FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION Sec 10-Rule 13
A STABLE OPHTHALMIC COMPOSITION OF MICONAZOLE
INDOCO REMEDIES LIMITED
Indian
R-92-93, T.T.C Industrial Area, Thane Belapur Road, Rabale MIDC
Navi Mumbai-400701
The following complete specification describes the invention and the manner in which it is to be performed.

Technical Field:
The present invention relates to a stable ophthalmic composition of miconazole. More specifically, the present invention relates to a miconazole ophthalmic composition and a method of preparing same, which is chemically stable at room temperature. Moreover, the composition of the present invention is stable and sterile aqueous ophthalmic composition.
Background of the Invention:
Miconazole is antifungal medicine, has chemical structure as below -

The chemical name of Miconazole is l-[2-(2,4-Dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]ethyl]-l//-imidazole. It is sparingly soluble drug.
Imidazole compounds have good antifungal effect and hence these compounds are used in drug therapy over the period of time due to its effective antifungal effect by inhibiting ergosterol biosynthesis. Most Fungal disorders are benign but can be life threatening in immunocompromised or malnourished populations. Since 1970's Miconazole is used as antifungal active ingredient, mostly in topical form however, now it is also available in oral dosage formulation. The miconazole is widely used in millions population, however fungal resistance to this drug is relatively low.

Imidazole including miconazole are primarily used for skin antifungal disorders and are effective against several strains of Candida, being primarily used in treatment of dermatophitosis, oral and vaginal candidiasis due to rapid antifungal action. US patent no. 4446145 discloses anti acne composition comprising combination of benzoyl peroxide and miconazole.
Miconazole dosage forms approved by USFDA are cream, tablet, suppository, injection, and ointment etc and scientist are now focusing on to develop miconazole containing new dosage form.
Tomoko Miyakubo, Daisuke Todokoro, Koichi Makimura and Hideo Akiyama in article titled as "Fungal keratitis caused by Didymella gardeniae (formerly Phoma gardeniae) successfully treated with topical voriconazole and miconazole" concluded that combination use of voriconazole and miconazole is effective in keratitis caused by D. gardenia.
Linda Gyanfosu, George Asumeng Koffuor, Samuel Kyei, Ben Ababio-Danso, Kwabena Peprah-Donkor, Wilson Bright Nyansah & Frederick Asare, "Efficacy and safety of extemporaneously prepared miconazole eye drops in Candida albicans-induced keratomycosis", provides result of extemporaneously prepared miconazole eye drops used in patients suffering from Candida albicans-induced keratomycosis and it was found that the extemporaneously prepared miconazole eye drops are effective and safe to use in keratomycosis.
C. Stephe n Foster, M.D. "Miconazole therapy for Keratomycosis", discloses study conducted on 7 patient, where patients are treated with topical and subconjunctival miconazole and it was found that miconazole didn't show ocular toxicity and subconjunctival miconazole was well tolerated. From this article author concluded that fungi could be eradicated with topical and subconjunctival miconazole and orally administered ketokonazole.

C. Stephen Foster, MD; Mary Stefanyszyn, MD, in article titled "Intraocular Penetration of Miconazole in Rabbits", discovered that penetration power of miconazole is far better than natamycin.
Eyes are important sense organs of Human body. There are many types of ocular disorder and disease, which often require treatment applied directly to the eye. In such cases, it is desired that the formulations have a suitable surface retention properties such as viscosity, so that the formulation stay on the eye for a suitable period of time to deliver desire medication. The ophthalmic drug delivery dosage forms are preferred for treatment of various conditions of eyes. But absorption of drug after administration of drop in eye is limited by unique mechanisms of eye & it's anatomy like drainage of the solution, lacrimation, sensitive to foreign particle & metabolism etc. Such factors affects therapeutic efficacy of dosage form by causing low absorption of drug from administration site. Many times it has been observed that continuous blinking of eye, drains out instilled solution from the eye, & hence increase the treatment duration & cost associated with it.
The inventors of the present invention have developed long retentive miconazole ophthalmic suspension based on the polymer platform system of carbopol and xanthan gum. Specifically, the inventors of the present invention have developed an ophthalmic suspension of miconazole using combination of carbopol and xanthan gum, optionally with benzalkonium chloride as preservative, and is stable at room temperature for period of 6 months.
Object of the Invention:
The main object of the present invention is to provide a long retentive miconazole ophthalmic suspension based on polymer platform system of carbopol and xanthan gum.

