A PROCESS OF FORMULATING OCUSERTS OF FLUCONAZOLE

Abstract

To formulate ocuserts of Fluconazole and to evaluate both physicochemical parameters of in vitro release and in vivo permeation. Several polymeric systems used to fabricate ocular inserts for better ocular bioavailability and retention of drug for which gelling systems have shown advantages of convenient administration and increased contact time. Fluconazole ocular inserts were prepared by using Poly vinyl alcohol and Hydroxy propyl methyl cellulose as film forming polymers and Propylene glycol as plasticizer. Total six formulations were prepared by solvent casting technique and characterized thickness, weight variation, drug content, moisture loss, moisture absorption, folding endurance, surface pH, ocular irritation study, in vitro and in vivo release studies. The in vitro release studies were carried out by putting excised goat cornea between donor and receptor compartment of Franz diffusion cell. The research findings Hydroxy Propyl methyl cellulose is a good film forming hydrophilic polymer and is a promising agent for ocular delivery. Incorporation of propylene glycol enhances the permeability and thus therapeutic levels of the drug could be achieved. In vivo release profile indicated that drug release was less compared to in vitro release, and there was complete absence of eye irritation and redness of the rabbit eye. Further future work will be progressed to establish the therapeutic utility of these systems by pharmacokinetic and pharmacodynamic studies in human beings.

Specification

Description:FIELD OF INVENTION:
[0001] The main object of this invention is to formulate ocuserts of Fluconazole and to evaluate both physicochemical parameters of in vitro release and in vivo permeation more particularly using Polyvinyl Alcohol, Poly vinyl pyrrolidine and Propylene Glycol.

BACKGROUND:
[0002] Fluconazole is a prescription drug indicated for the treatment and prophylaxis of fungal infections where other antifungals have failed or are not tolerated (e.g. due to adverse effects), including Candidiasis caused by susceptible strains of Candida, Tinea corporis, Tinea cruris or Tinea pedis, Onychomycosis and Cryptococcal meningitis. Ocular therapy in the fungal infections would be significantly improves if the precorneal residence time of drugs could be increased. Successful results have been obtained with inserts and collagen shields. Several polymeric systems are investigated to fabricate ocular inserts for better ocular bioavailability and retention of drugs.

[0003] In the present research it was aim to prepare and evaluate ocular films containing fluconazole along with film forming polymers namely; Poly vinyl alcohol and Hydroxy propyl methyl cellulose at different concentrations with better bioavailability and longer duration of action. Fluconazole, a synthetic antifungal agent, is a triazole derivative used in the treatment of a wide range of fungal infections and it belongs to class II of biopharmaceutical classification system (BCS) having low water solubility.

SUMMARY:
[0004] The polymeric systems used to fabricate ocular inserts for better ocular bioavailability and retention of drug for which gelling systems have shown advantages of convenient administration and increased contact time. Fluconazole ocular inserts were prepared by using Poly vinyl alcohol and Hydroxy propyl methyl cellulose as film forming polymers and Propylene glycol as plasticizer. Total six formulations were prepared by solvent casting technique and characterized thickness, weight variation, drug content, moisture loss, moisture absorption, folding endurance, surface pH, ocular irritation study, in vitro and in vivo release studies. The in vitro release studies were carried out by putting excised goat cornea between donor and receptor compartment of Franz diffusion cell.

[0005] The research findings that the Hydroxy Propyl methyl cellulose is a good film forming hydrophilic polymer and is a promising agent for ocular delivery. Incorporation of propylene glycol enhances the permeability and thus therapeutic levels of the drug could be achieved. In vivo release profile indicated that drug release was less compared to in vitro release, and there was complete absence of eye irritation and redness of the rabbit eye. Further future work will be progressed to establish the therapeutic utility of these systems by pharmacokinetic and pharmacodynamic studies in human beings.

DESCRIPTION OF TABLES & FIGURES:
[0006] Table (0001): illustrates formulation of Fluconazole Ocuserts.

[0007] Table (0002): illustrates standard Calibration Curve of Fluconazole.

[0008] Table (0003): illustrates thickness of Patch.

[0009] Table (0004): illustrates weight Variation of Patch.

[0010] Table (0005): illustrates moisture Uptake & Moisture Loss.

[0011] Table (0006): illustrates folding Endurance.

[0012] Table (0007): illustrates surface pH of the films.

[0013] Table (0008): illustrates uniformity of fluconazole content.

[0014] Table (0009): illustrates cumulative percent release of fluconazole.

[0015] Figure (0001): illustrates UV Spectra of Fluconazole.

[0016] Figure (0002): illustrates standard Calibration Curve of Fluconazole in pH 7.4 STF.

[0017] Figure (0003): illustrates IR Spectra of Fluconazole + HPMC.

[0018] Figure (0004): illustrates IR Spectra of Fluconazole + PVA.

[0019] Figure (0005): illustrates preparation of Fluconazole Ocuserts

[0020] Figure (0006): illustrates in vivo cumulative percent drug release

DETAILED DESCRIPTION:
[0021] Fluconazole was procured from Hetero Drugs, Hyderabad, Polyvinyl Alcohol, Poly vinyl pyrrolidine K30, HPMC K-100 and Propylene Glycol. All other chemicals were pharmaceutical grade and used without further modification.

