FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION Sec 10-Rule 13
A STABLE OPHTHALMIC COMPOSITION.
INDOCO REMEDIES LIMITED
Indian
R-92-93, T.T.C Industrial Area, Thane Belapur Road, Rabale MIDC
Navi Mumbai-400701

Technical Field:
The present invention relates to a stable ophthalmic composition. Specifically, the invention relates to a prostaglandin ophthalmic composition and a method of preparing same, which is chemically stable at room temperature, and free of quaternary ammonium salt. More specifically, the present invention relates to a travoprost ophthalmic composition and a method of preparing same, which is chemically stable at room temperature, and free of quaternary ammonium salt. Moreover, the composition of the present invention is a stable and sterile aqueous ophthalmic composition.
Back2round of the Invention:
Travoprost is a prostaglandin analogue having the following formula:

It is fluprostenol isopropyl ester, also known under the chemical name propane-2-yl
(Z)-7-[(lR52R,3R,5S)-3,5-dihydroxy-2-[(lE,3R)-3-hydroxy-4-[3-
(trifluoromethyl)phenoxylbut-l-enyl]cyclopentyl]hept-5-enoate, also known under the
chemical name isopropyl (Z)-7-[(lR,2R,3R,5S)-3,5-dihydroxy-2-[(lE,3R)-3-hydroxy-
4-[(α,α,α-trinuoro-m-tolyI)oxy]-l-butenyl]cyclopentyl]-5-heptenoate.
The formula of travoprost is C26H35 F3O6and it has a molecular weight of 500.55.
Travoprost is widely used for reducing intraocular pressure (IOP) in patients affected with glaucoma and ocular hypertension. It exerts its ocular hypotensive effect through the prostaglandin FP receptors, located in the ciliary muscle and the trabecular meshwork. Alcon Laboratories Inc. markets Travoprost ophthalmic solution 0.004% in several countries across the globe. However, Alcon's formulation is not the same in all

countries. In USA and Canada, the formulation contains sofzia preservative system and is marketed under the brand name Travatan Z®. In Europe and Australia, the preservative is polyquaternuim-1 and in South Africa, the preservative is Benzalkonium Chloride.
US Patent 8323630 titled 'Self- preserved aqueous pharmaceutical composition' claims sofzia preservative system. The '630 discloses that Zinc present in the sofzia preservative system interacts with 12- hydroxyl stearic acid and tends to form precipitate on storage. Thus the shelf life granted to Alcon for this formulation during initial review was only 14 months.
One of the prostaglandin derivatives, latanoprost (Xalatan®) which is approved by USFDA was developed by Pfizer. It is supplied as sterile, isotonic, buffered aqueous solution with a pH of 6.7 and an osmolality of approximately 267mOsmol/kg. Each ml of Xalatan® contains 50µg of latanoprost. Benzalkonium chloride 0.02% is added as preservative. It also contains Sodium chloride, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate and water for injection .The formulation must be kept away from light and unopened bottles are stored under refrigeration at approximately 5 C. Bottle may be kept at 40 C for less than 8 days during shipment. Once a bottle is opened for use, it may be stored at room temperature up to 25 °C for 6 weeks.
The prime disadvantage of travoprost is its low water solubility. Thus a solubilizing step has to be incorporated in the manufacturing process to improve the solubility or by increasing the concentration of the preservative used for example benzalkonium chloride, however, it has adverse reactions due to use of higher concentration.
Preservatives are an important component of ophthalmic preparations, providing antimicrobial activity in the bottle and preventing microbial spoilage of the product and / or decomposition of active drug. The most common preservative used in ophthalmic