Another object of the present invention is to provide a stable ophthalmic composition comprising miconazole, carbopol, xanthan gum and pharmaceutically acceptable excipients.
Another object of the present invention is to provide a stable ophthalmic composition comprising miconazole, carbopol xanthan gum, benzalkonium chloride and pharmaceutically acceptable excipients.
Yet another object of the present invention is to provide a stable ophthalmic composition comprising miconazole, combination of carbopol and xanthan gum, benzalkonium chloride and pharmaceutically acceptable excipients.
Further object of the present invention is to provide a stable ophthalmic composition comprising miconazole, combination of carbopol and xanthan gum, benzalkonium chloride and pharmaceutically acceptable excipients using low density polyethylene (LDPE) containers.
Further object of the present invention is to provide a stable ophthalmic suspension comprising miconazole that is stable at room temperature.
Further object of the present invention is to provide method for preparation of a stable ophthalmic composition comprising miconazole.
Yet further object of the present invention is to provide stable ophthalmic suspension comprising miconazole, with its ability to provide sustained release, which results in less frequent administration and enhancing patient compliance.
Yet further object of the present invention is to provide a stable ophthalmic composition comprising miconazole for treating fungal keratitis and other fungal infections.

Summary of the Invention:
The present invention relates to a miconazole ophthalmic composition and a method of preparing same, which is chemically stable at room temperature. The composition of the present invention is stable and sterile ophthalmic composition.
Specifically, the present invention relates to an ophthalmic suspension comprising miconazole, combination of xanthan gum and carbopol as viscosity enhancing agents, with benzalkonium chloride as preservative.
Detailed Description:
The present invention relates to a stable ophthalmic composition comprising miconazole and a method of preparing same, which is chemically stable at room temperature.
Thus, in one aspect, the present invention provides a stable ophthalmic composition comprising miconazole, viscosity enhancing agents and pharmaceutically acceptable excipients.
Combinations of polymers have been used in ophthalmic compositions. Certain combinations of polymers are known to provide synergistic effects on viscosity and in some cases, even a phase transition from a liquid to a gel. For example, US. Patent number. 4136173 discloses ophthalmic compositions containing a combination of xanthan gum and locust bean gum.
One approach to achieve a target viscosity in a topically administrable ophthalmic composition might involve simply adding a sufficient amount of one polymeric ingredient. Often, however, it is desirable to minimize the total amount of polymeric additives in topically administrable ophthalmic compositions. A mixed polymer system containing more than one polymer can significantly enhance the viscosity and lubrication property of a composition while minimizing total polymer concentration and cost of materials.

The inventors of the present invention made intensive studies by focusing attention on the fact that when an eye drop is instilled into eye, it comes into contact with the precorneal tear film on the ocular surface, in particular mucin in the precorneal tear film.
Systematic approach of quality by design was used to identify the synergistic polymer platform system. By use of quality by design interaction amongst various polymers can be studied in more effective way with less number of experiments. As these polymer platform systems are to be used in ophthalmic formulation which needs to be sterile, hence, many times sterilization process, buffers, or pH adversely or synergistically impact these polymers. Therefore the impact of all sterilization process & pH or buffers data becomes useful for further formulation development activity.
Following polymers were studied at 0.5% concentration.
Table 1: Viscosity of single polymer (0.5%).

Sr. No. Polymer Viscosity (cps)
1. Sodium carboxy methyl cellulose (Sodium CMC) 4.78
2 Hydroxy propyl methyl cellulose (Hypromellose 2910) 6.79
3 Gellan gum (Kelcogel CG-LA) 616
4 Carbomer homopolymer Type B (carbopol) 31.8
5 Methyl Cellulose 1.92
6 Guar gum 123
7 Xanthan gum (Xantural-75) 2753
Each polymer was dissolved in purified water under stirring to get 0.5% aqueous solution. Viscosity of these aqueous solution was measured by using appropriate spindle and rpm. Polymers with viscosity more than 100 cps were chosen for further i.e gellan gum, guar gum, xanthan gum were selected. In addition to this carbopol was selected because after neutralization of carbopol it would result higher viscosity.

The design of experiment was employed systematically to identify the synergistic polymeric combination.
Mixture design was selected as this design is used when the response changes as function of the relative proportion of the component.
Table 2: Mixture design for polymer mixture.