[0022] To formulate ocuserts of Fluconazole and to evaluate both physicochemical parameters of in vitro release and in vivo permeation. Several polymeric systems used to fabricate ocular inserts for better ocular bioavailability and retention of drug for which gelling systems have shown advantages of convenient administration and increased contact time. Fluconazole ocular inserts were prepared by using Poly vinyl alcohol and Hydroxy propyl methyl cellulose as film forming polymers and Propylene glycol as plasticizer. Total six formulations were prepared by solvent casting technique and characterized thickness, weight variation, drug content, moisture loss, moisture absorption, folding endurance, surface pH, ocular irritation study, in vitro and in vivo release studies. The in vitro release studies were carried out by putting excised goat cornea between donor and receptor compartment of Franz diffusion cell.

[0023] Formulation F5 shows a maximum cumulative percentage drug release of 69.02 % at the end of 2 hours through excised goat cornea. In vivo release profile indicated that drug release was less compared to in vitro release, and there was complete absence of eye irritation and redness of the rabbit eye. It can be concluded that Hydroxy Propyl methyl cellulose is a good film forming hydrophilic polymer and is a promising agent for ocular delivery.

[0024] Solubility Analysis: Pre-formulation solubility analysis was done, which included the selection of suitable solvent system to dissolve the drug as well as various excipients.

[0025] Melting Point Determination: Melting point determination of the obtained drug sample was done; as it is a first indication of purity of the sample. The presence of relatively small amount of impurity can be detected by lowering as well as widening in the melting point range.

[0026] Determination of λmax: An accurately weighed 10 mg of Fluconazole was transferred in a 100 ml volumetric flask. To the flask stimulated tear fluid was added in small proportion so as to dissolve fluconazole. The volume was made up to 100 ml with stimulated tear fluid (STF) pH 7.4 to get a concentration of 100μg/ml. 20 μg/ml solution of Fluconazole was prepared in dilution. The resulting solution was scanned in UV-Vis spectrophotometer from 400- 200 nm to determine the λmax.

[0027] Construction of calibration curve: Weigh accurately 100 mg of Fluconazole& transfer into 100 ml volumetric flask & make up the final volume with pH 7.4 STF. From the stock solution different concentrations (1 - 10 µg/ml) were prepared by transferring suitable volume into the 10 ml volumetric flasks. Each concentration sample was taken & the absorption was measured at 260 nm by using UV spectrophotometer by using pH 7.4 STF as a blank. The graph was plotted by taking concentration versus absorption and the plot appeared as a straight line, the linearity was determined by using y= mx +c formula.

[0028] The compatibility of drug with the excipient used was studied by Fourier transform infrared (FTIR) spectroscopy. The FTIR spectrums of Fluconazole and Formulation (F-5) blend were studied by using FTIR spectrophotometer using the KBr disk method. The scanning range was 500 to 4000 cm−1, and the resolution was 1 cm−1. This spectral analysis was employed to check the compatibility of drugs with the polymers used.

[0029] PREPARATION OF OCUSERTS: The polymeric drug reservoir films were prepared by dissolving 3, 4, and 5.0 % of PVA in 1/3rd volume of double distilled water. Along with this 300 mg of Fluconazole was separately dissolved in remaining amount of water and then it was poured to the polymeric solution. The solution was stirred using magnetic stirrer at 100 rpm. Then propylene glycol (6 % w/w) was incorporated to the above solution under same stirring conditions. After complete mixing the solution was cast 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).

[0030] The cast solution was allowed to evaporate by placing it inside a hot air oven maintained at 37 ± 2◦C, 30 ± 0.5% of RH for 24 hours. After drying the medicated films of 1 cm2 diameter each containing 15 mg of drug were cut using a stainless-steel borer, which is previously sterilized. Similar procedure was carried out for the preparation of HPMC patches.

[0031] EVALUATION: The ocuserts were evaluated for their physical characters such as shape, colour, texture, and appearance.Thickness: Ocuserts were evaluated for the thickness using a verniercaliper a least count of 0.01mm at different spots of the patches. The average of 6 readings was taken at different points and the mean thickness was calculated. The standard deviations (SDs) in thickness were computed from the mean value.

[0032] Weight variation: Six ocuserts were taken from each batch and their individual weights were determined by using a digital electronic balance of Shimadzu Corporation, Japan. The mean and standard deviation (S.D) were then calculated.

[0033] Moisture Uptake: The percentage moisture uptake test was carried out to check physical stability or integrity of ocuserts. Ocuserts were weighed and placed in a desiccator containing 100ml of saturated solution of Aluminium chloride by which a humidity of 79.5% RH was maintained. After three days the ocuserts were taken out and reweighed, the percentage moisture uptake was calculated by using formula.
% Moisture Uptake= Final Weight - Initial Weight × 100
Initial Weight

[0034] Moisture Loss: The percentage moisture loss was carried out to check integrity of the ocuserts at dry condition. Ocuserts were weighed and kept in a desiccator containing anhydrous Calcium chloride. After 3 days, the ocuserts were taken out and reweighed; the percentage moisture loss was calculated using the formula.
% Moisture Loss = Initial Weight - Final Weight × 100
Initial Weight

[0035] Folding Endurance: The folding endurance is expressed as the number of times the insert is folded at the same place, either to break the specimen or to develop visible cracks [7]. The specimen was folded in the centre, between the fingers and the thumb and then opened. This was termed as one folding. The process was repeated till the insert showed breakage or cracks in centre of insert. The total folding operations were named as folding endurance value.

