of complexation of Acylocvir with b-cyclodextrin is shown in Fig.3.

Absorption spectrum of pure drug was scanned between 200 - 400 nm with 10 mg concentration prepared in pH 7.4 phosphate buffer saline. The absorption maximum was noted at 254.48 nm. Also the lmax for FLUCONAZOLE-b-CD complex was determined by similar procedure and was found to be at 255.19 nm as shown in Fig. 4 and 5.

Accurately weighed amounts of the powdered drug Fluconazole and complex, each containing 100 mg equivalent of FLUCONAZOLE, were used in triplicate to carry out the studies on an enhancement of dissolution rate of Fluconazole. The USP XXIII-8 station Dissolution Rate Test Apparatus was employed for the purpose. Tabulations are given in table 7 & 8. Comparative plot of cum. percentage drug release Vs time mode for pure drug and complex is shown in Fig. 6.

Table 2 shows results of drug content uniformity of each formulation. Three replicates of each batch were analyzed; mean and standard deviations were calculated.

Also a plot of comparative percentage drug content between different formulations is depicted in Fig. 7.

The percentage drug retained and cumulative percentage drug release by the each film in the in vitro release studies was based on the mean content of the drug present in the respective films.

Thickness of all formulations in triplicate was measured by using dial caliper. The mean values and standard deviations were noted down as given in table 10.

The uniformity of weight is calculated for three different films of each formulation, mean and standard deviation was noted, results are given in table 10.

Since the films were folded manually, the films were folded on an average of more than 90 times and shown in table 10.

Each formulation in triplicate was analyzed for percentage moisture absorption studies and the results are tabulated in table 11.

The percentage moisture loss was calculated for three different films of each formulation from AO-I to AO-IX. The results were given in table 11.

The water uptake and swelling behavior studies were conducted on drug reservoir membranes formulated using HPMC-K4M swelling polymer. The results were shown in table 12.

All the nine formulations prepared were subjected to in vitro drug release studies. These studies were conducted using oscillating water bath at 32 ± 10C. In the present work the in vitro studies were carried out in triplicate. For different time intervals the samples were withdrawn, and analyzed for absorbance using UV-spectroscopy. Cumulative drug release in milligrams is calculated. Cumulative percent drug release and cumulative percentage drug retained were calculated on the basis of mean amount of Fluconazole present in the respective films.

The results obtained from in vitro drug release studies were plotted adopting five different mathematical models of data treatment.

The release data obtained for formulations F-1 to F-2 were tabulated in Table 13 to 21 and the plotted graphs for all the models were shown in Fig. 8- 22. The kinetic values obtained for all the models were depicted from table 22-27.

In vivo drug release studies were performed on New Zealand strain albino rabbits. Out of nine formulations F-4 and F-5 were subjected to in vivo drug release studies, because these two formulations shown good linearity, maximum release rate and fulfilled many requirements of the optimized formulations. Hence they were considered as the formulations of choice for in vivo studies.

The study was carried out in triplicate for different time intervals, samples were withdrawn and cumulative amount of drug release were calculated by subtracting drug remaining from the main content of respective insert and on the basis of the amount of drug release percentage of FLUCONAZOLE was calculated. The results obtained for the formulations F-4 and F-5 were tabulated in table 28 and 29. Figure 23 shows the in vivo cumulative percentage drug released as a function of time for the formulations F-4 and F-5.

Scattered graph based on correlation between in vitro/in vivo cum. Percentage drug release for both the formulations were shown in figure 24 & 25.

Stability studies were carried out to predict the degradation that may occur over prolonged periods of storage at normal shelf condition for formulations F-4. The results of the stability studies, which were conducted for 30 days, are given in the table 31. Table 32 gives the parameters obtained from the stability studies data. The shelf life of the fabricated device was calculated based on these parameters.