Run Gellan gum (Kekogel CG-LA) (%) Carbopo Guar gum
(%) Xanthan gum
(Xantural-75)
(%) Viscosity (cps)
1 0.000 0.000 0.250 0.250 2816
2 0.250 0.250 0.000 0.000 14.7
3 0.000 0.000 0.000 0.500 2753
4 0.000 0.250 0.250 0.000 105.4
5 0.125 0.125 0.125 0.125 1575
6 0.063 0.063 0.063 0.313 7828
7 0.063 0.313 0.063 0.063 166.8
8 0.000 0.500 0.000 0.000 21.1
9 0.500 0.000 0.000 0.000 235
10 0.250 0.000 0.000 0.250 1773
11 0.125 0.125 0.125 0.125 1356
12 0.063 0.063 0.313 0.063 2615
13 0.167 0.000 0.167 0.167 342.5
14 0.250 0.000 0.250 0.000 82
15 0.000 0.250 0.250 0.000 192.8
16 0.000 0.250 0.000 0.250 6284
17 0.313 0.063 0.063 0.063 91.6
18 0.000 0.000 0.500 0.000 4.26
19 0.000 0.250 0.000 0.250 7453
Optimal mixture design with cubic mixture model was used to study interaction. Total 19 experimental runs were conducted with 2 center point and 2 replicate. Total concentration for mixture was kept 0.5%.

Aqueous solutions were prepared by dissolving polymer in purified water under stirring. Viscosity of these aqueous solution was measured by using appropriate spindle
and rpm.
In polynomial equation positive sign represents a synergistic effect, while negative sign indicates antagonistic effect. The negative coefficient of A and B in the model refers to decrease in viscosity due to for gellan gum and guar gum combination. Highest positive coefficient of 5.81 between B and D showed the highest viscosity which in turn means maximum synergy. + 2.38 × A + 1.33 × B + 0.64 × C + 3.45 × D - 2.88 × AB+ 1.68 × AC+1.40 × AD+4.21 ×BC + 5.81 × BD + 5.68 × CD+66.99 × ABC -120.27 × ABD - 15.95 × ACD + 86.54 × BCD - 11.55 BD (B-D). Where A, B, C and D are concentration of gellan gum, carbopol, guar gum and xantural 75 respectively. The regression analysis of the data obtained from the experimental runs generated the following polynomial equations in which the model F ratio was statistically significant at a < 0.05 with Adj - R2 value in the range close to 1 with a statistically non-significant lack of fit at α>0.05.
Table No. 3: Statistical analysis of experimental design.

Response Model p value Adj - R2 Lack of fit test p value
Viscosity (cps) < 0.0001 0.9949 0.1063
Table no 3 depicts that model is significant. Table no 3 suggest that less than 0.01 % chance that the model F value of polynomial equation occurred due to noise. The p value for lack of fit was not significant, indicating that this model equations fitted the data well. Therefore reduced cubic mixture model could adequately describe the data and could be employed to arrive at synergy.
From the viscosity study data it can be concluded that four polymer combination system and carbopol & xanthan gum (0.250:0.250) gave the best synergy. The viscosity

of synergistic combination even at lower concentration (0.250:0.250) was higher than the viscosity of individual polymer at higher concentration (0.500). Further to identify the best ratio of binary polymer combination carbopol & Xanthan gum again mixture design was applied.
Mixture I-optimal design, quartic model was used for this study. 10 experimental runs were conducted. Total concentration for mixture was kept 0.5%.
Table No. 4: Experimental run along with viscosity data.

Run Carbopol Xanthan gum (Xantural 75) Viscosity (cps)
1 0.000 0.500 2615
2 0.500 0.000 13.8
3 0.500 0.000 13.9
4 0.375 0.125 1380
5 0.250 0.250 7483
6 0.125 0.375 14607
7 0.250 0.250 7138
8 0.167 0.333 14297
9 0.333 0.167 2681
10 0.000 0.500 2289
The regression analysis of the data obtained from the experimental runs generated the following polynomial equations in which the model F ratio was statistically significant at a < 0.05 with Adj - R2 value in the range close to 1. Polynomial equation generated is as below
+ 1.14 x A + 3.39 x B + 6.42 x AB + 0.33 x AB(A -B) + 4.05 x AB (A-B) Ʌ 2 where A and B are concentration of carbopol and xanthan gum respectively.
It was concluded that carbopol & xanthan gum in ratio of 0.125: 0.375 showed the best synergy.

According to preferred embodiment, the present invention provides a stable ophthalmic composition comprising miconazole, carbopol, xanthan gum and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising miconazole, carbopol, xanthan gum, benzalkonium chloride and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising miconazole, benzalkonium chloride and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising miconazole, combination of carbopol and xanthan gum, benzalkonium chloride and pharmaceutically acceptable excipients, wherein the composition is sterile.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising miconazole, combination of carbopol and xanthan gum, benzalkonium chloride, monobasic sodium phosphate, dibasic sodium phosphate, propylene glycol, sodium hydroxide and purified water.
According to one embodiment, one of the most important feature of the present invention is to provide a stable ophthalmic composition comprising miconazole by using a combination of viscosity enhancing agent such as carbopol and xanthan gum in the ratio of 1:3 respectively.
According to another embodiment, the present invention provides a stable ophthalmic composition comprising miconazole, combination of sodium alginate and carrageenan, benzalkonium chloride and pharmaceutically acceptable excipients.

