NZ751841B2

NZ751841B2 - Stable povidone-iodine compositions with steroids or non-steroidal anti-inflammatories

Info

Publication number: NZ751841B2
Application number: NZ751841A
Authority: NZ
Inventors: Joseph Capriotti; Bo Liang; C Michael Samson; Jason Stein; Michael Weiser
Original assignee: Takeda Pharmaceutical Company Limited
Priority date: 2011-05-12
Filing date: 2012-05-11
Publication date: 2020-11-03

Abstract

Disclosed are stable ophthalmic composition, comprising: 0.3 to 1% (w/w) polyvinylpyrrolidinone-iodine complex; 0.05 to 2% (w/w) non-steroidal anti-inflammatory drug (NSAID) bromfenac, or 0.05 to 2% (w/w) steroid selected from the group consisting of prednisolone acetate, loteprednol etabonate, difluprednate, hydrocortisone acetate, and combinations thereof; 0.005% to 0.02% (w/w) EDTA; 0.01 to 0.5% (w/w) sodium chloride; 0.02 to 0.1% (w/w) tyloxapol as a co-solvent/surfactant; 0.5% to 2% (w/w) sodium sulfate; and 0.1 to 0.5% (w/w) hydroxyethylcellulose as a viscosity increasing agent. These ophthalmic compositions are suitable for the treatment and/or prophylaxis of an eye disorder selected from the group consisting of a microorganism infection of at least one tissue of the eye, conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis and herpesvirus-related keratitis. fluprednate, hydrocortisone acetate, and combinations thereof; 0.005% to 0.02% (w/w) EDTA; 0.01 to 0.5% (w/w) sodium chloride; 0.02 to 0.1% (w/w) tyloxapol as a co-solvent/surfactant; 0.5% to 2% (w/w) sodium sulfate; and 0.1 to 0.5% (w/w) hydroxyethylcellulose as a viscosity increasing agent. These ophthalmic compositions are suitable for the treatment and/or prophylaxis of an eye disorder selected from the group consisting of a microorganism infection of at least one tissue of the eye, conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis and herpesvirus-related keratitis.

Description

TITLE Stable Povidone-Iodine Compositions with Steroids or Non-Steroidal Anti-Inflammatories This application is a divisional of New Zealand patent application 734781, which is itself a divisional of New Zealand patent application 717573, which is itself a divisional of New Zealand patent application 617433, which is the national phase entry in New Zealand of PCT international application , published as WO2012/155062, incorporated herein by reference in its entirety.

BACKGROUND Topical corticosteroids are routinely used to control ocular inflammation. Their mechanism of action involves the inhibition of the immune response and the subsequent tissue destruction that exuberant inflammation may cause. Corticosteroid has the undesirable side effect of limiting the body's intrinsic ability to fight infection. In fact, inopportune steroids usage can worsen the course of an infection secondary to mycobacteria, virus, or fungus. Thus, the use of a combined antimicrobial-steroid medication in ocular infections is recommended only under careful observation of a trained ophthalmologist because of these significant risks. In fact, TOBRADEX (Alcon), the most commonly prescribed combination ophthalmic antimicrobial- steroid drug, specifically lists `viral disease of the cornea and conjunctiva, mycobacteria infection, and fungal infection` as absolute contraindications to its use. Clearly, these combination drugs were not intended to be used in the face of infectious conjunctivitis in which bacterial infection cannot be confirmed.

In U.S. Patent 7,767,217, it is shown that under certain specific conditions, dexamethasone can be combined with povidone-iodine (PVP-I) to form an effective antimicrobial-steroid pharmaceutical composition. However, it is also shown that most preparations which combine PVP-I (or iodine) with a steroid suffer from instability due, in part, to reactivity of the iodine with the steroid. In fact, U.S. Patent 3,886,268 demonstrates the well- known instability of steroid-iodine combinations.

BRIEF SUMMARY In a first aspect, the present invention provides an ophthalmic composition suitable for topical administration to an eye, comprising a) povidone-iodine in a concentration between 0.01% and 10%, and b) a steroid, wherein the steroid is prednisolone acetate, loteprednol etabonate or difluprednate, and wherein after a period of one month after mixing the povidone-iodine and the steroid, the steroid concentration is at least 95% by weight of the steroid starting concentration.

In a further aspect, the present invention provides use of an ophthalmic composition according to the first aspect for the manufacture of a medicament for the treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye.

In another aspect, the present invention provides use of povidone-iodine and a steroid, wherein the steroid is prednisolone acetate, loteprednol etabonate or difluprednate, in the manufacture of a topical medicament for treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye, wherein the concentration of povidone- iodine in the medicament is between 0.01% and 10%, and wherein after a period of one month after mixing the povidone-iodine and the steroid, the steroid concentration in the medicament is at least 95% by weight of the steroid starting concentration.

