Patent Description:
Hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester has the following formula (I)
<CHM>.

The compound has shown to be effective as intra ocular pressure (IOP)-lowering agent (<NPL>). <CIT>) discloses the use of Hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester for treating glaucoma and ocular hypertension.

<NPL> discloses non-clinical pharmacological studies showing the greater IOP-lowering efficacy of NCX <NUM> than that of equimolar doses of bimatoprost. Both compounds were dissolved in a vehicle containing <NUM>% Tween <NUM>, <NUM>% BAK, <NUM>% glycerine and <NUM>% EDTA.

Topical instillation is the most widely preferred non-invasive route of drug administration for treating elevated intraocular pressure. Most of the conventional dosage forms of the marketed IOP-lowering drugs are eye-drops in the form of aqueous solution, nonetheless the ocular bioavailability of the active principle is very low with topical drop administration. Numerous anatomical and physiological factors limit the ocular absorption of topically applied ophthalmic drugs such as rapid precorneal drug elimination due to solution drainage and systemic absorption from the conjunctival sac and the corneal epithelial barrier. An important target of pharmaceutical formulations is to enable the IOP-lowering drug to penetrate inside the eye after either a unique or multiple topical drops instillations. It is known that the ocular residence time of a drug at the ocular surface is very short after a single drop application. As a result, several formulation approaches are generally used for providing more opportunities to the drug penetrating inside the eye after an application at the ocular surface, and ideally with the lowest dose regimen like once a day, in order to foster patient treatment adherence.

One approach is to increase the ophthalmic solution viscosity. As a result, the solution will have a better adherence to the ocular surface and increasing the ocular residence time of the drug at this side accordingly. As a result, an increase amount of the drug is supposed to penetrate inside the eye. For example, <CIT> discloses a delivery system consisting in a gelling system based on the combination of two gelling agents to obtain a non-viscous polymeric delivery system that ensures the desired level of viscosity and to potentiate the solubility of the active ingredient, prostaglandin.

Another approach is to leverage an ocular penetration enhancing agent. One of the most famous penetration enhancer is the benzalkonium chloride (BAK). In the field of the prostaglandin ophthalmic formulation, this approach has been leveraged by Allergan with their product Lumigan®. A first product generation has been commercialized where the Bimatoprost prostaglandin was formulated at the <NUM>% dose. In this product, the benzalkonium chloride dose was <NUM>%. This benzalkonium chloride dose was mainly used for ensuring the antimicrobial protection of the solution. Some year later, a second generation of the Lumigan® has been commercialized with the same efficacy of the old product generation. This new formulation has a lower concentration of bimatoprost (<NUM>%) but has a <NUM>-fold increase in the amount of benzalkonium chloride (<NUM>%) compared to the original formulation (<NUM>%).

Another technical challenge of the ophthalmic pharmaceutical formulations is to stabilize the active principle. It can be noted that some eye drop formulations require cold storage to preserve their active ingredient level, thereby entailing drawbacks of poor usability; for example, for multi-dose bottles the content of active ingredient may decrease during the time if the preparation is not properly stored at a low temperature. Eye drops for the treatment of glaucoma or ocular hypertension are often prescribed for aged persons who could have difficulties to apply the "cold" requirement for drug storage.

<CIT>) discloses ophthalmic solutions stable at room temperature containing a prostaglandin and Solutol® HS15 (macrogol <NUM>-hydroxystearate) without an antimicrobial preservative.

<CIT> discloses that Solutol® HS15 is able to solubilize some prostaglandins like Latanoprost and confers stability at ambient temperature of the solution in absence of quaternary ammonium agent, like benzalkonium chloride, used there as the usual solubilizer for such a prostaglandin analog. More specifically, it confers prostaglandin analog solution stability to the packaging, in particular to LDPE type plastic packaging of European Pharmacopoeia (EP) quality. Therefore Solutol® HS15 can be used as an alternative to polysorbate <NUM> as solubilizing agent.

The test examples of <CIT> disclose ophthalmic "vehicles" containing Solutol® HS15 (<NUM>%), phosphate buffer, sorbitol and EDTA.

<CIT>) discloses formulations containing macrogol <NUM>-hydroxystearate and a preservative agent. This patent application discloses that the use of macrogol <NUM>-hydroxystearate (Solutol® HS15) as surfactant in place of polyethoxylated surfactants (polysorbate <NUM>/Tween® <NUM>) has several advantages such as solubility enhancement of the API, improved stability of APIs susceptible to degradation by oxidation mechanisms, improved preservative effectiveness of benzalkonium chloride, improved tolerability for ophthalmic use.

<CIT>) discloses that the addition of a stabilizing amount of polyglycol ester of <NUM>-hydroxystearic acid (Solutol® HS15) to an ophthalmic composition comprising one or more prostaglandin derivatives reduces the sorption of the prostaglandin derivatives to the polyethylene containers and that the addition of a small amount of oil further reduces the sorption of prostaglandin derivatives onto the low density polyethylene containers.

) discloses the use of Solutol® HS15 as absorption enhancer to improve the systemic absorption of topically applied therapeutic agents through the mucosal membranes of the nasal cavity, buccal cavity and respiratory tract. However the mechanisms of the adsorption of an active principle applied topically to mucosal membranes are different from the pathway employed by a therapeutic agent applied topically to cross the eye; indeed the therapeutic agent must cross anatomical barriers that are inherent and unique to ocular anatomy (i.e. cornea epithelium, conjunctiva and sclera) and bypass protective mechanisms of the eye (i.e. blinking, tear film turnover and drainage).

