FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION Sec 10-Rule 13
A STABLE OPHTHALMIC COMPOSITION OF POSACONAZOLE
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 posaconazole. More specifically, the present invention relates to a posaconazole ophthalmic composition and a method of preparing same, which is chemically stable at room temperature. Moreover, the composition of the present invention is a stable and sterile aqueous ophthalmic composition.
Background of the Invention:
Posaconazole is chemically designated as:
4-[4-[4-[4-[[(3R,5R)-5-(2,4-difluorophenyl)tetrahydro-5-(1H-1,2,4-triazol-1-ylmethyl)-3-furanyl]methoxy]phenyl]-1-piperazinyl]phenyl]-2-[(lS,2S)-1-ethyl-2-hydroxypropyl]-2,4-dihydro-3H-l,2,4-triazol-3-one and has the following structural formula:

Posaconazole is a second generation triazole approved by USFDA in 2006. It results from an improvement in the molecule itraconazole and is primarily indicated for the treatment of invasive fungal infections in onco-hematological patients. It is available as injection, oral suspension and delayed release tablet. Gastrointestinal complaints are the only adverse effects reported to date. It has a broad spectrum activity against Candida species; Aspergillus species and Fusarium species, among others. Posaconazole is effective against most agents resistant to Itraconazole and Fluconazole. Experience of its use in ocular infections is still limited, however, the initial results are encouraging.
McCartney DL, Beatty RF, et.al. Successful treatment of resistant ocular fusariosis with posaconazole, American Journal of Ophthalmology, 2007; page 222-227,

discloses a series of three cases of Fusarium keratitis progressing to endophthalmitis unresponsive to treat with oral and topical voriconazole, a rapid therapeutic response to posaconazole was observed.
Sponso et.al., Ocular and systemic posaconazole treatment of invasive Fusarium solani keratitis and endophthalmitis, British Journal of Ophthalmology, 2002, page 829-830, discloses a case of keratitis by Fusarium solani resistant to Amphotericin B and Natamycin but successfully treated with oral posaconazole 200 mg 4 times along with topical application.
Altun A, Kurna A, et.al., Effectiveness of posaconazole in Recalcitrant Fungal Keratitis Resistant to conventional antifungal drugs, Case Reports in Ophthalmological medicine, volume 14, article ID 701653 discloses success of posaconazole in two cases with recalcitrant fungal keratitis non-responsive to voriconazole, fluconazole, amphotericin B and natamycin. The response to topical and systemic posaconazole was remarkable. The results were achieved by administration of oral suspension in eye. However, the oral pharmaceutical formulation i.e. oral suspension could damage eye as it is not designed for ophthalmic use.
Hitherto, there is no ophthalmic formulation available for topical administration in the eye.
Thus, there exists a need to develop an effective, stable, yet comfortable and safe posaconazole formulations for ophthalmic administration for the treatment of fungal keratitis and other fungal infections.
Such formulation for administration directly to the eye would be advantageous over systemic oral formulations due to faster action and avoidance of the side effects associated with systemic administration.
Instilled ophthalmic formulations may not be completely available for eliciting therapeutic action because of various reasons such as rapid tear drainage, blinking