[0036] Surface pH Determination: Inserts were left to swell for 5 hours on agar plate prepared by dissolving 2% (m/v) agar in warm simulated tear fluid under stirring and then pouring the solution into Petri dish till gelling at room temperature. The surface pH was measured by means of a pH paper placed on the surface of swollen patch.

[0037] Uniformity of fluconazole content: The ocuserts were placed in 5ml of pH 7.4 STF and were shaken in orbital shaker incubator at 50 rpm to extract the drug from ocuserts. After incubation for 24 h, the solution was filtered through a 0.45 μm filter and the filtrate was suitably diluted with STF solution. The absorbance of the resulting solution was measured at 260 nm.

[0038] In Vitro trans corneal permeation Studies: Whole eyeball of goat was transported from local butcher shop and cornea was carefully isolated along with 2 to 4 mm of surrounding tissue and was washed with cold normal saline free from proteins. Isolated cornea was mounted between clamped donor and receptor compartments of Franz diffusion.

[0039] A strip of film (1cm) placed in donor compartment and the solution (pH 7.4 STF) in receptor compartment were stirred at 100 rpm by using magnetic stirrer and the temperature was maintained at 370 C±50 C. At 15, 30, 45, 60-, 90-, 120- and 150-min time intervals 0.5 ml of test sample was removed from the receptor and the volume was replaced by adding fresh buffer.

[0040] The test samples were filtered and the absorbance of each sample was measured at 260 nm by using UV spectrophotometer and reagent blank (pH 7.4 STF). The absorbance was converted into concentration by using the standard curve. The release rates were calculated and the graphs were plotted taking time on X- axis & cumulative amount on Y- axis.

[0041] Ocular Irritation: The potential ocular irritation and/or damaging effects of the ocusert under test were evaluated by observing them for any redness, inflammation, or increased tear production. Formulation was tested on five 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 were observed up to 12 hours.

[0042] In Vivo Drug Release Study: Out of six batches of formulations F-5 was taken for in vivo study on the basis of in vitro drug release studies. The ocuserts were sterilized by using UV radiation before in vivo study. After sterilization, ocuserts were transferred into polyethylene bag with the help of forceps inside the sterilization chamber itself. Albino rabbits of either sex (New-Zealand strain), weighing between 2.5-3.0 kg, were used for the experiment.

[0043] The ocuserts containing Fluconazole were taken for in vivo study was placed into the lower conjunctival cul-de-sac. The ocuserts were inserted into each of the six rabbits and at the same time the other eye of six rabbits served as control. Ocuserts were removed carefully at 30, 60, 90, 120, and 150 minutes and analysed for drug content. The drug remaining was subtracted from the initial drug content of ocuserts that will give the amount of drug released in the rabbit eye.
, Claims:Claims:
I/We Claim:
1.A method of evaluation of atorvastatin calcium liquisolid tablets and comparing the dissolution
data with marketed tablet, comprising:
an accurately weighed 10 mg of fluconazole was transferred in a 100 ml volumetric flask;
whereby to the flask stimulated tear fluid was added in small proportion so as to dissolve
fluconazole;
the volume was made up to 100 ml with stimulated tear fluid (stf) ph 7.4 to get a concentration of 100μg/ml;
a 20 μg/ml solution of fluconazole was prepared in dilution;
the resulting solution was scanned in uv-vis spectrophotometer from 400- 200 nm to determine the λmax. weigh accurately 100 mg of flucoazole and transfer into 100 ml volumetric flask & make up the final volume with ph 7.4 stf;
whereby from the stock solution different concentrations (1-10 μg/ml) were prepared by
transferring suitable volume into the 10 ml volumetric flasks;
each concentration sample was taken & the absorption was measured at 260 nm by using uv
spectrophotometer by using ph 7.4 stf as a blank;
the blank polymeric patches were prepared using pva and hpmc alone by solvent casting technique.
the polymeric drug reservoir films were prepared by dissolving 3, 4, and 5.0 % of pva in 1/3rd
volume of double distilled water;
wherein along with this 300 mg of fluconazole was separately dissolved in remaining amount of water and then it was poured to the polymeric solution;
the solution was stirred using magnetic stirrer at 100 rpm;
whereby propylene glycol (6 % w/w) was incorporated to the above solution under same stirring conditions;
whereby after complete mixing the solution was cast in petri dish 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 ± 2◦c, 30 ± 0.5% of rh for 24 hours; and whereby after drying the medicated films of 1 cm2 diameter each containing 15 mg of drug were cut using a stainless-steel borer, which is previously sterilized. similar procedure was carried out for the preparation of HPMC patches.

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