Macrogol <NUM> hydroxystearate (Kolliphor® HS15, from BASF, formerly known as Solutol® HS15) is the main excipient of the formulation of the present invention; as reported above, there are several prior art documents that discloses the use of macrogol <NUM> hydroxystearate as excipient for ophthalmic composition.

The term "macrogol <NUM> hydroxystearate" refers to a mixture of mainly monoesters and diesters of <NUM>-hydroxystearic acid and macrogols obtained by the ethoxylation of <NUM>-hydroxystearic acid. Macrogol <NUM> hydroxystearate is also known as <NUM>-hydroxystearic acid polyethylene glycol copolymer, polyethylene glycol-<NUM>-hydroxystearate and polyethylene glycol <NUM><NUM>-hydroxystearate. USP-NF listed this compound as Polyoxyl <NUM> hydroxystearate too.

As reported above, several prior art documents disclose the use of macrogol <NUM> hydroxystearate as excipient for ophthalmic compositions, however, as per today, there is only one eyedrop approved in Europe including macrogol <NUM> hydroxystearate and a prostaglandin analog. This drug has been registered by Rafarm S. A under the name of Provastor® and it contains Travoprost as a prostaglandin analog and BAK as antimicrobial preservative system.

Hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester is a viscous oil having a minimum aqueous solubility of <NUM>/ml therefore aqueous pharmaceutical compositions of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester must contain solubilizing agents that increase the water solubility of the compound and allow achieving therapeutically active concentrations of the compound.

<CIT> discloses a formulation containing hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl] -<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester in a vehicle comprising polysorbate <NUM> (Tween® <NUM>) <NUM>%, benzalkonium chloride <NUM>%, citrate buffer, water and having pH <NUM>.

Polysorbate <NUM> has been extensively used as excipient for ophthalmic compositions. For example, the product Rescula®, sold by Novartis, combines unoprostone with a mixture of benzalkonium chloride and polysorbate <NUM> at <NUM>% by weight of the solution.

The present invention relates to an ophthalmic aqueous composition in the form of solution comprising hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, and macrogol <NUM> hydroxystearate, wherein macrogol <NUM> hydroxystearate is preferably the only solubilizing agent.

The ophthalmic aqueous compositions of the invention provide a higher ocular permeability of the active principle hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester compared to known formulation, so allowing an enhanced ocular absorption of the therapeutic active compound.

The invention also provides ophthalmic aqueous compositions in the form of solutions comprising hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester that are both chemically and physically stable on storage at room temperature.

The present invention provides an ophthalmic aqueous composition in the form of solution comprising <NUM>% to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, from <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate, wherein macrogol <NUM> hydroxystearate is preferably the only solubilizing agent.

Another embodiment is an ophthalmic aqueous composition in the form of solution comprising <NUM>% to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, from <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate, wherein macrogol <NUM> hydroxystearate is preferably the only solubilizing agent.

A preferred embodiment of the present invention provides an ophthalmic aqueous composition in the form of solution comprising from <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, from <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate and a pharmaceutically acceptable aqueous vehicle, wherein macrogol <NUM> hydroxystearate is preferably the only solubilizing agent.

In the field of ophthalmology, and in particular in aqueous ophthalmic compositions, solubilizing agents are compounds which improve the dissolution of a biologically active component with relatively low water solubility.

Preferably the amount of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester in the ophthalmic solution is from <NUM>% w/w to <NUM>% w/w, most preferably is <NUM>% w/w, <NUM>% w/w or <NUM>% w/w.

The ophthalmic aqueous solution may contain benzalkonium chloride (BAK) as antimicrobial preservative agent in an amount from <NUM>% w/w to <NUM>% w/w and an edetate salt (EDTA), such as ethylenediaminetetraacetic acid disodium salt, in an amount from <NUM>% w/w to <NUM>% w/w as antimicrobial preservative aid agent; preferably the amount of benzalkonium chloride (BAK) is from <NUM>% w/w to <NUM>% w/w and the amount of the edetate salt (EDTA) is <NUM>% w/w; most preferably the amount of benzalkonium chloride (BAK) is <NUM>% w/w and the amount of the edetate salt (EDTA) is <NUM>% w/w.

The aqueous ophthalmic composition of the invention further includes a buffer selected from: sodium dihydrogen phosphate, disodium hydrogen phosphate heptahydrate, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate, boric acid and salts thereof, acetates such as sodium acetate and mixtures thereof preferably. Preferably, the buffer of the aqueous ophthalmic composition of the invention is a mixture of sodium phosphate dibasic heptahydrate and boric acid or a mixture of citric acid and sodium phosphate dibasic heptahydrate. The preferred pH for the formulation is <NUM>. This pH has been found as being the optimal pH for enabling both a suitable formulation stability over long term storage conditions and an appropriate ocular tolerance when formulations are delivered on the eye surface.

The pH of the aqueous ophthalmic composition is adjusted preferably in a range of from <NUM> to <NUM>, more preferably at pH <NUM>.