of eye, lower residence time of ophthalmic formulation in eye and lower cul-de sac volume.
The inventors of the present invention have developed long retentive posaconazole ophthalmic suspension based on the polymer plattorm system of sodium alginate and carrageenan. Specifically, the inventors of the present invention have developed an ophthalmic suspension of posaconazole using combination of carrageenan and sodium alginate, optionally with benzalkonium chloride as preservative, and is stable at room temperature for a period of 6 months.
Object of the Invention:
The main object of the present invention is to provide a long retentive posaconazole ophthalmic suspension based on polymer platform system of sodium alginate and carrageenan.
Another object of the present invention is to provide a stable ophthalmic composition comprising posaconazole, sodium alginate, carrageenan and pharmaceutically acceptable excipients.
Another object of the present invention is to provide a stable ophthalmic composition comprising posaconazole, sodium alginate, carrageenan, benzalkonium chloride and pharmaceutically acceptable excipients.
Yet another object of the present invention is to provide a stable ophthalmic composition comprising posaconazole, combination of sodium alginate and carrageenan, benzalkonium chloride and pharmaceutically acceptable excipients.
Further object of the present invention is to provide a stable ophthalmic composition comprising posaconazole, combination of sodium alginate and carrageenan, 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 posaconazole that is stable at room temperature.
Further object of the present invention is to provide method for preparation of a stable ophthalmic composition comprising posaconazole.
Yet further object of the present invention is to provide stable ophthalmic suspension comprising posaconazole, 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 posaconazole for the treatment of fungal keratitis and other fungal infections.
Summary of the Invention:
The present invention relates to a posaconazole ophthalmic composition and a
method of preparing same, which is chemically stable at room temperature. The
composition of the present invention is a stable and sterile aqueous ophthalmic
composition.
Specifically, the present invention relates to an ophthalmic suspension comprising
posaconazole, combination of carrageenan and sodium alginate as viscosity
enhancing agents, with benzalkonium chloride as preservative.
Detailed Description:
The present invention relates to a stable ophthalmic composition comprising
posaconazole 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 posaconazole, viscosity enhancing agents and pharmaceutical^ 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 sutticient amount ot 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. Hence the impact of all sterilization process & pH or buffers data becomes useful for further formulation development activity.
Following polymers were studied at 1.0% concentration. Table 1: Viscosity of single polymer (1.0%).

Sr. No. Polymer Viscosity (cps)
1. Hydroxy ethyl cellulose (Natrosol 250 G pharm) 20.3
2 Carrageenan (Gelcarin PH-812) 697.4
3 Polycarbophil 4439
4 Polyvinyl alcohol 26-88 2.31

5 Povidone K30 1.8
6 Sodium alginate (Manukol LKX) 107.7
7 Sodium alginate (Protanal LFR 5/60) 4.03
8 Locust bean gum (Viscogum) 184.2
9 Pullulan 2.12
1.0% aqueous solution was prepared by dissolving polymers, each separately, in
purified water under stirring. Viscosity of these aqueous solutions was measured by
using appropriate spindle and rpm. Polymers of more than 100 cps viscosity i.e.
carrageenan, sodium alginate (Manukol LKX), polycarbophil, locust bean gum
were selected for further interaction study.
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 4 polymer mixture.

Run Polycarbophil Sodium alginate (Manukol LKX) Locust bean
gum
(Viscogum) Carrageenan (Gelacrin PH-812) Viscosity
1 0.000 0.500 0.000 0.500 6903
0 1.000 0.000 0.000 0.000 1587
3 0.000 0.000 1.000 0.000 9.02
4 0.000 0.500 0.500 0.000 30.1
5 0.250 0.250 0.250 0.250 1500
6 0.125 0.125 0.625 0.125 623.9
7 0.000 0.000 0.500 0.500 418.9
8 0.125 0.125 0.125 0.625 16834
9 0.000 0.500 0.000 0.500 8408
10 0.333 0.000 0.333 0.333 2754

11 0.500 0.500 0.000 0.000 405.9
12 0.500 0.000 0.000 0.500 623.9
13 0.625 0.125 0.125 0.125 200.4
14 0.000 1.000 0.000 0.000 77.7
15 0.000 0.500 0.500 0.000 88.6
16 0.000 0.000 0.000 1.000 925.8
17 0.500 0.000 0.500 0.000 483.5
18 0.250 0.250 0.250 0.250 346.4
19 0.125 0.625 0.125 0.125 129.9
Optimal mixture design with quadratic model was used to study interaction. Total
19 experimental runs were conducted with center point and 2 replicate. Total
concentration for mixture was kept 1.0%
Aqueous solutions were prepared by dissolving polymer in purified water under
stirring. Viscosity of these aqueous solutions were measured by using appropriate
spindle and rpm.
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 a statistically non-significant lack of fit at a > 0.05 .
Table No.3 Statistical analysis of experimental design.

Response Model p value Adequate precision Lack of fit test p value
Viscosity (cps) 0.0109 10.277 0.0600
Table no.3 suggest that model is significant. The p value for lack of fit was not significant, indicating that this model equation fitted the data well. Adequate precision of 10.277 indicates adequate model discrimination. Therefore quadratic model could adequately describe the data and could be employed to arrive at synergy.