compositions is benzalkonium chloride (BAC). It is cationic-surfactant having anti-bacterial properties. Benzalkonium chloride has been found to increase the corneal penetration of a number of drugs by emulsification and disruption of cell junctions in the corneal epithelium. Benzalkonium chloride works by adhering to the cell membrane of microorganism and increases their permeability which leads to cell lysis. It has been recognized that mammalian cells are unable to neutralize BAC and corneal epithelium is damaged by its entrance. This induces cytotoxic damage to conjuctivial cells and stroma.
Monocots by spectrum Thea laboratories was the first latanoprost formulation to be marketed without preservatives. Clear sterile ophthalmic drops of 0.05ml / day contain 50µg of latanoprost per ml of eye drop solution which is 0.005%. They are provided in polyethylene containers of 0.2ml to avoid contamination. However, the above mentioned formulation is single dose.
The prostaglandin derivatives are known to be unstable either because of absorption or adsorption or degradation, when store in polyethylene containers. US Patent No. 6235781 discloses that aqueous prostaglandin compositions packaged in polypropylene containers are more stable than those packaged in polyethylene containers. The '781 further teaches that the stability of prostaglandin formulations is affected by polyethylene containers as compared to polypropylene containers at different stability conditions.
Polypropylene is known to be stronger, stiffer, and more high temperature resistant than low density polyethylene. However, polypropylene has a poor resistance to oxidizing agents such as oxygen and acids, which can lead to fissures and yellowing of the plastic. Also polypropylene does not provide superior flexibility and processability as compared to polyethylene and hence it is not a first choice of material of containers for sterile compositions. Also polypropylene is not a cost effective option as compared to polyethylene.

Further PCT application No. WO2002022106 discloses that unless refrigerated (2-8°C), lipid soluble prostaglandin derivatives and analogues show unacceptable stability in standard low density polyethylene containers. The requirement that the ophthalmic preparation should be refrigerated greatly reduces the availability of the treatment to those in under developed countries. Furthermore, even wherever available, refrigeration of the preparation increases the cost of the treatment to the patient, and thus, further reduces its availability to those in need.
The need of the hour is to develop a travoprost ophthalmic solution without using a quaternary ammonium preservative, which should be chemically stable over time at room temperature, for a period of between 18 and 24 months and compatible to the packaging in which it is stored, i.e. low-density polyethylene (LDPE) material.
Surprisingly, the inventors of the present invention have found that combination of Poysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15) as solubilisers along with potassium sorbate as preservative solved the problem of adsorption of travoprost to the polyethylene containers, and is stable at room temperature for a period between 18 to 24 months.
Object of the Invention:
The main object of the present invention is to provide a stable ophthalmic composition comprising travoprost, using low density polyethylene (LDPE) bottles and without using a quaternary ammonium preservative.
Another object of the present invention is to provide a stable ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients.
Another object of the present invention is to provide a stable ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients, wherein the composition is free of quaternary ammonium preservative.

Further object of the present invention is to provide a stable ophthalmic composition comprising travoprost, potassium sorbate, polysorbate 80, Macrogol 15 hydroxystearate (Solutol HS 15) and pharmaceutically acceptable excipients, wherein the composition is free of quaternary ammonium preservative.
Yet another object of the present invention is to provide a stable ophthalmic composition comprising travoprost, potassium sorbate, combination of polysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15), and pharmaceutical^ acceptable excipients, wherein the composition is free of quaternary ammonium preservative.
Yet another object of the present invention is to provide a stable ophthalmic composition comprising travoprost, potassium sorbate, combination of polysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15), and pharmaceutically acceptable excipients, using low density polyethylene (LDPE) containers, wherein the composition is free of quaternary ammonium preservative.
Another object of the present invention is to provide a stable ophthalmic composition comprising travoprost that shows no sorption to the low density polyethylene containers (LDPE).
Yet another object of the present invention is to provide a stable ophthalmic solution comprising travoprost that is stable at room temperature.
Further object of the present invention is to provide method for preparation of a stable ophthalmic composition comprising travoprost,
Further object of the present invention is to provide a method for preparation of a stable ophthalmic composition comprising travoprost, which do not require any controls of nitrogen flushing during manufacturing or filling and showed no detectable level of 15-keto impurity on stability.

Further object of the present invention is to provide a stable ophthalmic composition comprising travoprost for the treatment of elevated intraocular pressure.
Summary of the Invention:
The present invention relates to a travoprost ophthalmic composition and a method of preparing same, which is chemically stable at room temperature, compatible to the low-density polyethylene (LDPE) packaging material, and free of quaternary ammonium salt. The composition of the present invention is a stable and sterile aqueous ophthalmic composition.
The present invention also relates to a preservative free aqueous ophthalmic composition comprising travoprost and pharmaceutically acceptable excipients.
Detailed Description:
The present invention relates to a stable ophthalmic composition comprising travoprost and a method of preparing same, which is chemically stable at room temperature, and free of quaternary ammonium preservative.
Thus, in one aspect, the present invention provides a stable ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients, wherein the composition is free of quaternary ammonium preservative.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising travoprost, potassium sorbate, combination of polysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15) and pharmaceutically acceptable excipients.