The aqueous ophthalmic composition of the invention may include a pH-adjusting agent selected from hydrochloric acid, citric acid, phosphoric acid, acetic acid, tartaric acid, sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium bicarbonate; preferably the aqueous ophthalmic composition of the invention contains hydrochloric acid and or sodium hydroxide in an amount to adjust the pH in a range from <NUM> to <NUM>; more preferably at pH <NUM>.

The ophthalmic aqueous solution may contain tonicity agents used for adjusting the osmolality of the formulation and targeting required isotonicity. Preferred tonicity agents are sodium chloride, sorbitol, glycerin (or glycerol) and mannitol. Isotonicity is set-up at 300mOsm/kg, but a broader range of <NUM> to <NUM> mOsm/kg is usually acceptable for formulating ophthalmic solutions. Preferably, osmolality of the solution is within the range of <NUM> to <NUM> mOsm/kg.

The ophthalmic aqueous solution of the invention may contain a viscosity-adjusting agent used for improving the contact between the solution and the eye and fostering an improved spreading of the product on the ocular surface. Preferred viscosity-adjusting agents are cellulose derivative polymers like carboxymethyl cellulose or hydroxypropyl methyl cellulose, hyaluronic acid, polyvinyl alcohol, carboxylic acid polymers like carbomers or polycarbophils. Most preferably, the viscosity-adjusting agent is hydroxypropyl methyl cellulose at a concentration less than <NUM>% w/w. The viscosity of the ophthalmic aqueous solution is adjusted between <NUM> and <NUM>. s for Newtonian solutions when using cellulose derivatives, but can be higher with high viscosity solution with non-Newtonian / pseudoplastic rheological behaviors, typically obtained when using carboxylic polymers.

Another embodiment of the invention provides an ophthalmic aqueous composition in the form of solution comprising <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w to <NUM>% w/w benzalkonium chloride, <NUM>% w/w to <NUM>% w/w ethylenediaminetetraacetic acid disodium salt dihydrate, sodium phosphate dibasic heptahydrate and boric acid and water, wherein the pH of the ophthalmic solution is <NUM> and wherein macrogol <NUM> hydroxystearate is the only solubilizing agent.

Another embodiment of the invention provides an ophthalmic aqueous composition in the form of solution comprising <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w to <NUM>% w/w benzalkonium chloride, <NUM>% w/w to <NUM>% w/w ethylenediaminetetraacetic acid disodium salt dihydrate, sodium phosphate dibasic heptahydrate and boric acid and water, wherein the pH of the ophthalmic solution is <NUM> and wherein macrogol <NUM> hydroxystearate is the only solubilizing agent; preferably the amount of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester in the ophthalmic solution is from <NUM>% w/w to <NUM>% w/w, most preferably is <NUM>% w/w, <NUM>% w/w or <NUM>% w/w; optionally the ophthalmic solution further comprises HCl <NUM> / NaOH <NUM> to adjust the pH to pH <NUM>.

Another embodiment of the invention provides an ophthalmic aqueous composition in the form of solution comprising <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w benzalkonium chloride, <NUM>% w/w ethylenediaminetetraacetic acid disodium salt dihydrate, sodium phosphate dibasic heptahydrate and boric acid and water, wherein the pH of the ophthalmic solution is <NUM> and wherein macrogol <NUM> hydroxystearate is the only solubilizing agent.

Another embodiment of the invention provides an ophthalmic aqueous composition in the form of solution comprising <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w benzalkonium chloride, <NUM>% w/w ethylenediaminetetraacetic acid disodium salt dihydrate, sodium phosphate dibasic heptahydrate and boric acid and water, wherein the pH of the ophthalmic solution is <NUM> and wherein macrogol <NUM> hydroxystearate is the only solubilizing agent; preferably the amount of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester in the ophthalmic solution is <NUM>% w/w to <NUM>% w/w, most preferably is <NUM>% w/w, <NUM>% w/w or <NUM>% w/w; optionally the ophthalmic solution further comprises HCl <NUM> / NaOH <NUM> to adjust the pH to pH <NUM>.

Another embodiment of the invention provides an ophthalmic aqueous composition in the form of solution consisting of: <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% to <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w benzalkonium chloride, <NUM>% w/w ethylenediaminetetraacetic acid disodium salt dihydrate, <NUM>% w/w sorbitol, <NUM>% w/w sodium phosphate dibasic heptahydrate (Na<NUM>HPO<NUM> <NUM><NUM>O), <NUM> % w/w boric acid and water, wherein the pH of the ophthalmic solution is <NUM> and wherein macrogol <NUM> hydroxystearate is the only solubilizing agent.

Another embodiment of the invention provides an ophthalmic aqueous composition in the form of solution consisting of: <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w benzalkonium chloride, <NUM>% w/w ethylenediaminetetraacetic acid disodium salt dihydrate, <NUM>% w/w sorbitol, <NUM>% w/w sodium phosphate dibasic heptahydrate (Na<NUM>HPO<NUM> <NUM><NUM>O), <NUM> % w/w boric acid and water, wherein the pH of the ophthalmic solution is <NUM> and wherein macrogol <NUM> hydroxystearate is the only solubilizing agent; preferably the amount of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester in the ophthalmic solution is from <NUM>% w/w to <NUM>% w/w, most preferably is <NUM>% w/w, <NUM>% w/w or <NUM>% w/w; optionally the ophthalmic solution further comprises HCl <NUM> / NaOH <NUM> to adjust the pH to pH <NUM>.