A positive sign represents a synergistic effect, while a negative sign indicates antagonistic effect. The negative coefficient of A and D in the model refers to decrease in viscosity, for polycarbophil and carrageenan combination. Highest positive coefficient of 4.54, between B and D showed the highest viscosity which in turn means maximum synergy between sodium alginate and carrageenan. +3.21x A + 1.78 x B + 1.10 x C + 2.98 x D + 0.16 x AB + 2.34 x AC - 1.24 x AD + 1.98 x BC + 4.54 x BD + 2.56 x CD, where A, B, C and D are concentration of polycarbophil, sodium alginate, locust bean gum and carrageenan respectively. From the viscosity data it was concluded that four polymer combination system and sodium alginate and carrageenan (0.500: 0.500) system gave the best synergy. The viscosity of synergistic combination even at lower concentration (0.500: 0.500) was higher than the viscosity of individual polymer at higher concentration (1.00). Further to identify the best ratio of binary polymer combination (sodium alginate & carrageenan), again mixture design was applied. Table No. 4: Experimental run along with viscosity data.

Run Sodium alginate (%w/w) Carrageenan (%w/w) Viscosity (cps)
1 0.742 0.258 64
0 0.500 0.500 1377
3 0.000 1.000 1212
4 1.000 0.000 1188
5 1.000 0.000 82.4
6 0.249 0.751 6194
7 1.000 0.000 81.3
8 0.000 1.000 951.8
9 0.500 0.500 1215
Mixture I- optimal design, Quartic model was used for this study. Nine experimental runs were conducted. Total concentration for mixture was kept at 1.0%

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
+ 1.91 x A + 3.08 x B -7.67 x AB (A-B) -3.56 x AB (A-B)^2, where A and B are concentration of sodium alginate and carrageenan respectively.
[able no.5 suggest that model is significant. Adequate precision ratio of 107.83 indicates adequate model discrimination. Therefore quartic model could adequately describe the data and could be employed to arrive at synergy. A positive sign represents a synergistic effect, while a negative sign indicates antagonistic effect. Maximum synergy observed in 0.25 to 0,750 ratio of sodium alginate to carrageenan respectively.
It was concluded that sodium alginate and carrageenan in ratio of 0.250: 0.750 (1:3) showed the best synergy.
According to preferred embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole, sodium alginate, carrageenan and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole, combination of sodium alginate and carrageenan, benzalkonium chloride and pharmaceutical^ acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole, benzalkonium chloride and pharmaceutical^ acceptable excipients.

According to one embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole, combination of sodium alginate and carrageenan, benzalkonium chloride and pharmaceutically acceptable excipients, wherein the composition is sterile.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole, combination of sodium alginate and carrageenan, benzalkonium chloride, monobasic sodium phosphate, dibasic sodium phosphate, propylene glycol, polysorbate, 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 posaconazole by using a combination of viscosity enhancing agent such as sodium alginate and carrageenan in the ratio of 1:3 respectively.
According to another embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole, combination of carbopol and xanthan gum, benzalkonium chloride and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole, combination of carbopol and xanthan gum, benzalkonium chloride, monobasic sodium phosphate, dibasic sodium phosphate, propylene glycol, polysorbate, sodium hydroxide and purified water.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
1 % to 7 % (w/v) posaconazole,
0.01 % to 0.5 % (w/v) benzalkonium chloride,
0.1 % to 0.75 % (w/v) sodium alginate,
0.1 % to 1.3 % (w/v) carrageenan,
0.001 % to 0.5 % (w/v) monobasic sodium phosphate,
0.001 % to 1 % (w/v) dibasic sodium phosphate,

0.3 % to 2 % (w/v) propylene glycol, and, 0.01 % to 0.2 % polysorbate.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
1% to 7 % (w/v) posaconazole,
0.01 % to 0.5 % (w/v) benzalkonium chloride,
0.1 % to 0.75 % (w/v) sodium alginate,
0.1 % to 1.3 % (w/v) carrageenan,
0.001 % to 0.5 % (w/v) monobasic sodium phosphate,
0.001 % to 1% (w/v) dibasic sodium phosphate,
0.3 % to 2 % (w/v) propylene glycol, and,
0.01 % to 0.2 % polysorbate,
wherein the composition has pH of about 6 to 7.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
1 % to 7 % (w/v) posaconazole,
0.01 % to 0.5 % (w/v) benzalkonium chloride,
0.1 % to 0.75 % (w/v) sodium alginate,
0.1 % to 1.3 % (w/v) carrageenan,
0.001 % to 0.5 % (w/v) monobasic sodium phosphate,
0.001 % to 1 % (w/v) dibasic sodium phosphate,
0.3 % to 2 % (w/v) propylene glycol, and,
0.01 % to 0.2 % polysorbate,
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:

4 % (w/v) posaconazole,
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 %(w/v) dibasic sodium phosphate,
1.50 % (w/v) propylene glycol, and,
0.1 % (w/v) polysorbate.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
4 % (w/v) posaconazole,
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 % (w/v) dibasic sodium phosphate,
1.50 % (w/v) propylene glycol, and,
0.1 % (w/v) polysorbate.
wherein sodium alginate and carrageenan 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:
4 % (w/v) posaconazole,
0.025 % (w/v) benzalkonium chloride,
0.125% (w/v) carbopol,
0.750 % ( w/v) xanthan gum,
0.1 % (w/v) monobasic sodium phosphate,
0.05 % (w/v) dibasic sodium phosphate,
1.60 % (w/v) propylene glycol, and,
0.1 % (w/v) polysorbate.

The pharmaceutically acceptable excipients contained in the aqueous ophthalmic composition of the present invention include preservative, viscosity enhancing agents, buffering agents, surfactants, osmotic agent, solubilisers, 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 posaconazole and pharmaceutically acceptable excipients. In one embodiment, the present invention relates to a preservative free stable ophthalmic composition comprising posaconazole, combination of sodium alginate and carrageenan, 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, 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
Solubilisers that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to polysorbate 80, Poloxamer 407, Propylene glycol, cyclodextrin 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 posaconazole is between 1 % to 7 % (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 1 % to 7 % posaconazole, from about 0.01% to 5% preservative, from about 0.1 % to 2% viscosity enhancing agent, 0.01 to 2 % buffer, from about 0.3% to 4% osmotic agent, and from about 0.01 % to 2 % solubiliser.
According to another aspect, the present invention provides a method for the preparation of a stable ophthalmic composition comprising posaconazole and pharmaceutically acceptable excipients.
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, propylene glycol and polysorbate;

e) preparing a vehicle by mixing said buffer phase and said solution and filtration using polyethersulfone filter;
f) sterilizing posaconazole by dry heat sterilization;
g) preparing a drug phase by aseptically mixing said dry heat sterilized posaconazole and said vehicle;
h) aseptically mixing said drug phase and said dispersion to prepare a homogeneous ophthalmic suspension of posaconazole.
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 by autoclaving at 121°C for 15 minutes;
c) preparing a buffer phase comprising of at least one buffer;
d) preparing a solution comprising of at least one preservative, at least one
osmotic agent, and at least one solubiliser;
e) preparing a vehicle by mixing said buffer phase and said solution and filtration using polyethersulfone filter;
f) sterilizing posaconazole by dry heat sterilization;
g) preparing a drug phase by aseptically mixing said dry heat sterilized posaconazaole and said vehicle;
h) aseptically mixing said drug phase and said dispersion to prepare a homogeneous ophthalmic suspension of posaconazole.
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, propylene glycol and polysorbate;
e) preparing a vehicle by mixing said buffer phase and said solution and filtration using polyethersulfone filter;
f) sterilization of posaconazole by dry heat sterilization;
g) preparing a drug phase by aseptically mixing said dry heat sterilized posaconazole and said vehicle;
h) aseptically mixing said drug phase and said dispersion to prepare a homogeneous ophthalmic suspension of posaconazole.
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, dibasic sodium phosphate;
e) preparing a solution comprising of benzalkonium chloride, propylene glycol and poysorbate;
f) preparing a vehicle by mixing said buffer phase and said solution, and filtration using polyethersulfone filter;
g) sterilizing posaconazole by dry heat sterilization;
h) preparing a drug phase by aseptically mixing said dry heat sterilized
posaconazole and said vehicle; i) aseptically mixing said drug phase and said dispersion to prepare a
homogeneous ophthalmic suspension of posaconazole.
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 posaconazole and pharmaceutically acceptable
excipients.
In ono 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 posaconazole 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 posaconazole, benzalkonium chloride and pharmaceutically acceptable excipients.
According to another aspect, the present invention provides a stable ophthalmic composition comprising posaconazole 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 posaconazole and pharmaceutically acceptable excipients packaged in glass containers.
In one embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole and pharmaceutically acceptable excipients packaged in polypropylene containers.
In one embodiment, the present invention provides a stable ophthalmic composition comprising posaconazole 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 Posaconazole 4
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 1.50
8 Polysorbate 80 0.1
9 Sodium hydroxide q.s
10 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.
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, polysorbate 80
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 dry heat sterilized
Posaconazole and vehicle of step (d).
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.