According to one embodiment, the present invention provides a stable ophthalmic composition comprising miconazole, combination of sodium alginate and carrageenan, benzalkonium chloride, monobasic sodium phosphate, dibasic sodium phosphate, propylene glycol, sodium hydroxide and purified water.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
0.5% to 2 % (w/v) miconazole,
0.01 % to 0.5 % (w/v) benzalkonium chloride,
0.05 % to 0.38 % (w/v) carbopol,
0.05 % to 0.7% (w/v) xanthan gum,
0.001 % to 0.5 % (w/v) monobasic sodium phosphate,
0.001 % to 1% (w/v) dibasic sodium phosphate and
0.3 % to 2.5 % (w/v) propylene glycol.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
0.5% to 2 % (w/v) miconazole,
0.01 % to 0.5 % (w/v) benzalkonium chloride,
0.05 % to 0.38 % (w/v) carbopol,
0.05 % to 0.7% (w/v) xanthan gum,
0.001 % to 0.5 % (w/v) monobasic sodium phosphate,
0.001 % to 1% (w/v) dibasic sodium phosphate and
0.3 % to 2.5 % (w/v) propylene glycol,
wherein the composition has pH of about 6 to 7.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
0.5% to 2 % (w/v) miconazole,
0.01 % to 0.5 % (w/v) benzalkonium chloride,

0.05 % to 0.38 % (w/v) carbopol,
0.05 % to 0.7% (w/v) xanthan gum,
0.001 % to 0.5 % (w/v) monobasic sodium phosphate,
0.001 % to 1% (w/v) dibasic sodium phosphate and
0.3 % to 2.5 % (w/v) propylene glycol.
wherein the composition has pH of about 6 to 7 and stable over time at room
temperature, and compatible to the low-density polyethylene (LDPE) packaging
material.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
1 % (w/v) miconazole,
0.025 % (w/v) benzalkonium chloride,
0.125% (w/v) carbopol,
0.375% (w/v) xanthan gum,
0.1 % (w/v) monobasic sodium phosphate,
0.05 % to ]% (w/v) dibasic sodium phosphate and
1.92 % (w/v) propylene glycol.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
1 % (w/v) miconazole,
0.025 % (w/v) benzalkonium chloride,
0.125% (w/v) carbopol,
0.375% (w/v) xanthan gum,
0.1 % (w/v) monobasic sodium phosphate,
0.05 % to 1% (w/v) dibasic sodium phosphate and
1.92 % (w/v) propylene glycol.
wherein carbopol and xanthan gum are in the ratio of 1:3 and the composition has pH
of about 6 to 7.

According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
1 % (w/v) miconazole,
0.025 % (w/v) benzalkonium chloride,
0.250 % (w/v) sodium alginate,
0.750 % (w/v) carrageenan,
0.1 % (w/v) monobasic sodium phosphate,
0.05 % to 1% (w/v) dibasic sodium phosphate and
1.92 % (w/v) propylene glycol.
The pharmaceutically acceptable excipients contained in the aqueous ophthalmic composition of the present invention include preservative, viscosity enhancing agents, buffering agents, surfactants, osmotic agent, chelating agents, vehicles and other conventional agents that may be typically used in formulating an ophthalmic composition.
The ophthalmic composition of the present invention may optionally contain a preservative that is conventionally used in ophthalmic compositions such as eye drops. The preservatives that can be used in the aqueous ophthalmic composition include, without limitation, hydrogen peroxide, biquanides, sorbic acid, potassium sorbate, boric acid, borate, chlorohexidine, zinc chloride, propylene glycol, purite, polyquad, benzalkonium chloride, or the like.
Accordingly, the present invention relates to a preservative free aqueous ophthalmic suspension comprising miconazole and pharmaceutically acceptable excipients.
In one embodiment, the present invention relates to a preservative free stable ophthalmic composition comprising miconazole, combination of carbopol and xanthan gum, and pharmaceutically acceptable excipients.