Specific examples of the ophthalmic aqueous composition in the form of solution of the invention are:.

In the above ophthalmic aqueous compositions hydrochloride acid or sodium hydroxide may be used as pH adjusting agents.

The above reported ophthalmic aqueous solutions may be packaged in low density polyethylene (LDPE) primary containers, usually a multidose ophthalmic bottles with cap an dropper tip enabling to deliver calibrated drops with a controlled drop size. Example of such primary containers can be Rispharm® bottles from Berry-Plastics, Boston Round® from Amcor, <NUM> pieces bottles from either Gerresheimer, Philips-Medisize, Bormioli or equivalent.

The ophthalmic aqueous composition in the form of solution may be provided as anti-microbial preservative free ophthalmic aqueous solutions.

An embodiment of the invention provides an anti-microbial preservative free ophthalmic aqueous composition in the form of solution comprising <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate, sodium phosphate dibasic heptahydrate and boric acid and water, wherein the pH of the ophthalmic solution is <NUM>, further comprising a tonicity agent and optionally a further pH-adjusting agent, wherein macrogol <NUM> hydroxystearate is the only solubilizing agent.

A specific example of preservative free ophthalmic aqueous composition in the form of solution is the following composition consisting of: <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w sorbitol, <NUM>% w/w sodium phosphate dibasic heptahydrate, <NUM> % w/w boric acid and water and having pH <NUM>.

Other specific examples of preservative free ophthalmic aqueous composition in the form of solution are the following compositions:.

The antimicrobial preservative free ophthalmic aqueous compositions of the invention are packaged in containers that prevent microbial contamination of the formulations even after multiple uses or the antimicrobial preservative free ophthalmic aqueous compositions are packaged in unit dose containers which are sterile in the unopened form.

Such antimicrobial preservative free formula can be packaged either in a single-dose or unit dose LDPE primary packaging or inside a preservative free multidose container system.

Usually, the preservative free ophthalmic aqueous compositions of the invention are packed in monodose containers intended for single-use by the patient. In another embodiment, the antimicrobial preservative free ophthalmic aqueous compositions of the invention are packed in preservative free multidose containers that enable the formulation to be kept germ-free even after multiple uses by the patient. Examples of preservative free multidose containers are: OSD® from Aptar, Novelia® from Nemera, <NUM>® from Aeropump, or equivalent devices).

The macrogol <NUM> hydroxystearate used in the invention is the commercially available Kolliphor® HS15, formerly known as Solutol® HS15 and listed at the USP as polyoxyl-<NUM> hydroxystearate too; it consists of polyglycol mono- and di-esters of <NUM>-hydroxystearic acid and of about <NUM>% of free polyethylene glycol.

The aqueous ophthalmic compositions in the form of solution of the invention showed efficacy in reducing intraocular pressure, therefore they may be used in the treatment of ocular hypertension, glaucoma or in a method of reducing intraocular pressure.

Another object of the invention relates to an ophthalmic aqueous composition in the form of solution according to the invention for use in the treatment of ocular hypertension, glaucoma or in a method of reducing intraocular pressure.

Another object of the invention relates to the above defined ophthalmic aqueous compositions in the form of solution for use in the treatment of ocular hypertension, glaucoma or for reducing intraocular pressure.

Another embodiment of the invention relates to an ophthalmic aqueous composition in the form of solution comprising <NUM>% to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, from <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate wherein macrogol <NUM> hydroxystearate is the only solubilizing agent, for use in the treatment of ocular hypertension, glaucoma or for reducing intraocular pressure.

Another embodiment of the invention relates to a method of treating ocular hypertension or glaucoma or to a method of reducing intraocular pressure comprising administering to a patient in need thereof a therapeutically effective amount of the above defined ophthalmic aqueous composition in the form of solution.

Another embodiment of the invention relates to a method of treating ocular hypertension or glaucoma or to a method of reducing intraocular pressure comprising administering to a patient in need thereof a therapeutically effective amount of an ophthalmic aqueous composition in the form of solution comprising <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, from <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate and a pharmaceutically acceptable aqueous vehicle, wherein macrogol <NUM> hydroxystearate is the only solubilizing agent.

Another embodiment of the invention relates to a method of treating ocular hypertension or glaucoma or to a method of reducing intraocular pressure comprising administering to a patient in need thereof a therapeutically effective amount of an ophthalmic aqueous composition in the form of solution comprising from <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w benzalkonium chloride, <NUM>% w/w ethylenediaminetetraacetic acid disodium salt dihydrate, sodium phosphate dibasic heptahydrate and boric acid and water, wherein the pH of the ophthalmic solution is <NUM> and wherein macrogol <NUM> hydroxystearate is the only solubilizing agent; preferably the amount of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester in the ophthalmic solution is from <NUM>% w/w to <NUM>% w/w, most preferably is <NUM>% w/w, <NUM>% w/w or <NUM>% w/w; optionally the ophthalmic solution further comprises HCl <NUM> / NaOH <NUM> to adjust the pH to pH <NUM>.