Example 2

Sr.No Ingredients % w/v
1 2 Posaconazole 4

Benzalkonium chloride 0.025
3 Carbopol 974 P 0.125
4 Xanthan gum 0.750
5 Monobasic sodium phosphate dihydrate 0.1
6 Dibasic sodium phosphate dihydrate 0.05
7 Propylene glycol 1.60
8 Polysorbate 80 0.1
9 Sodium hydroxide q.s
10 Water for Injection q.s
Manufacturing Process:
a. Xanthan gum was sterilized by ethylene oxide.
b. Carbopol was sterilized by autoclaving at 121°C for 15 minutes;
c. A homogenous dispersion was prepared by adding carbopol and xanthan
gum in water for injection.
d. Buffer phase was prepared by adding monobasic sodium phosphate and
dibasic phosphate in water for injection.
e. A solution was prepared by mixing benzalkonoium chloride, polysorbate 80
and propylene glycol in water for injection.
f. A vehicle was prepared by mixing step (b) buffer phase and step (c) solution
and filtered using polyethersulfone filter.
g. Drug phase was prepared by aseptically mixing dry heat sterilized
Posaconazole and vehicle of step (d).
h. Drug phase of step (e) and homogenous dispersion of step (a) was mixed. i. Volume make up was done & pH was adjusted to 6-7 using sodium hydroxide.

Sterilization Method for Posaconazole:
For sterilization of posaconazole degradation product was found to be higher in case of gamma sterilization. Impurities were found to be similar for steam, dry heat & ethylene oxide sterilization. Steam sterilization resulted in hard cake formation due to high solid content. Hence dry heat sterilization was selected for further formulation development. Comparative impurities profile for various sterilization techniques is summarized in table no 6. Table 6:

Impurity Limits Initial ETO DHS Gamma Steam
(%) befor sterili 160°C/ sterilizat sterilizatio
e
sterili
zation zation 2 hrs ion (2.5 KG) n 121°C for 15 minutes
Tosylated NMT 0.12 0.12 0.12 0.12 0.07
compound 0.12
Hydroxyl NMT ND ND ND ND ND
Triazole 0.12
Deshydroxy NMT 0.05 0.05 0.05 0.05 0.03
posaconazole 0.12
Bezylated NMT 0.01 0.01 0.01 0.01 0.01
posaconazole 0.12
Single max. NMT 0.03 0.03 0.03 0.19 0.02
unspecified 0.10 of
impurity each
Total 1.0 0.32 0.35 0.33 0.67 0.2
impurities
NMT : Not more than ND: Not detected

One batch of Posaconazole suspension was manufactured as per formula in
Example 1 at 90% concentration of benzalkonium chloride in order to assess its
preservative effectiveness as per USP.
The results of Antimicrobial efficacy test (AET) for 90% preservative concentration
is tabulated in table 7.
Table 7:

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

After 7 After 14 Limit: No

days days increase from
(Limit: (Limit: 14 days
Bacteria NLT 1) NLT 3)
Escherichia 4.02 5.64 No increase Complies
coli ATCC
8739
Pseudomon 2.98 4.87 No increase Complies
as
aeruginosa
ATCC 9027
Staphylococ 3.05 6.12 No increase Complies
cus aureus
ATCC 6538
Yeasts and Log of Log of Log of viable
Molds viable viable count count at 28 days USP
count at 7 days at 14 days compliance