According to one embodiment, the present invention provides a stable ophthalmic composition comprising travoprost, potassium sorbate, combination of polysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15) and pharmaceutically acceptable excipients, wherein the composition is free of quaternary ammonium preservative,
According to one embodiment, the present invention provides a stable ophthalmic composition comprising travoprost, potassium sorbate, combination of polysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15) and pharmaceutically acceptable excipients, wherein the composition is sterile and free of quaternary ammonium preservative.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising travoprost, potassium sorbate, combination of polysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15), propylene glycol, and sodium chloride, hydrochloric acid and purified water.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising travoprost, potassium sorbate, combination of polysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15), propylene glycol, and sodium chloride, hydrochloric acid and purified water, wherein the composition is sterile and free of quaternary ammonium preservative.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising;
0.002% to 0.15% (w/v) travoprost,
0.4 % to 0.5 % (w/v) potassium sorbate,
0.05 % to 0.15 % (w/v) polysorbate 80
0.05 % to 0.15 % (w/v) Macrogol 15 hydroxystearate,
0.7 % to 0.8 % (w/v) propylene glycol, and,
0.3 % to 0.4 % (w/v) sodium chloride.

According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
0.002% to 0.15% (w/v) travoprost,
0.4 % to 0.5 % (w/v) potassium sorbate,
0.05 % to 0.15 % (w/v) polysorbate 80
0.05 % to 0.15 % (w/v) Macrogol 15 hydroxystearate,
0.7 % to 0.8 % (w/v) propylene glycol, and,
0.3 % to 0.4 % (w/v) sodium chloride,
wherein the composition has pH of about 5.7.
According to one embodiment, the present invention provides a stable ophthalmic composition comprising: 0.002% to 0.15% (w/v) travoprost, 0.4 % to 0.5 % (w/v) potassium sorbate, 0.05 % to 0.15 % (w/v) polysorbate 80 0.05 % to 0.15 % (w/v) Macrogol 15 hydroxystearate, 0.7 % to 0.8 % (w/v) propylene glycol, and, 0.3 % to 0.4 % (w/v) sodium chloride,
wherein the composition has pH of about 5.4 to 6 and stable over time at room temperature, for a period of between 18 and 24 months and compatible to the low-density polyethylene (LDPE) packaging material.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
0.004% (w/v) travoprost,
0.47% (w/v) potassium sorbate,
0.1% (w/v) polysorbate 80,
0.1% (w/v) Macrogol 15 hydroxystearate,
0.75% (w/v) propylene glycol,

0.35% (w/v) sodium chloride,
hydrochloric acid,
and purified water, wherein the composition has pH of about 5.7.
According to one embodiment, the present invention provides a stable ophthalmic
composition comprising:
0.003% (w/v) travoprost,
0.47% (w/v) potassium sorbate,
0.1%(w/v)polysorbate80,
0.1% (w/v) Macrogol 15 hydroxystearate,
0.75% (w/v) propylene glycol,
0.35% (w/v) sodium chloride,
hydrochloric acid, and purified water.
The pharmaceutically acceptable excipients contained in the aqueous ophthalmic composition of the present invention include tonicity agents, buffering agents, chelating agents, surfactants, solubilisers, viscosity imparting 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, or the like.
In one aspect, the present invention provides an aqueous ophthalmic composition without using a preservative.