The present study is aimed to develop and prepare ocular drug delivery system in the form of ocular inserts useful in the treatment of herpes simplex keratitis using Fluconazole as a choice of drug.

Preformulation studies were found to show confirmative results. Figure 5 shows the standard calibration curve with a good reproducibility, having a slope 0.0549 and with regression value 0.9997.

The absorbance maximum for pure drug Fluconazole is found to be at 254.48 nm, whereas the binary system of FLUCONAZOLE-b-CD showed the lmax at 255.19 nm.

Rf value of 0.81 was obtained for pure drug. Whereas Rf value for A0-V and AO-VIII were 0.84 and 0.80 respectively by thin layer chromatographic compatibility studies. The Rf values of the drug and medicated formulations were almost similar which inferred for compatibility of formulations with its excipients.

The lmax was found to be almost similar i.e.254.93 nm for medicated formulation extracts, which signifies that, there is no possible interference of excipients in absorbance pattern of the drug.

The I.R.spectra recorded for formulations were taken as qualitative tool in order to assess the change in peaks, pattern of curve etc. No major differences were observed in the IR spectrum of the pure drug and medicated formulations.

The cum.percent drug release for pure drug Fluconazole was found to be 34.27% whereas 61.18% was found with the binary system. This clearly indicates the enhancement in dissolution rate of poorly soluble drug Fluconazole when complexed with b-cyclodextrin.

For the various formulations the drug content uniformity was found to be between 0.675±0.012 to 0.689±0.006mg. Formulations AO-V & AO-VIII showed the maximum and uniform percent drug content of 99.85 and 99.49%.

The mean film thickness (n=3) was almost uniform in all the nine formulations, it was found to vary between 0.151±0.001 to 0.171±006. The little variation was observed with formulations, AO-VI and AO-IX might be due to the more concentration of rate controlling membrane.

The values of uniformity of weight found to vary from 13.73±0.141 to 18.59±0.224. Formulations AO-I and AO-IX showed good uniformity in weight.

The values for folding endurance ranging from 87.9 to 103.6.And no cracks were observed. Formulations AO-I & AO-VI showed maximum folding endurance.

The highest moisture absorption was marked from formulations AO-VII (11.83±0.346). This may be due to the presence of larger concentration of hydrophilic polymer HPMC-K4M.

It was observed that there was no change in integrity in all the formulations. The moisture loss for all the formulations varied between 6.61 ± 0.241 to 11.61 ±0.092. Formulation AO-I showed the maximum amount of moisture loss in dry conditions might be due to presence of hydrophobic polymer cellulose acetate phthalate. And low concentrations of polymers in overall preparation.

Water uptake was found to be maximum with 2% HPMC K4M concentration due to the presence of more concentration of swellable polymer. After complete hydration, moderate gelling and swelling of the polymer was observed and had a diameter of approximately 10.2 mm.

All the nine formulations were subjected to in vitro drug release studies. The overall cumulative % drug release for formulations, AO-I to AO-IX was found to be from 78.61, 94.64, 95.11, 92.00, 98.31, 97.67, 97.58, 98.03 & 92.50% respectively at the end of 20th hour. The data obtained were grouped in five models of the mathematical treatment.

Based on the highest regression value (r), which is nearing to unity, the formulations AO-II, IV, VI, VII & VIII followed Higuchi-Matrix kinetics. This suggests that the drug release by swellable polymer matrix through the diffusion of tear fluids. Formulations AO-I, III, V and IX were best fitted into Peppas model with an 'n' values 0.5011, 0.7704, 0.7536 & 0.7793 respectively. This indicates that the release by non-Fickian-diffusion mechanism. Cum % drug release for AO-V and AO-VIII was found to be 98.31%, 98.03%, and is depicted complete and more controlled fashion of all the other formulations. Therefore, it was ascertained that the drug release could be more controlled by using 5% concentration of CAP, because above which the polymer concentration will restrict the drug release with therapeutic point of view.