Another embodiment of the invention relates to a method of treating ocular hypertension or glaucoma or to a method of reducing intraocular pressure comprising administering to a patient in need thereof a therapeutically effective amount of an ophthalmic aqueous composition in the form of solution consisting of: from <NUM>% w/w to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, <NUM>% w/w macrogol <NUM> hydroxystearate, <NUM>% w/w benzalkonium chloride, <NUM>% w/w ethylenediaminetetraacetic acid disodium salt dihydrate, <NUM>% w/w sorbitol, <NUM>% w/w sodium phosphate dibasic heptahydrate, <NUM>% w/w boric acid and water, and having pH <NUM>; preferably in the ophthalmic aqueous composition in the form of solution used in above method the amount of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester is <NUM>% w/w to <NUM>% w/w, most preferably is <NUM>% w/w, <NUM>% w/w or <NUM>% w/w; optionally the above ophthalmic aqueous composition in the form of solution further include HCl <NUM> / NaOH <NUM> to adjust the pH to <NUM>.

The ophthalmic compositions of the invention may be administered as an eye drop for treating a chronic ophthalmic disease such as glaucoma or ocular hypertension. The ophthalmic solution is intended to be generally administered once per day in each eye, on a daily frequency.

Another embodiment of the invention relates to a process for manufacturing the ophthalmic aqueous solution of the invention, the process comprises the following steps:.

The above reported order of addition of the excipients must be respected in order to comply with the pH and the tonicity requirements, pH <NUM> and 300mOsm/kg.

Step <NUM>) Preparation of the bulk ophthalmic solution by adding the API concentrated solution of Step <NUM> into the manufacturing tank containing the aqueous solution of Step <NUM> and water for injection up to the targeted final weight.

Optionally the pH of the bulk ophthalmic solution is adjusted to pH <NUM> with sodium hydroxide <NUM> or hydrochloric acid <NUM>.

Step <NUM>) Sterilization of the bulk ophthalmic solution by filtering the bulk ophthalmic solution of Step <NUM> through a Polyethersulfone (PES) filters having pore size of about <NUM>.

Step <NUM>) Optionally, the bulk ophthalmic solution is filled in low density polyethylene (LDPE) ophthalmic primary containers.

Optionally, when the ophthalmic aqueous solution further includes a viscosity-adjusting agent, in Step <NUM>) the viscosity agent is added in the manufacturing tank containing the water for injection as first component and, once it is fully dissolved, the other excipients are added.

Another embodiment of the invention relates to a process for manufacturing of antimicrobial preservative free ophthalmic aqueous solution of the invention, the process comprises the following steps:.

When the ophthalmic aqueous solution further includes a viscosity-adjusting agent, in Step <NUM>) the viscosity agent is added in the manufacturing tank containing the water for injection as first component and, once it is fully dissolved, the other excipients are added.

The ophthalmic composition ingredients are listed below:.

<NUM> of water for injection at about <NUM> was added to <NUM>,<NUM> of Polyoxyl <NUM> hydroxystearate pre-weighted in a container which was placed in a hot water bath until polyoxyl <NUM> hydroxystearate was completely melted. The melted polyoxyl <NUM>-hydrostearate / water mixture was added to a <NUM> batch can containing <NUM> of NCX-<NUM>.

The <NUM> batch can was thermostated at <NUM> with a water bath and the API solution was stirred until all ingredients were fully dissolved and maintained under continuous stirring until it was added to the solution of remaining excipients vehicle.

<NUM> of water for injection were poured in a manufacturing tank made of stainless steel (<NUM> grade) and cooled between <NUM> to <NUM>. About <NUM> liters of this water for injection was pulled from the tank and stored in another container for use during the preparation.

The following compounds were added according exactly the following order to the manufacturing tank containing the water under continuous stirring; each compound was fully dissolved before adding the next compound:.

The API concentrated solution was transferred into the manufacturing tank; the <NUM> batch can was rinsed with water for injection and the rinses were added to the bulk to complete the transfer. Water for injection was added to the manufacturing tank to adjust a final targeted weight of <NUM>.

The ophthalmic solution was sterilized by redundant filtration through <NUM> Polyethersulfone (PES) filters (Supor®).

After the filtration step, the ophthalmic solution was filled into LDPE multidose containers of appropriate volume under a grade A environment according to conventional aseptic process practices.

Ophthalmic formulations of the invention stored in multidose Low Density Poly-Ethylene (LDPE) containers sterilized with different sterilization methods were tested for stability The stability of ophthalmic formulations containing NCX <NUM>, <NUM>% w/w were evaluated at <NUM>, at initial, at <NUM> months and at <NUM> months, <NUM> months (long term storage condition) and for accelerated stability at <NUM>, at not more than <NUM>% relative humidity (RH), at initial, at <NUM> months and at <NUM> months.

Results are shown in Tables <NUM> to <NUM>.

The results of the stability tests at <NUM> (Table <NUM> and <NUM>) and <NUM> (Table <NUM> and <NUM>) demonstrated good stability of the ophthalmic solutions according to the invention, so that the ophthalmic solutions filled in a LDPE primary containers are expected to have a product shelf-life of at least <NUM> month in storage conditions at ambient temperature.

Primary container: Transparent LDPE / Pre-sterilized by radiation (gamma rays / 25kGy)
Primary container configuration: <NUM> fill in a <NUM> bottle.