Viscosity enhancing agents that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to hydroxy ethyl cellulose, carrageenan, polycarbophil, polyvinyl alcohol, povidone, sodium alginate, locust bean gum, pullulan, sodium carboxy methyl cellulose, hydroxyl propyl methyl cellulose, gellan gum, methyl cellulose, guar gum, carbomer homopolymer, xanthan gum and combinations thereof.
Buffering agents that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to monobasic sodium phosphate dihydrate, dibasic sodium phosphate dihydrate, acetic acid, hydrochloric acid, sodium carbonate, sodium hydroxide and combinations thereof.
Osmotic agents that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to propylene glycol, sodium chloride, potassium chloride, mannitol, glycerin sorbitol, dextrose and combinations thereof.
According to the present invention, the ophthalmic composition can be formulated as suspension, ointment, gel, emulsion and other dosage forms for topical administration, preferably suspension.
In the context of the present invention, the concentration of miconazole is between 0.5% to 4% (w/v), or any specific value within the said range.
In accordance to the present invention, the pharmaceutical composition contains in weight by volume from about 0.5% to 4 % (w/v) miconazole, from about 0.01% to 5% preservative, from about 0.1 % to 1% viscosity enhancing agent, 0.01 to 2 % buffer and from about 0.3% to 4% osmotic agent.
According to another aspect, the present invention provides a method for the preparation of a stable ophthalmic composition comprising miconazole and pharmaceutically acceptable excipients.

In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic composition, comprising:
a) sterilization of xanthan gum by ethylene oxide;
b) sterilization of carbopol by autoclaving at 121°C for 15 minutes;
c) preparing a homogenous dispersion comprising of carbopol and xanthan gum;
d) preparing a buffer phase comprising of monobasic sodium phosphate and dibasic sodium phosphate;
e) preparing a solution comprising of benzalkonium chloride and propylene glycol.
f) preparing a vehicle by mixing said buffer phase and said solution, and filtration using polyethersulfone filter;
g) sterilizing miconazole by ethylene oxide sterilization;
h) preparing a drug phase by aseptically mixing said ethylene oxide sterilized
miconzaole and said vehicle; i) aseptically mixing said drug phase and said dispersion to prepare a
homogeneous ophthalmic suspension of miconzaole.
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic composition, comprising:
a) preparing a homogenous dispersion comprising of at least one viscosity enhancing agent;
b) sterilization of the said dispersion;
c) preparing a buffer phase comprising of at least one buffer;
d) preparing a solution comprising of at least one preservative and at least one osmotic agent;
e) preparing a vehicle by mixing said buffer phase and said solution and filtration using polyethersulfone filter;
t) sterilizing miconazole by ethylene oxide sterilization;

g) preparing a drug phase by aseptically mixing said sterilized miconazole and
said vehicle; h) aseptically mixing said drug phase and said dispersion to prepare a
homogeneous ophthalmic suspension of miconazole.
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic composition, comprising:
a) preparing a homogenous dispersion comprising of sodium alginate and carrageenan;
b) sterilization of the said dispersion by autoclaving at 121°C for 15 minutes;
c) preparing a buffer phase comprising of monobasic sodium phosphate, dibasic sodium phosphate;
d) preparing a solution comprising of benzalkonium chloride and propylene glycol;
e) preparing a vehicle by mixing said buffer phase and said solution and filtration using polyethersulfone filter;
f) sterilizing miconazole by ethylene oxide sterilization;
g) preparing a drug phase by aseptically mixing said ethylene oxide sterilized miconazole and said vehicle;
h) aseptically mixing said drug phase and said dispersion to prepare a homogeneous ophthalmic suspension of miconazole.
According to another aspect, the present invention provides a method of prevention or treatment of an ocular disease by administering to an affected eye a stable ophthalmic composition comprising miconazole and pharmaceutically acceptable excipients.
In one embodiment, the present invention provides a method of treating fungal keratitis and other fungal infections by administering to an affected eye an aqueous ophthalmic composition comprising miconazole and pharmaceutically acceptable excipients.

In one embodiment, the present invention provides a method of treating fungal keratitis and other fungal infections by administering to an affected eye an aqueous ophthalmic composition comprising miconazole, benzalkonium chloride and pharmaceutically acceptable excipients.
According to another aspect, the present invention provides a stable ophthalmic composition comprising miconazole and pharmaceutically acceptable excipients packaged in glass containers, or polypropylene containers, or polyethylene containers.
In one embodiment, the present invention provides a stable ophthalmic composition comprising miconazole and pharmaceutically acceptable excipients packaged in glass containers.
In one embodiment, the present invention provides a stable ophthalmic composition comprising miconazole and pharmaceutically acceptable excipients packaged in polypropylene containers.
In one embodiment, the present invention provides a stable ophthalmic composition comprising miconazole and pharmaceutically acceptable excipients packaged in polyethylene containers.
Accordingly, the following examples are intended to illustrate the present invention, but are not intended to limit the scope of the invention.
Example 1