Accordingly, the present invention relates to a preservative free aqueous ophthalmic composition comprising travoprost and pharmaceutically acceptable excipients.
In one embodiment, the present invention relates to a preservative free aqueous ophthalmic composition comprising travoprost, combination of polysorbate 80 and Macrogol 15 hydroxystearate (Solutol HS 15), and pharmaceutically acceptable excipients.
Tonicity agents that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, sodium bromide, mannitol, glycerol, sorbitol, propylene glycol, dextrose sucrose, and combinations thereof. Tonicity agent is used to make the composition isotonic with respect to the ophthalmic fluids present in the human eye.
Solubilisers that can be used in the aqueous ophthalmic composition of the present invention include, but are not limited to, propylene glycol, macrogol 15 hydroxystearate, polyethylene glycol, polypropylene glycol, polysorbate, tyloxapol, povidone, polyvinyl alcohol, hypromellose, polaxamer, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl-15- hydroxystearate, glycerine, ethanol, dimethyl acetamide, dimethyl sulfoxide, PEG 300 caprylic/capric glycerides, PEG 300 oleic glycerides, miglyol, soluplus, sorbitan monooleate, sodium lauryl sulfate, docusate sodium, polyoxyalkyl ethers, polyoxyalkyl phenyl ethers, caprylocaproyl polyoxyl 8- glycerides, Kolliphor, caprylyl capryl glucoside, heptyl glucosides, and lauroyl macrogol- 32 glycerides, macrogol glycerol hydroxystearate and glyceryl laurate and combinations thereof.
Macrogol 15 Hydroxystearate (Solutol HS 15), a non-ionic solubilizer, was developed by BASF, to fulfill an unmet need for a safe and effective excipient for parenteral, ophthalmic and solid oral dose formulations containing poorly soluble active pharmaceutical ingredients (APIs). Chemically, Solutol HS 15 is composed of
11

polyglycol mono- and di-esters of 12-hydroxy-stearic acid and contains approximately 30% polyethylene glycol. Solutol HS 15 is listed as Macrogol 15 hydroxystearate in the European Pharmacopoeia (PhEur). The excipient has been used in approved drugs in some countries, including Canada and Argentina, and also in the Inactive ingredient guide published by US FDA for ophthalmic use.
Macrogol 15 Hydroxystearate is also known as 12-hydroxyoctadecanoic acid polymer with a- hydro-cjo-hydroxypoly(oxy-1, 2-ethanediyl); 12-hydroxystearic acid polyethylene glycol copolymer; Macrogol 15 hydroxystearas; polyethylene glycol-15-hydroxystearate; and polyethylene glycol 660 12-hydroxystearate.
The aqueous ophthalmic composition of the present invention comprise a pharmaceutically acceptable buffering agent selected from the group consisting of acetic acid, hydrochloric acid, sodium carbonate and sodium hydroxide. The buffering agent is used in the composition in an amount so as to produce a pH ranging from about 5.4 to about 6.0.
The ophthalmic composition may optionally contain a suitable water soluble polymers
which includes, but are not limited to of methylcellulose, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl
ethylcellulose, sodium carboxymethylcellulose, polyvinyl alcohol,
polyvinylpyrrolidone, polyethylene glycol, poly(methyl methacrylate), polycarbophil, gelatin, alginate, poly(acrylic acid), polyethylene oxide and chitosan, and combinations thereof.
The ophthalmic composition may optionally contain a suitable chelating agent. The chelating agent generally used is disodium salt of ethylene diamine tetraacetic acid (EDTA).

According to the present invention, the ophthalmic composition can be formulated as solution, suspension, ointment, gel, emulsion and other dosage forms for topical administration, preferably solution.
In the context of the present invention, the concentration of travoprost is between 0.002% to 0.15% (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 0.002 % to 0.15 % travoprost, from about 0.4 % to 0.5 % preservative, from about 0.8% to 1.5 % solubiliser and from about 0.3 % to 0.4 % tonicity agent.
According to another aspect, the present invention provides a method for the preparation of a stable ophthalmic composition comprising travoprost, potassium sorbate 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 vehicle comprising of at least one solubiliser and at least one cosolubiliser.
b) Travoprost is dissolved in vehicle solution of step a).
c) Preparing a solution comprising at least one tonicity agent and at least one preservative agent
d) Mixing of step (b) vehicle and step (c) solution, followed by pH adjustment and making up of volume using purified water.
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic composition, comprising:
a) Preparing a vehicle comprising of at least one solubiliser and at least one cosolubiliser.