Primary container: LDPE / Pre-sterilized by Ethylene Oxide
Primary container configuration: <NUM> fill in a <NUM> bottle.

As a conclusion, the ophthalmic solution from the present invention can be packaged in LDPE primary containers, whatever is the pre-sterilization mode of such containers. Multidose primary containers can be sterilized either by gamma radiation or ethylene oxide gas. Single-use or unit dose containers produced by BFS® (blow-fill-seal) technology provides native LDPE containers, naturally sterile, and such LDPE material preparation is appropriate for packaging the preservative free ophthalmic solution from the present invention too.

Antimicrobial effectiveness tests were performed to assess the ability of the ophthalmic solutions according to the present invention to meet the antimicrobial preservative efficacy criteria.

Ophthalmic solutions containing Edetate disodium <NUM>% (w/w) (see Table <NUM>) and different concentrations of benzalkonium chloride (see Table <NUM>) were tested. The ophthalmic compositions were prepared by applying the process disclosed in Example1. The tests were performed according to the procedure for the performance of the test disclosed in United States Pharmacopoeia, Monograph <<NUM>>, "Antimicrobial Effectiveness Testing" (AET). The success of this USP test is equivalent of matching the European Pharmacopoeia criteria B.

The results reported in Table <NUM> showed that the ophthalmic compositions containing benzalkonium chloride in a range from <NUM>% (w/w) to <NUM>% (w/w) met the criteria for microbial effectiveness and passed the Antimicrobial Effectiveness Testing (AET).

NCX <NUM> = Hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester.

To assess the effect of EDTA as antimicrobial preservative aid, the efficacy to meet the preservative criteria of an ophthalmic composition containing benzalkonium chloride (<NUM> % w/w) and Edetate disodium (<NUM>% w/w) (Formulation A - Table <NUM>) and of an ophthalmic composition containing benzalkonium chloride (<NUM> % w/w) but not Edetate disodium (Formulation B - Table <NUM>) were assessed.

The results showed that the presence of Edetate disodium is required to meet the preservative criteria defined in the US Pharmacopoeia monograph <<NUM>> or European Pharmacopoeia criteria B.

<CIT> discloses that polyoxyl <NUM> hydroxystearate improves antimicrobial preservative effectiveness allowing reducing the dose of the antimicrobial preservative agent benzalkonium chloride for matching USP <<NUM>> or EP-criteria B. The preservative studies carried out with the ophthalmic solutions of the invention showed that polyoxyl <NUM> hydroxystearate does not improve the antimicrobial preservative efficacy of benzalkonium chloride and that for ensuring antimicrobial preservation efficacy_of the ophthalmic solutions must contain an amount of benzalkonium chloride that must be higher than <NUM>% w/w in the presence of EDTA. <NUM>% w/w. The most appropriate antimicrobial preservative dose target was achieved with a mixture of <NUM>% w/w benzalkonium chloride and <NUM>% w/w EDTA.

Hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester is a dual-acting prostaglandin analog derivative that combines the pharmacological activity of bimatoprost with nitric oxide. Bimatoprost free acid is one of the active metabolites of Hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester; in this study bimatoprost free acid level was used as marker to compare the ocular penetration of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester after topical instillation of an aqueous ophthalmic solution of the invention with respect to a commercially available eyedrop containing bimatoprost and a reference formulation containing hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester.

The objective of this study was to comparatively assess the amounts of bimatoprost free acid in aqueous humor samples taken following instillation of the tested aqueous ophthalmic solutions.

Two different aqueous ophthalmic solutions containing hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,SR)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester (Formulations <NUM> and <NUM>) and a commercially available eyedrop containing bimatoprost (Formulation <NUM>) were evaluated in a rabbit ocular pharmacokinetic study.

The two different aqueous solutions comprising hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester are an ophthalmic solution according to the invention (Formulation <NUM>), and the formulation disclosed in <CIT> (Formulation <NUM>).

Table <NUM> reports the vehicles of the two aqueous ophthalmic solutions containing hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester <NUM>% w/w.

The concentration <NUM>% w/w of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester is equimolar with respect to the moles of bimatoprost of the commercially available eyedrop (Formulation <NUM>).

Macrogol <NUM>-hydroxystearate is the commercially available Kolliphor® HS15 (BASF). Polysorbate <NUM> is the commercially available Polysorbate <NUM> Super Refined® (Croda).

Groups of <NUM> male naive Dutch Belted Rabbits were assigned to the study and administered either with Formulations <NUM> and <NUM> or Formulation <NUM> by instillation to each eye at nominal target doses of <NUM>µg/eye for Formulations <NUM> and <NUM> and <NUM>µg/eye for bimatoprost solution. An additional group of <NUM> males was employed to provide blank control matrix (aqueous humor) for bio-analytical purposes. During the study, body weight measurements and clinical observations were performed and at pre-determined time points, animals were sacrificed for the purposes of aqueous humor (AH) harvesting at <NUM>, <NUM>, <NUM> and <NUM>. No clinical signs associated with dosing where observed during the study.

The results reported in Table <NUM> showed that the aqueous ophthalmic solution according to the invention (Formulation <NUM>), demonstrated greater exposure to bimatoprost acid (in terms of Cmax and AUC values) compared with that achieved following administration of the reference formulation (Formulation <NUM>) and the commercially available eyedrop (Formulation <NUM>).