Limit No No increase No increase
increas e form '0'hr form '0'hr form '0'hr
Candida No No increase No increase Complies
albicans increas
ATCC e
10231
Aspergillus No No increase No increase Complies
brasiliensis increas
ATCC e
16404
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 hour 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 standard protocol. 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 posaconazole ophthalmic formulation. Another group of fungi induced infected animals (six) was kept as positive control. A dose of two drops of the test formulation (examplel) was instilled into eyes of each rat twice a 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
8.
Statistical 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 8: Score study data

Parameters Chemosis Eyelid membra
nes (hypera
emia) Corneal
membrane
opacity Corneal reflex Blindness
vison impaired/
not impaired
Animal number

Positive control 1 5 5 5 2 VM

2 4 4 5 1 VM

3 5 5 4 2 VM

4 5 5 4 1 VM

5 2 5 5 2 VM

6 5 4 5 2 VM
Average 4.33 4.67 4.67 1.67 .
Test
formulati
on 1 1 3 1 4 VM

2 2 2 2 3 NI

3 1 1 2 5 NI

4 2 2 1 4 NI

5 2 1 2 5 NI

6 1 1 1 5 VM
Average 1.50 1.67 1.50 4.33
p value -
VM : vision impaired NI: Vision not impaired

Accelerated stability studies
Finished product formulation of different pH (6.0, 6.5 and 7.0) was filled in low density polyethylene bottles with high density cap closure. Optimum pH (6.5) 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/25 ± 5 % RH for 6 months.
Posaconazole assay and impact of pH & sterilization of container closure system on assay:
Across the pH range 6.0, 6.5 and 7.0 (low, optimum and high) the posaconazole 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 and ethylene oxide sterilization (ETO) of container closure did not show much difference in degradation product on stability. Table 9 shows pH impact on posaconazole suspension in stability.
Table 9 : Impact of pH on Posaconazole ophthalmic suspension

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
Posacon
azole 90.0-110.0% 93.8 100.0 101.2 93.3 101.1 98.7
Assay of BKC 90.0-110.0% 99.7 100.9 99.6 97.8 98.8 97.5
Related substance s
Single Unkno wn impurit
y NMT 0.20% 0.05 0.06 0.05 0.06 0.06 0.05

Total NMT 0.17 0.18 0.17 0.21 0.26 0.25
impurit ies 1.0%
Table 10 : Impact of container closure sterilization on Posaconazole ophthalmic suspension

Test Limits Initial 6 M 40°C/25%RH

Gamma ETO Gamma ETO
Assay of 90.0- 99.8 100.0 98.8 101.1
Posaconazole 110.0%
Assay of 90.0- 101.8 100.9 103.7 98.8
BKC 110.0%
Related substa nces
Single NMT 0.05 0.06 0.05 0.06
Unknown 0.20%
impurity
Total NMT 0.15 0.18 0.22 0.24
impurities 1.0%

We Claim:
1. A stable ophthalmic composition comprising posaconazole, viscosity enhancing agents and pharmaceutically acceptable excipients.
2. The stable ophthalmic composition of claim 1, wherein posaconazole is used in the range of 1% to 7%.
3. The stable ophthalmic composition of claim 1, wherein the viscosity enhancing agents are sodium alginate and carrageenan gum.
4. The stable ophthalmic composition of claim 3, wherein sodium alginate are carrageenan gum are in the ratio of 1:3.
5. The stable ophthalmic composition of claim 1, wherein pharmaceutically acceptable excipients are selected from group comprising of preservatives, buffering agents, osmotic agents, solubilisers and combinations thereof.
6. 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.
7. 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
8. 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.

9. The stable ophthalmic composition of claim 1, wherein solubiliser is selected
from group comprising of polysorbate, 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.
10. 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 by autoclaving at 121°C for 15 minutes;
c) preparing a buffer phase comprising of at least one buffer;
d) preparing a solution comprising of at least one preservative, at least one osmotic agent, and at least one solubiliser;
e) preparing a vehicle by mixing said buffer phase and said solution and filtration using polyethersulfone filter;
f) sterilizing posaconazole by dry heat sterilization;
g) preparing a drug phase by aseptically mixing said dry heat sterilized posaconazole and said vehicle;
h) aseptically mixing said drug phase and said dispersion to prepare a homogeneous ophthalmic suspension of posaconazole.