In an embodiment, disclosed herein is an ophthalmic composition suitable for topical administration to an eye, effective for treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye, comprising povidone-iodine in a concentration between 0.01% and 10%, and a steroid selected from the group consisting of prednisolone acetate, loteprednol etabonate, difluprednate, hydrocortisone acetate, and combinations thereof.

In an embodiment, the povidone-iodine is between 0.1% and 2.5% by weight. In an embodiment, the povidone-iodine is between 0.5% and 2% by weight. In an embodiment, the total weight of the povidone-iodine and the steroid is between 0.1% and 4.5% in the solution. In an embodiment, the steroid is at a concentration of between 0.01 and 2%. In an embodiment, the steroid is at a concentration of between 0.05 and 1%.

In an embodiment, disclosed herein is a pharmaceutical composition comprising povidone-iodine in a concentration between 0.01% and 10%, and a steroid selected from the group consisting of prednisolone acetate, loteprednol etabonate, difluprednate, and combinations thereof, wherein the steroid is at a concentration of between 0.05 and 1%. In an embodiment, the PVP-I is at a concentration of about 0.4%. In an embodiment, the steroid is at a concentration selected from the group consisting of about 0.1%, about 0.05% and about 0.005%.

In an embodiment, an ophthalmic composition further comprises a topical anesthetic which relieves pain. In an embodiment, a topical anesthetic is selected from the group consisting of proparacaine, lidocaine, tetracaine and a combination thereof.

In an embodiment, an ophthalmic composition further comprises a penetration enhancer which enhances the penetration of povidone-iodine into the tissues of the eye. In an embodiment, a penetration enhancer is a topical anesthetic.

In an embodiment, an ophthalmic composition further comprises an antimicrobial preservative. In an embodiment, the antimicrobial preservative is selected from the group consisting of benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, EDTA, sorbic acid, Onamer M and a combination thereof. In an embodiment, the antimicrobial preservative is at a concentration of about 0.001% to 1.0% by weight in said solution.

In an embodiment, an ophthalmic composition further comprises a co-solvent/surfactant.

In an embodiment, the co-solvent/surfactant is selected from the group consisting of polysorbate , polysorbate 60, polysorbate 80, Pluronic F-68, Pluronic F-84, Pluronic P-103, cyclodextrin, tyloxapol and a combination thereof. In an embodiment, the co-solvent/surfactant is at a concentration of about 0.01% to 2% by weight in said composition.

In an embodiment, an ophthalmic composition further comprises viscosity increasing agent. In an embodiment, the viscosity increasing agent is selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose, and a combination thereof. In an embodiment, the viscosity increasing agent is at a concentration of about 0.01% to 2% by weight in said solution.

In an embodiment, an ophthalmic composition suitable for topical administration to an eye, effective for treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye, comprises povidone-iodine in a concentration between 0.01% and %, and bromfenac. In an embodiment, an ophthalmic composition comprises: 0.3 to 1% (w/w) polyvinylpyrrolidinone-iodine complex; 0.05 to 2% (w/w) bromfenac; 0.005% to 0.02% (w/w) EDTA; 0.01 to 0.5% (w/w) sodium chloride; 0.02 to 0.1% (w/w) tyloxapol; 0.5% to 2% (w/w) sodium sulfate; and 0.1 to 0.5% (w/w) hydroxyethylcellulose.

In an embodiment, an ophthalmic composition is in the form of a solution, suspension, emulsion, ointment, cream, gel, or a controlled-release/sustain-release vehicle.

In an embodiment, a microorganism treated or prevented by prophylaxis using a composition encompassed herein is selected from the group consisting of bacteria, viruses, fungi, and amoebae. In an aspect, bacteria is mycobacteria.

In an embodiment, a disorder treated using an ophthalmic composition encompassed herein is selected from the group consisting of a microorganism infection of at least one tissue of the eye, conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis and herpesvirus-related keratitis.

In an embodiment, an ophthalmic composition is used for prophylaxis of infection following corneal abrasion or ocular surgery.

In an embodiment, an ophthalmic composition comprises: 0.3 to 1% (w/w) polyvinylpyrrolidinone-iodine complex; 0.05 to 2% (w/w) steroid; 0.005% to 0.02% (w/w) EDTA; 0.01 to 0.5% (w/w) sodium chloride; 0.02 to 0.1% (w/w) tyloxapol; 0.5% to 2% (w/w) sodium sulfate; and 0.1 to 0.5% (w/w) hydroxyethylcellulose; wherein the steroid is selected from the group consisting of prednisolone acetate, loteprednol etabonate, difluprednate, hydrocortisone acetate, and combinations thereof.