Cmax is the maximum concentration that bimatoprost free acid achieved in the aqueous humor. AUC (area under the curve) represents the total amount of bimatoprost free acid in the aqueous humor over time that is available to produce a biological effect.

The aim of this study was to assess the aqueous humor content of bimatoprost free acid following topical ocular dosing with three different aqueous ophthalmic solutions containing hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester (<NUM>% w/w) in three different vehicles reported in Table <NUM>.

Vehicle <NUM> and <NUM> contain polysorbate <NUM> that is the solubilizer agent of the formulation disclosed in the prior art document <CIT>; vehicle <NUM> differs from vehicle <NUM> in that it contains benzalkonium chloride whereas vehicle <NUM> does not contains benzalkonium chloride.

Vehicle <NUM> contains a mixture of polysorbate <NUM> and macrogol <NUM>-hydroxystearate.

Table <NUM> reports the vehicles of the aqueous ophthalmic solutions containing hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester <NUM>% w/w.

An additional reference formulation was tested; this formulation is the commercially available eyedrop containing active bimatoprost <NUM>%.

Forty animals were included in the study. They were allocated into four groups of <NUM> animals/group and were administered with the above reported tested aqueous ophthalmic solutions
All animals included in the study were administered by ocular instillation of both eyes by means of a graduated pipette at a volume of <NUM>µL/eye. Two animals per formula were sacrificed at <NUM> (pre-dose), <NUM>, <NUM>, <NUM> and <NUM>, and aqueous humor samples were obtained immediately from both eyes.

The pharmacokinetic data reported in Table <NUM> showed that the exposure to bimatoprost acid (in terms of Cmax and AUC values) of the aqueous ophthalmic solutions containing Vehicle <NUM> or Vehicle <NUM> are equivalent whereas the aqueous ophthalmic solution containing Vehicle <NUM> showed a greater exposure (higher concentration of bimatoprost free acid in the aqueous humor) to bimatoprost free acid.

The lowest exposure parameters for bimatoprost acid were obtained in group of the Reference formulation.

Moreover the results demonstrated that benzalkonium chloride did not exert any effect on the ocular penetration of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester hexanoate up to a <NUM>% w/w concentration, indeed the pharmacokinetic data are considered equivalent for the two aqueous ophthalmic solutions containing Vehicles <NUM> or <NUM>.

In conclusion, the results of the above reported studies demonstrated that macrogol <NUM>-hydroxystearate is able to enhance the ocular penetration of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester. The enhanced absorption is a surprising effect because it does not depend on the known solubilizing activity of macrogol <NUM>-hydroxystearate since in all the tested aqueous ophthalmic solutions the therapeutic active compound was solubilized.

Indeed the solubility of hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester in aqueous solutions at pH <NUM> containing <NUM>% (w/w) of macrogol <NUM>-hydroxystearate (Kolliphor® HS15) or <NUM>% (w/w) polysorbate <NUM> (Tween® <NUM>) are <NUM>% (w/w) and <NUM>% (w/w) respectively, and so considered basically equivalent, therefore in all the tested vehicles hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester was solubilized.

Moreover, the results of Study <NUM> showed that benzalkonium chloride did not have effect on the ocular bioavailability of the active compound; indeed vehicle <NUM>, which contains polysorbate <NUM> and benzalkonium chloride, and vehicle <NUM>, which contains polysorbate <NUM> but not benzalkonium chloride, showed equivalent concentration of bimatoprost free acid in the aqueous humor.

In this study the efficacies in lowering intraocular pressure (IOP) in ocular normotensive Beagle dogs of an ophthalmic aqueous composition according to the present invention were assessed.

The tested aqueous compositions were administered by topical route to the conjunctival sac of both eyes of Beagle dogs twice a day, at approximately <NUM>-hour intervals, for <NUM> days. Two groups, each containing three males and three female of Beagle dogs, were included:.

Each animal received <NUM>µL/eye of the indicated substances on each treatment.

The first dose of the day was administered between <NUM> and <NUM> am, every day approximately at the same time.

The test item was placed by means of an automatic pipette in the conjunctiva of both eyes of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item.

Intraocular pressure was measured by an electronic tonometer, before the administration and at <NUM>-<NUM> hours after the daily dose on treatment day <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

Pupils were instilled with oxybuprocaine hydrochloride (Prescaina® <NUM>%) or oxybuprocaine hydrochloride, tetracaine hydrochloride (Colircusi Doble® Anestésico) eye drops before measuring the intraocular pressure.

The results are reported in Table <NUM> as IOP change versus baseline.

The aqueous compositions of the invention were effective in lowering IOP in ocular normotensive dogs. Furthermore, repeated daily dosing of the aqueous compositions of the invention resulted in sustained IOP lowering activity over time.

The stability of an ophthalmic formulation of the invention containing <NUM>% w/w NCX <NUM> stored in <NUM> Gamma-rays sterilized LDPE bottles was evaluated at <NUM>, at initial, at <NUM> months and at <NUM> months and for accelerated stability at <NUM>, at not more than <NUM>% relative humidity (RH), at initial, at <NUM> months and at <NUM> months.

The ophthalmic formulation was prepared according to method described in Example <NUM>.