b) Travoprost is dissolved in vehicle solution of step a).
c) Preparing a solution comprising at least one tonicity agent and at least one preservative agent.
d) Mixing of step (b) vehicle and step (c) solution, followed by pH adjustment and making up of volume using purified water;
wherein the said process is devoid of nitrogen flushing during manufacturing.
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic composition, comprising:
a) Preparing a vehicle comprising combination of solubilisers and at least one cosolubiliser.
b) Travoprost is dissolved in vehicle solution of step a).
c) Preparing a solution comprising at least one tonicity agent and at least one preservative agent.
d) Mixing of step (b) vehicle and step (c) solution, followed by pH adjustment and making up of volume using purified water.
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic composition, comprising:
a) Preparing a vehicle comprising of polysorbate 80, 15 hydroxystearate (Solutol HS 15) and propylene glycol in water.
b) Travoprost is dissolved in vehicle solution of step a).
c) Preparing a solution comprising sodium chloride and potassium sorbate in water.
d) Mixing of step (b) vehicle and step (c) solution, followed by pH adjustment and making up of volume using purified water.
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic composition, comprising:
14

a) Preparing a vehicle comprising of at least one solubiliser and at least one cosolubiliser.
b) Travoprost is dissolved in vehicle solution of step a).
c) Preparing a solution comprising at least one tonicity agent and at least one preservative agent.
d) Mixing of step (b) vehicle and step (c) solution, followed by pH adjustment and making up of volume using purified water;
wherein the said process is devoid of nitrogen flushing during manufacturing and showed no detectable levels of 15-keto impurity even at accelerated conditions upto 6 months at pH 5.7
In one embodiment, the present invention provides a method for the preparation of a stable ophthalmic composition, comprising:
a) Preparing a vehicle comprising of polysorbate 80, Macrogol 15 hydroxystearate (Solutol HS 15) and propylene glycol in water.
b) Travoprost is dissolved in vehicle solution of step a).
c) Preparing a solution comprising sodium chloride and potassium sorbate in water,
d) Mixing of step (b) vehicle and step (c) solution, followed by pH adjustment and making up of volume using purified water;
wherein the said process is devoid of nitrogen flushing during manufacturing and showed no detectable levels of 15-keto impurity even at accelerated conditions upto 6 months at pH 5.7
15- keto impurity is an oxidative impurity and controlling this impurity at low levels require extensive controls on the oxygen content of the solution during manufacturing as well as nitrogen purging in the bottles during filling operations.

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 travoprost, potassium sorbate and pharmaceutically acceptable excipients.
In one embodiment, the present invention provides a method of treating elevated intraocular pressure by administering to an affected eye an aqueous ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients.
In one embodiment, the present invention provides a method of treating elevated intraocular pressure in patients with open angle glaucoma by administering to an affected eye an aqueous ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients.
In one embodiment, the present invention provides a method of treating elevated intraocular pressure in patients with ocular hypertension by administering to an affected eye an aqueous ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients.
According to another aspect, the present invention provides a stable ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients packaged in glass containers, or polypropylene containers, or polyethylene containers.
in one embodiment, the present invention provides the aqueous ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients packaged in glass containers, or polypropylene containers, or polyethylene containers with or without oxygen scavenger.

In one embodiment, the present invention provides the aqueous ophthalmic composition comprising travoprost potassium sorbate and pharmaceutically acceptable excipients packaged in glass containers.
In one embodiment, the present invention provides the aqueous ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients packaged in polypropylene containers.
In one embodiment, the present invention provides the aqueous ophthalmic composition comprising travoprost. potassium sorbate 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 mg/ml
1 Travoprost 0.04
2 Potassium Sorbate 4.7
3 Sodium Chloride 3.5
4 Propylene glycol 7.5
5 Polysorbate 80 1.0
6 Solutol HS 15 1.0
7 0.1N HCL q.s
8 Purified water q.s
1. Travoprost was dissolved in a stock solution of propylene glycol, Solutol HS 15 and polysorbate 80 in water.
2. A solution of sodium chloride and potassium sorbate in water was prepared.
3. The solution of step 2 was added to stock solution of step 1 and mixed well.
4. pH was adjusted with 0.1N HC1 between 5.4 and 6.0