Sr.No Ingredients % w/v
1 Miconazole 1
2 Benzalkonium chloride 0.025
3 Carbopol 974 P 0.125
4 Xanthan gum 0.375
5 Monobasic sodium phosphate dihydrate 0.1

6 Dibasic sodium phosphate dihydrate 0.05
7 Propylene glycol 1.92
8 Sodium hydroxide q.s
9 Water for Injection q.s
Manufacturing Process:
a. A homogenous dispersion was prepared by adding Carbopol 974 P in water for
injection.
b. A dispersion was sterilized by autoclaving at 121°C for 15 minutes.
c. A homogeneous dispersion was prepared by adding ethylene oxide sterilized
xanthan gum in water for injection.
d. A final polymer dispersion phase was prepared by adding Carbopol 974 P
dispersion of step b to xanthan gum dispersion of step c)
e. A buffer phase was prepared by adding monobasic sodium phosphate, dibasic
sodium phosphate dihydrate in water for injection;
f. A solution of benzalkonium chloride was prepared by adding to water for
injection; then preservative solution was added to buffer phase of step e).
g. A propylene glycol was added to buffer phase of step f).
h. A vehicle is prepared by filtering buffer phase of step g) through
polyethersulfone filter, i. A miconazole was sterilized by Ethylene oxide. j. Drug phase was prepared by adding miconazole of step i) to buffer phase of
step h) and further homogenization. k. Drug phase of step j) and polymer dispersion phase of step (d) was mixed. 1. Volume make up was done & pH was adjusted to 6-7 using sodium hydroxide.

Example 2

Sr.No Ingredients % w/v
1 Miconazole 1
2 Benzalkonium chloride 0.025
3 Sodium alginate 0.250
4 Carrageenan 0.750
5 Monobasic sodium phosphate dihydrate 0.1
6 Dibasic sodium phosphate dihydrate 0.05
7 Propylene glycol 1.92
8 Sodium hydroxide q.s
9 Water for Injection q.s
Manufacturing Process:
a. A homogenous dispersion was prepared by adding sodium alginate and
carrageenan in water for injection, and autoclaved further at 121 °C for 15
minutes.
b. Buffer phase was prepared by adding monobasic sodium phosphate and dibasic
phosphate in water for injection.
c. A solution was prepared by mixing benzalkonoium chloride and propylene
glycol in water for injection.
d. A vehicle was prepared by mixing step (b) buffer phase and step (c) solution
and filtered using polyethersulfone filter.
e. Drug phase was prepared by aseptically mixing ethylene oxide sterilized
miconazole and vehicle of step (d) and further homogenization.
f. Drug phase of step (e) and homogenous dispersion of step (a) was mixed.
g. Volume make up was done & pH was adjusted to 6-7 using sodium hydroxide.

Sterilization Method for Miconazole;
Based on degradation data miconazole ethylene oxide sterilization was preferred over gamma sterilization for formulation development. Comparative impurity profiles for various sterilization techniques is summarized in table no 5. Table 5:

Impurity Limit Miconazole untreated Miconazole ethylene oxide sterilized Miconazole
gamma
sterilized
Miconazole impurity I NMT 0.15% 0.01 0.01 0.08
Miconazole impurity II NMT 0.10% ND ND ND
Miconazole impurity III NMT 0.10% ND ND ND
Miconazole impurity IV NMT 0.15% 0.01 0.005 0.04
Miconazole impurity V NMT 0.15% ND ND ND
Any other unspecified impurity NMT 0.10% of each 0.01 0.02 0.08
Total impurity NMT 1.0% 0.04 0.06 0.37
NMT: Not more than, ND: Not detected
From above table it is concluded that ethylene oxide sterilization process is better than gamma sterilization process.
Preservative Efficacy Test:
One batch of miconazole suspension was manufactured as per formula 1 at 90% concentration of benzalkonium chloride in order to assess its preservative effectiveness as per USP.