In an embodiment, an ophthalmic composition comprises: 0.4% (w/w) polyvinylpyrrolidinone-iodine complex; 0.1% (w/w) steroid; 0.01% (w/w) EDTA; 0.3% (w/w) sodium chloride salt; 0.05% (w/w) tyloxapol; 0.2% (w/w) sodium sulfate; and 0.25% (w/w) hydroxyethylcellulose; wherein the steroid is selected from the group consisting of prednisolone acetate, loteprednol etabonate, difluprednate, hydrocortisone acetate, and combinations thereof.

In an embodiment, an ophthalmic composition retains 95% of its polyvinylpyrrolidinone- iodine and 95% of its steroid after a period of 1 month. In an embodiment, an ophthalmic composition retains 90% of its polyvinylpyrrolidinone-iodine and 90% of its steroid after a period of 3 months. In an embodiment, an ophthalmic composition retains 90% of its polyvinylpyrrolidinone-iodine and 90% of its steroid after a period of 1 month.

In an embodiment, an ophthalmic composition retains 95% of its polyvinylpyrrolidinone- iodine and 95% of its NSAID after a period of 1 month. In an embodiment, an ophthalmic composition retains 90% of its polyvinylpyrrolidinone-iodine and 90% of its NSAID after a period of 3 months. In an embodiment, an ophthalmic composition retains 90% of its polyvinylpyrrolidinone-iodine and 90% of its NSAID after a period of 1 month.

In an embodiment, an ophthalmic composition comprising polyvinylpyrrolidinone-iodine (PVP-I) and at least one steroid retains about 89% of its PVP-I after a period of 1 month, about 90% of its PVP-I after a period of 1 month, about 91% of its PVP-I after a period of 1 month, about 92% of its PVP-I after a period of 1 month, about 93% of its PVP-I after a period of 1 month, about 94% of its PVP-I after a period of 1 month, about 94% of its PVP-I after a period of 1 month, about 95% of its PVP-I after a period of 1 month, about 96% of its PVP-I after a period of 1 month, about 97% of its PVP-I after a period of 1 month, about 98% of its PVP-I after a period of 1 month, or about 99% of its PVP-I after a period of 1 month.

In an embodiment, an ophthalmic composition comprising polyvinylpyrrolidinone-iodine (PVP-I) and at least one NSAID retains about 89% of its PVP-I after a period of 1 month, about 90% of its PVP-I after a period of 1 month, about 91% of its PVP-I after a period of 1 month, about 92% of its PVP-I after a period of 1 month, about 93% of its PVP-I after a period of 1 month, about 94% of its PVP-I after a period of 1 month, about 94% of its PVP-I after a period of 1 month, about 95% of its PVP-I after a period of 1 month, about 96% of its PVP-I after a period of 1 month, about 97% of its PVP-I after a period of 1 month, about 98% of its PVP-I after a period of 1 month, or about 99% of its PVP-I after a period of 1 month.

In an embodiment, an ophthalmic composition comprising polyvinylpyrrolidinone-iodine (PVP-I) and at least one steroid retains about 89% of its PVP-I after a period of 3 months, about 90% of its PVP-I after a period of 3 months, about 91% of its PVP-I after a period of 3 months, about 92% of its PVP-I after a period of 3 months, about 93% of its PVP-I after a period of 3 months, about 94% of its PVP-I after a period of 3 months, about 94% of its PVP-I after a period of 3 months, about 95% of its PVP-I after a period of 3 months, about 96% of its PVP-I after a period of 3 months, about 97% of its PVP-I after a period of 3 months, about 98% of its PVP-I after a period of 3 months, or about 99% of its PVP-I after a period of 3 months.

In an embodiment, an ophthalmic composition comprising polyvinylpyrrolidinone-iodine (PVP-I) and at least one NSAID retains about 89% of its PVP-I after a period of 3 months, about 90% of its PVP-I after a period of 3 months, about 91% of its PVP-I after a period of 3 months, about 92% of its PVP-I after a period of 3 months, about 93% of its PVP-I after a period of 3 months, about 94% of its PVP-I after a period of 3 months, about 94% of its PVP-I after a period of 3 months, about 95% of its PVP-I after a period of 3 months, about 96% of its PVP-I after a period of 3 months, about 97% of its PVP-I after a period of 3 months, about 98% of its PVP-I after a period of 3 months, or about 99% of its PVP-I after a period of 3 months.

In an embodiment, an ophthalmic composition comprising PVP-I and at least one steroid retains about 89% of its at least one steroid after a period of 1 month, about 90% of its at least one steroid after a period of 1 month, about 91% of its at least one steroid after a period of 1 month, about 92% of its at least one steroid after a period of 1 month, about 93% of its at least one steroid after a period of 1 month, about 94% of its at least one steroid after a period of 1 month, about 94% of its at least one steroid after a period of 1 month, about 95% of its at least one steroid after a period of 1 month, about 96% of its at least one steroid after a period of 1 month, about 97% of its at least one steroid after a period of 1 month, about 98% of its at least one steroid after a period of 1 month, or about 99% of its at least one steroid after a period of 1 month.