5. The solution was aseptically filtered through 0.22 micron polyethersulfone membrane (PES) and filled in low density polyethylene vials (LDPE) fitted with LDPE droppers and high density polyethylene cap (HDPE). Example 2

Sr.No Ingredients mg/ml
1 Travoprost 0.03
2 Potassium Sorbate 4.7
3 Sodium Chloride 3.5
4 Propylene glycol 7.5
5 Polysorbate 80 1.0
6 Solutol HS 15 1.0
7 0.1N HCL q.s
8 Purified water q.s
1. Travoprost was dissolved in a stock solution of propylene glycol, Solutol HS 15 and polysorbate 80 in water.
2. A solution of sodium chloride and potassium sorbate in water was prepared.
3. The solution of step 2 was added to stock solution of step 1 and mixed well.
4. pH was adjusted with 0.1N HC1 between 5.4 and 6.0
5. The solution was aseptically filtered through 0.22 micron polyethersulfone
membrane (PES) and filled in low density polyethylene vials (LDPE) fitted with LDPE
droppers and high density polyethylene cap (HDPE).
Two batches of Travoprost were manufactured as per formula in Example 1 at 100% and 80% concentration of potassium sorbate in order to assess its preservative effectiveness as per USP.
The results of Antimicrobial efficacy test (AET) for 100% and 80% preservative concentration are tabulated in tables 1 and 2 respectively.

Table I: Summary results of Antimicrobial effectiveness at 100% concentration of Potassium Sorbate.

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

After 7 days After 14 days Limit: No
(Limit: NLT 1) (Limit: NLT 3) increase from 14 days
Bacteria
Escherichia coli 4.25 6.53 No increase Complies
ATCC 8739
Pseudomonas 4.27 6.48 No increase Complies
aeruginosa
ATCC 9027
Staphylococcus 4.26 6.52 No increase Complies
aureus ATCC
6538
Yeasts and Logs of viable Log of viable Log of viable USP
Molds count at 7 days count at 14 days count at 28 days compliance
(Limit: No (Limit: No (Limit: No

increase form increase form increase form
'0' hour) '0' hour) '0' hour)
Candida No increase No increase No increase Complies
albicans ATCC
10231
Aspergillus No increase No increase No increase Complies
brasiliensis
ATCC 16404

Table 2: Summary results of Antimicrobial effectiveness at lower concentration of Potassium Sorbate (80% of potassium sorbate in example 1)

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

After 7 days After 14 days Limit: No increase
Bacteria (Limit: NLT 1) (Limit: NLT 3) from 14 days
Escherichia coli 3.98 6.53 No increase Complies
ATCC 8739
Pseudomonas 4.03 6.48 No increase Complies
aeruginosa
ATCC 9027
Staphylococcus 4.00 6.52 No increase Complies
aureus ATCC
6538
Yeasts and Logs of viable Log of viable Log of viable USP
Molds count at 7 days count at 14 days count at 28 days compliance
(Limit: No (Limit: No (Limit: No

increase form increase form increase form
'0' hour) '0' hour) '0' hour)
Candida No increase No increase No increase Complies
albicans ATCC
10231
Aspergillus No increase No increase No increase Complies
brasiliensis
ATCC 16404
Test results of both formulations one containing 100% of potassium sorbate and second one containing 80% of potassium sorbate levels in the formulation were within the USP acceptance criteria for all the specifies bacteria and yeasts and fungi. Thus, potassium sorbate in the formulation acts effectively as a preservative.

Sterilization of Container Closure system:
Before starting formal stability studies on the final formulation, it is important to know the impact of sterilization technique of the container closure system (CCS) on the product stability. The container closure system selected for Travoprost ophthalmic solution 0.004% is white opaque LDPE vials with transparent nozzles and HDPE caps. Sterilization of CCS using two different techniques was done i.e. Ethylene Oxide gaseous sterilization and Gamma irradiation. The finished product was filled in both these containers and charged on stability at accelerated conditions of 40°C/25% RH. Data is tabulated in Table 3.
Table 3: Effect of Sterilization of CCS on related substances of Travoprost ophthalmic solution 0.004% (Example 1)