The result of antimicrobial efficacy test (AET) for 90% preservative concentration is tabulated in table 6. Table 6:

Name of microbial culture
Bacteria Log reduction in viable count from initial calculated viable count at '0' hour Log of viable count at 28 days USP
complianc
e

After 7 days (Limit: NLT 1) After 14 days (Limit: NLT 3) Limit: No increase from 14 days

Escherichia coli ATCC 8739 5.62 5.62 No increase Complies
Pseudomonas 5.64 5.64 No increase Complies
aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538 3.80 5.65 No increase Complies
Yeasts and Molds Log of viable count at 7 days Log of viable count at 14 days Log of viable count at 28 days USP
complianc
e
Limit No increase form '0'hr No increase form '0'hr No increase form '0'hr

Candida albicans ATCC 10231 No increase No increase No increase Complies
Aspergillus brasiliensis ATCC 16404 No increase No increase No increase Complies
Preservative efficacy data was well within the USP acceptance criteria for all the specified bacteria and yeasts and fungi. Thus benzalkonium chloride in the formulation acts effectively as a preservative.
Ocular Irritation Studies:
Ocular irritation study was performed as per standard protocol. New Zealand white rabbits (three), each weighing about 2 to 3 kg were used for study. A dose of one drop

of the test formulation was instilled in to right eye of each rabbit. The left eye served as control. The eyes of the rabbits were carefully examined, observed at 1 hr and 24 hours, 48 hours and 72 hours post application and the observations extended to determine the reversibility or irreversibility till the end of the observation period of 7 days. Score methodology was used for evaluation of cornea opacity, iris, conjunctivae redness, chemosis for eye lids and/or nictating membranes.
Pharmacodynamic Studies (In-vivo antifungal efficacy studies):
Antifungal efficacy study was performed in as per protocol number MVC/IAEC/ 10 /2019 at Bombay veterinary college, Mumbai. Wistar rat (six) of both genders, each weighing about 150-250 g was used for study. The animals were housed in individual cages, and the experiments were conducted in a sanitized room at a temperature maintained around 25 °C. Immunosupression in all test groups animals were induced by cyclophosphamide marketed preparation. The optimized dose of the drug used was 8 mg/kg bodyweight for 15 consequent days through oral route. The suppressed animals showed the signs of decrease body weight dullness and other motor responses. Fungal infection was induced by inoculating live culture of Candida albicans species of 10-8 cfu/ml concentration. The initial marginal injury was done on eye lid membrane to hasten the infection. Further inflammation and all markers like mucous membranes, opacity of lens, etc were taken in to consideration before instillation of miconazole ophthalmic formulation. Another group of fungi induced infected animals (six) was kept as positive control. A dose of two drops of the test formulation was instilled in to eyes of each rat twice every day. The eyes of the rats were carefully examined, observed everyday post application and the observations extended till complete recovery of fungal infection had happened. Score methodology was used for evaluation of chemosis, eyelid membranes (hyperaemia), corneal membrane opacity, corneal reflex, blindness or vision impairment. A score of 0 to 5 was used for all physiological observations except for corneal reflex scale of 5-0 were used, which indicates 5 scale is normal reflex action.

Conclusion:
The test formulations were administered in to the infected eye twice a day of animals for 15 consecutive days. 80% animals showed recovery in one week time in test formulations and rest of the animals were treated for complete 15 days for healing the remnants of infections. Improvements in the clinical parameters post instillation suggesting the propensity of the prepared systems to sustain drug release with a minimal loss due to drainage. Gross examination of the ocular tissues showed that the formulations caused no undue irritation and no leakage of the developed polymer based formulation was observed from any part of the eye.
Score data for positive control & test formulation is presented in table number 5. Stastical analysis for positive control and test formulation was done using t test for all physiological parameters and differences were found to be statistically significantly at p<0.05.
Table 7:

Parameters Animal Positive control (infected eye) Miconazole suspensionl product) ophthalmic % (Test
Chemosis 1 5 2

2 4 2

3 5 3

4 5 3

5 2 2

6 5 2
Average ± SD 4.33 ±1.21 2.33 ± 0.52
Eyelid
membranes
(hyperaemia) 1 5 2

2 4 3

3 5 2

4 5 3

5 5 3

6 4 2
Average ± SD 4.67 ± 0.52 2.50 ± 0.55
1 5 3

Corneal
membrane
opacity 2 5 2

3 4 2

4 4 1

5 5 1

6 5 1
Average ± SD 4.67 ± 0.52 1.67 ± 0.82
Corneal reflex 1 2 4

2 1 3

3 2 3

4 1 4

5 2 4

6 2 5
Average ± SD 1.67 ± 0.52 3.83 ± 0.75
Blindness vision
impaired/not
impaired 1 VM NI