Primary container configuration: <NUM> fill in a <NUM> bottle.

The results of the stability tests at <NUM> (Table <NUM>) and at <NUM> (Table <NUM>) demonstrated that the ophthalmic formulations according to the invention were stable, so that the ophthalmic solutions are expected to have a product shelf-life of at least <NUM> months in storage conditions at ambient temperature when stored LDPE containers.

Preparation of an ophthalmic preservative free composition containing <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester (NCX470). The ophthalmic composition ingredients are listed below:.

<NUM> of water for injection at about <NUM> was added to <NUM> of Polyoxyl <NUM> hydroxystearate pre-weighted in a container which was placed in a hot water bath until polyoxyl <NUM> hydroxystearate was completely melted. The melted polyoxyl <NUM>-hydrostearate / water mixture was added to a <NUM> batch can containing <NUM> of NCX-<NUM>.

The following compounds were added according to the following order to the manufacturing tank containing the water under continuous stirring; each compound was fully dissolved before adding the next compound:.

The vessel containing the solution was rinsed multiple times with sufficient water for injection and the rinses were added to the manufacturing tank to complete the transfer.

The API concentrated solution was transferred into the manufacturing tank; the <NUM> batch can was rinsed multiple times with water for injection and the rinses were added to the bulk to complete the transfer.

Water for injection was added to the manufacturing tank to adjust a final targeted weight of <NUM>.

pH can be fine-tuned more precisely by using sodium hydroxide and/or hydrochloric acid.

The ophthalmic solution was sterilized by redundant filtration through <NUM> Polyethersulfone (PES) filters (Supor® from Pall).

After the filtration step, the ophthalmic solution was filled into a LDPE preservative free multidose primary container system (e.g. Aptar OSD® system) of appropriate volume under a grade A environment according to conventional aseptic process practices. As another preservative free primary container system option, the bulk sterile solution can be filled into single-dose primary containers, for example single-dose primary containers produced by BFS technology (Blow-Fill-Seal®/Rommelag).

The stability of preservative free ophthalmic formulations of the invention containing <NUM>% w/w NCX <NUM> and <NUM>% NCX <NUM> stored in <NUM> glass bottles was evaluated at <NUM> and at <NUM>, at different time points.

The ophthalmic formulations were prepared according to method described in Example <NUM>. In these formulations the buffer system is mixture of sodium phosphate dibasic heptahydrate and citric acid monohydrate and the tonicity agent is glycerol.

<NUM>% w/w NCX <NUM> ophthalmic formulation composition:.

Primary container configuration: <NUM> fill in a <NUM> glass bottle.

The stability results of the two ophthalmic formulations are reported in the below tables <NUM>-<NUM>; the results showed that the preservative free ophthalmic formulations of the invention were stable.

Preparation of an ophthalmic composition containing hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl] -<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester (NCX <NUM>) <NUM> % w/w and a viscosity-adjusting agent.

<NUM> of water for injection at about <NUM> was added to <NUM> of Polyoxyl <NUM> hydroxystearate pre-weighted in a container which was placed in a hot water bath until polyoxyl <NUM> hydroxystearate was completely melted. The melted polyoxyl <NUM>-hydrostearate / water mixture was added to a <NUM> batch can containing <NUM> of NCX-<NUM>. The <NUM> batch can was thermostated at <NUM> with a water bath and the API solution was stirred until all ingredients were fully dissolved and maintained under continuous stirring until it was added to the solution of remaining excipients vehicle.

<NUM> of water for injection were poured in a manufacturing tank made of stainless steel (<NUM> grade) at a temperature higher than <NUM>. About <NUM> liters of this water for injection was pulled from the tank and stored in another container for use during the preparation.

<NUM> of HPMC (Metolose®/Shin-Etsu) are introduced slowly inside the tank while mixing. The cellulose is introduced slowly over <NUM>. Once the polymer has been fully dispersed into the tank, to keep mixing for additional <NUM> at a temperature of at least <NUM>. After that holding time, to cool the bulk solution down to <NUM>-<NUM> temperature.

Then, the following compounds were added according exactly the following order to the manufacturing tank containing the water under continuous stirring; each compound was fully dissolved before adding the next compound:.

the vessel containing the benzalkonium chloride solution was rinsed multiple times with sufficient water for injection and the rinses were added to the manufacturing tank to complete the transfer.

The API concentrated solution was transferred into the manufacturing tank; the <NUM> batch can was rinsed with water for injection and the rinses were added to the bulk to complete the transfer. Water for injection was added to the manufacturing tank to adjust the final targeted weight of <NUM>.

Claim 1:
A preservative free ophthalmic aqueous composition in the form of solution comprising <NUM>% to <NUM>% w/w hexanoic acid, <NUM>-(nitrooxy)-, (<NUM>,2E)-<NUM>-[(1R,2R,<NUM>,5R)-<NUM>-[(2Z)-<NUM>-(ethylamino)-<NUM>-oxo-<NUM>-hepten-<NUM>-yl]-<NUM>,<NUM>-dihydroxycyclopentyl]-<NUM>-(<NUM>-phenylethyl)-<NUM>-propen-<NUM>-yl ester, from <NUM>% w/w to <NUM>% w/w macrogol <NUM> hydroxystearate, wherein macrogol <NUM> hydroxystearate is the only solubilizing agent.