Test Limits Initial 40°C/25%RH

2M ETO 2M Gamma
Assay of Travoprost 90.0-110.0% 100.5 97.7 95.7
Assay of Potassium Sorbate 90.0-110.0% 100.6 96.3 95.2
Related substances
Travoprost related compound A NMT 1.0% 0.07 0.52 0.55
5,6-trans isomer NMT 5.0% ND ND ND
15-keto derivative NMT 1.0% ND 0.32 0.53
Single maximum unknown NMT 1.0% 0.3 0.57 0.6
Total impurities NMT 5.5% 1.1 2.8 3.5
NM 1 - Not more than; ND - Not detected
Results of 2 months samples showed that product filled in gamma irradiated CCS showed significantly higher percentage of 15-keto derivative than those filled in ETO sterilized ones. This impurity is formed due to oxidation. The higher percentage of 15-keto impurity in Gamma sterilized CCS, could be due to residual free radicals generated

during sterilization of CCS triggering an oxidative reaction. Based on the data, ETO sterilized CCS were chosen for conducting stability studies as per ICH guidelines.
Effect of pH on the finished product stability:
The finished product (Example 1) was manufactured at four different pH to study effect on pH on impurity profile of the finished product, namely pH 5.0, pH 5.4, 5.7 and 6.0. Comparative data of initial time point and after six months at accelerated conditions of 40°C/25%RH is tabulated in Table 4.
Table 4: Effect of pH on related substances of Travoprost ophthalmic solution

Test Limits Initial 6 M 40°C/25%RH

pH
5.0 pH 5.4 pH
5.7 pH
6.0 pH
5.0 pH 5.4 pH
5.7 pH
6.0
Assay of Travoprost 90,0-110.0% 99.1 96.8 100.5 100.7 106.9 95.8 98.7 104.0
Assay of
Potassium
Sorbate 80.0-110.0% 99.8 100.5 100.4 101.6 85.4 87.3 97.7 103.1
Related substance.
Travoprost related compound A NMT 1.0% 0.08 ND ND ND 1.5 0.63 0.36 0.41
5,6-trans isomer NMT 5.0% ND ND ND ND 0.15 0.1 0.16 0.17
15-keto derivative NMT 1.0% ND 0.09 0.10 ND 0.19 ND ND 0.09
Single maximum unknown NMT 1.0% 5.7 BL OQ 0.11 0.15 7.9 0.36 0.54 ND
Total impurities NMT 5.5% 6.2 0.77 0.21 0.41 9.7 1.5 1.4 0.67
ND -Not detected; BLOQ - Below LOQ

The results of related substances at different pH of the formulation from pH 5.0 to 6,0 shows significant increase in the values of total impurities with decrease in pH. At pH 5.0, initial results for total impurities is higher than the limit of 5.5% due to significant increase in unknown impurity. However, there is no drop in assay of Travoprost and mass balance is not achieved. This could be due to different chromophores present in the unknown impurity giving very different response as compared to Travoprost in the chromatographic method. At pH 5.7 and pH 6.0, the results of related substances is satisfactory and well within the acceptance criteria. Potassium Sorbate is known to be most effective below pH 6.0. Hence the pH range selected for Travoprost ophthalmic solution is 5.4 to 6.0, with an optimum pH of 5.7.
Stability Data:
The formulation given in example 1 was filled in 5ml ETO sterilized low density polyethylene (LDPE) bottles fitted with LDPE nozzles and turquoise colored polypropylene caps and charged on accelerated stability conditions of 40° C / 25% RH at 2-8 °C as per ICH guidelines for semi-permeable containers. The brand product (available in the market 'Travatan Z' was also charged at accelerated stability conditions of 40° C / 25% RH. The results are summarized in Table 5.
Table 5: Comparison of stability data of marketed formulation and the formulation mentioned in example 1.