2 VM NI

3 VM VM

4 VM NI

5 VM NI

6 VM VM
Accelerated Stability Studies:
Finished product formulation of pH (6.0, 6.5 and 7.0) was filled in low density polyethylene bottles with high density cap closure. Optimum pH formulation was packed in gamma and ethylene oxide sterilized low density polyethylene bottles with high density cap closure to understand the impact on sterilization of container closure system. Samples were kept at stability studies as per internal conference of harmonization (ICH) guideline[8] for semipermeable container at 40 ± 2°C and 25 ± 5% RH and 25 ± 2°C and 40 ± 5% RH for 6 month.
Miconazole Assay and Impact Of pH and Sterilization Of Container Closure System On Assay
Across the pH range 6.0, 6.5 and 7.0 (low, optimum and high) the miconazole content was well within specification limit of 90.0 to 110.0% which indicates formulation remains stable across pH range of 6.0 to 7.0. Gamma sterilization of container closure

has more deleterious effect on stability; miconazole content was beyond the acceptance limit as compared to that of ethylene oxide sterilization (ETO). ETO sterilization is better choice for container closure. Figure 10 shows miconazole assay in stability. Table 8:

Test Limits Initial 6 M 40°C/25%RH

pH 6.0 pH 6.5 pH 7.0 pH 6.0 pH 6.5 pH
7.0
Assay of Miconazole 90.0 to 110.0
% 94.6 95.2 99.7 91.3 94.3 98.3
Assay of BKC 90.0 to
110.0
% 99.7 97.8 106.0 102.7 100.5 108.6
Related subs tances
Single
Unknown
impurity NMT 0.50% BLQ BLQ BLQ BLQ BLQ BLQ
Total impurities NMT 1.0% BLQ BLQ BLQ BLQ BLQ BLQ
Table 9 shows impact of container closure sterilization impact on miconazole ophthalmic suspension Table 9

Test Limits Initial 6 M 40°C/25%RH

ETO Gamma ETO Gamma
Assay of Miconazole 90.0 to 110.0% 95.2 95.8 94.3 85.7
Assay of BKC 90.0 to 110.0% 97.8 96.4 100.5 97.7
Related substances
Single Unknown impurity NMT 0.50% BLQ BLQ BLQ 0.23
Total impurities NMT 1.0% BLQ BLQ BLQ 0.34

We Claim:
1. A stable ophthalmic composition comprising miconazole, viscosity enhancing
agents and pharmaceutically acceptable excipients.
2. The stable ophthalmic composition of claim 1, wherein miconazole is used in the range of 0.5% to 2%.
3. The stable ophthalmic composition of claim 1, wherein the viscosity enhancing agents are carbopol and xanthan gum.
4. The stable ophthalmic composition of claim 3, wherein carbopol and xanthan gum are in the ratio of 1:3.
5. The stable ophthalmic composition of claim 1, wherein the viscosity enhancing agents are sodium alginate and carrageenan gum.
6. The stable ophthalmic composition of claim 1, wherein pharmaceutically acceptable excipients are selected from group comprising of preservatives, buffering agents, osmotic agents, solubilizers and combinations thereof.
7. The stable ophthalmic composition of claim 1, wherein preservative is selected from group comprising of hydrogen peroxide, biquanides, sorbic acid, potassium sorbate, boric acid, borate, chlorohexidine, zinc chloride, propylene glycol, purite, polyquad, benzalkonium chloride and combinations thereof.
8. The stable pharmaceutical composition of claim 1, wherein buffering agent is selected from group comprising of monobasic sodium phosphate dihydrate, dibasic sodium phosphate dihydrate, acetic acid, hydrochloric acid, sodium carbonate, sodium hydroxide and combinations thereof.
7. The stable ophthalmic composition of claim 1, wherein osmotic agent is selected from group comprising of propylene glycol, polyethylene glycol, polypropylene glycol, and combinations thereof.

8. The stable ophthalmic composition of claim I, wherein solubilizers is selected from group comprising of polylobate, macrogol 15 hydroxystearate, tyloxapol, povidone, polyvinyl alcohol, hypromellose, polaxamer, ethanol, dimethyl acetamide, dimethyl sulfoxide, PEG 300 caprylic/capric glycerides, sodium lauryl sulfate, caprylocaproyl polyoxyl 8- glycerides, glyceryl laurate and combinations thereof.
9. The method for the preparation of a stable ophthalmic composition of claim 1,
comprises of following steps:
a. preparing a homogenous dispersion comprising of at least one viscosity enhancing
agent;
b. sterilization of the said dispersion;
c. preparing a buffer phase comprising of at least one buffer;
d. preparing a solution comprising of at least one preservative and at least one
osmotic agent;
e. preparing a vehicle by mixing said buffer phase and said solution and filtration
using polyethersulfone filter;
f. sterilizing miconazole by ethylene oxide sterilization
g. preparing a drug phase by aseptically mixing said sterilized miconazole and said
vehicle;
h. aseptically mixing said drug phase and said dispersion to prepare a homogeneous ophthalmic suspension of miconazole.