Tests Limits Travatan Z Example 1

Initial 40° C / 25% RH 40° C / 25% RH

3M 6M Initial 3M 6M
% Assay of Travoprost 90.0-110.0 101.1 100.9 104.9 100.5 100.1 98.7
PH 5.4-6.0 5.67 NP 5.83 5.7 5.9 6.0
Osmolality (mOsmols/ Kg) 260-300 278 NP 283 278 284 284

Related Substances
Travoprost related compound A NMT
1.0% ND 0.06 0.15 ND 0.16 0.36
5,6- trans isomer NMT
5.0% 3.2 3.2 2.9 ND 0.16 0.16
15- keto derivative NMT 1.0% 0.11 0.14 0.17 0.10 ND ND
Single
maximum
unknown NMT 1.0% 0.97 3.6 6.4 0.11 0.31 0.54
Total Impurities NMT
5.5% 4.3 7.6 11.3 0.21 1.0 1.4
NP- Not detected; NP- Not performed; NMT: Not more than.
There is no significant difference in assay of Travoprost in the test product (example 1) as well as in the marketed product Travatan Z. Travoprost related compound A is the free acid degradant of Travoprost. It is formed by the hydrolytic cleavage of the isopropyl ester group from the Travoprost molecule. In the marketed product the free acid degradant increases from below detection level to 0.15% at 6 months accelerated conditions. However, in the test product, though the free acid impurity is slightly higher than the brand product and increases from below detection limit to 0.36% at 6 months accelerated conditions, it is still well within the USP acceptance criteria of not more than 1.0%. The 5,6-trans isomer is a degradation product formed by thermal conditions. The values of this impurity for test product is better than the marketed product, though both are within USP limits of 5.0%.
15-keto impurity is an oxidative impurity and controlling this impurity at low levels require extensive controls on the oxygen content of the solution during manufacturing as well as nitrogen purging in the bottles during filling operations. The marketed product Travatan Z contains specific resin, syndiotactic polypropylene to contain

adsorption of API and to control oxidation of Travoprost. The marketed product Travatan Z was assigned a shelf life of only 14 months in the initial approval and required storage at 2-25°C. Surprisingly, the test product (example 1) that was developed, did not require any controls of nitrogen flushing during manufacturing or filling and showed no detectable levels of 15- keto impurity even at accelerated conditions up to 6 months as optimum pH of 5.7

We Claim:
1. A stable ophthalmic composition comprising travoprost, potassium sorbate and pharmaceutically acceptable excipients.
2. The stable pharmaceutical composition of claim 1, wherein travoprost is used in the range of 0.002% to 0.15%.
3. The stable pharmaceutical composition of claim 1, wherein potassium sorbate is used
in the range of 0.4% to 0.5%
4. The stable pharmaceutical composition of claim 1, wherein pharmaceutically
acceptable excipients are selected from group comprising of tonicity agents,
solubilisers, buffering agents and combinations thereof.
5. The stable pharmaceutical composition of claim 4, wherein tonicity agent is selected from group comprising of sodium chloride, potassium chloride, calcium chloride, sodium bromide, mannitol, glycerol and combinations thereof.
6. The stable pharmaceutical composition of claim 4, wherein buffering agent is selected from group comprising of acetic acid, hydrochloric acid, sodium carbonate, sodium hydroxide and combinations thereof.
7. The stable pharmaceutical composition of claim 4, wherein solubiliser is selected
from group comprising of propylene glycol, macrogol 15 hydroxystearate,
polyethylene glycol, polypropylene glycol, polysorbate, tyloxapol, povidone,
polyvinyl alcohol, hypromellose, polaxamer, polyoxyl 35 castor oil, polyoxyl 40
hydrogenated castor oil, polyoxyl-15- hydroxystearate, glycerine, ethanol, dimethyl
acetamide, dimethyl sulfoxide, PEG 300 caprylic/capric glycerides, sodium lauryl
sulfate, caprylocaproyl polyoxyl 8- glycerides, caprylyl capryl glucoside, heptyl

glucosides, and lauroyl macrogol- 32 glycerides, macrogol glycerol hydroxystearate, glyceryl laurate and combinations thereof.
8. The method to prepare stable pharmaceutical composition of claim 1, comprises of following steps:
a) Preparing a vehicle comprising of at least one solubiliser and at least one cosolubiliser.
b) Travoprost is dissolved in vehicle solution of step a).
c) Preparing a solution comprising at least one tonicity agent and at least one preservative agent.
d) Mixing of step (b) vehicle and step (c) solution, followed by pH adjustment and making up of volume using purified water;
wherein the said method is devoid of nitrogen flushing and showed no detectable levels of 15-keto impurity at accelerated conditions upto 6 months at PH5.7