In an embodiment, an ophthalmic composition comprising PVP-I and at least one NSAID retains about 89% of its at least one NSAID after a period of 1 month, about 90% of its at least one NSAID after a period of 1 month, about 91% of its at least one NSAID after a period of 1 month, about 92% of its at least one NSAID after a period of 1 month, about 93% of its at least one NSAID after a period of 1 month, about 94% of its at least one NSAID after a period of 1 month, about 94% of its at least one NSAID after a period of 1 month, about 95% of its at least one NSAID after a period of 1 month, about 96% of its at least one NSAID after a period of 1 month, about 97% of its at least one NSAID after a period of 1 month, about 98% of its at least one NSAID after a period of 1 month, or about 99% of its at least one NSAID after a period of 1 month.

In an embodiment, an ophthalmic composition comprising PVP-I and at least one steroid retains about 89% of its at least one steroid after a period of 3 months, about 90% of its at least one steroid after a period of 3 months, about 91% of its at least one steroid after a period of 3 months, about 92% of its at least one steroid after a period of 3 months, about 93% of its at least one steroid after a period of 3 months, about 94% of its at least one steroid after a period of 3 months, about 94% of its at least one steroid after a period of 3 months, about 95% of its at least one steroid after a period of 3 months, about 96% of its at least one steroid after a period of 3 months, about 97% of its at least one steroid after a period of 3 months, about 98% of its at least one steroid after a period of 3 months, or about 99% of its at least one steroid after a period of 3 months.

In an embodiment, an ophthalmic composition comprising PVP-I and at least one NSAID retains about 89% of its at least one NSAID after a period of 3 months, about 90% of its at least one NSAID after a period of 3 months, about 91% of its at least one NSAID after a period of 3 months, about 92% of its at least one NSAID after a period of 3 months, about 93% of its at least one NSAID after a period of 3 months, about 94% of its at least one NSAID after a period of 3 months, about 94% of its at least one NSAID after a period of 3 months, about 95% of its at least one NSAID after a period of 3 months, about 96% of its at least one NSAID after a period of 3 months, about 97% of its at least one NSAID after a period of 3 months, about 98% of its at least one NSAID after a period of 3 months, or about 99% of its at least one NSAID after a period of 3 months.

In an embodiment, an ophthalmic composition is an aqueous solution.

In an embodiment, a method is provided for treating and/or prophylaxis of an eye disorder or a microorganism infection of at least one tissue of the eye comprising the step of administering one of more doses of an ophthalmic composition encompassed herein to the eye.

In an embodiment, the prophylaxis is prophylaxis of infection following corneal abrasion or ocular surgery. In an embodiment, the eye disorder is selected from the group consisting of a microorganism infection of at least one tissue of the eye, conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis and herpesvirus-related keratitis. In an embodiment, the microorganism is a bacteria, virus, fungi, or amoebae. In an embodiment, the bacteria is mycobacteria.

In an embodiment, in a method of treatment, the sum of the povidone-iodine and the steroid is between 0.001 mg to 5 mg per dose. In an embodiment, in a method of treatment, each dose is between 10 microliters to 200 microliters. In an embodiment, in a method of treatment, each dose is between 50 microliters to 80 microliters. In an embodiment, in a method of treatment, the administering step comprises administering a composition encompassed herein to an eye one to four times a day. In an embodiment, in a method of treatment, the administering step comprises administering a composition encompassed herein to an eye one to twenty-four times a day. In an embodiment, in a method of treatment, the method includes storing the composition for at least one month, at least three months, at least six months, or at least 1 year before the administration step.

In the description in this specification reference may be made to subject matter which is not within the scope of the appended claims. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the appended claims.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this specification and claims which include the term “comprising”, other features besides the features prefaced by this term in each statement can also be present. Related terms such as “comprise” and “comprises” are to be interpreted in similar manner.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an image depicting the HPLC-UV/(+)ESI-MS and MS/MS spectral data of dexamethasone phosphate.

Figure 2 is an image depicting the HPLC-UV/(+)ESI-MS and MS/MS spectral data of prednisolone acetate.

Figure 3 is an image depicting the HPLC-UV/(+)ESI-MS and MS/MS spectral data of loteprednol etabonate.

Figure 4 is an image depicting the HPLC-UV/(+)ESI-MS and MS/MS spectral data of difluprednate.

Figure 5 is an image depicting the HPLC/UV chromatograms of PVP-I at the concentration of 200 µg/mL for dexamethasone sodium phosphate.

Figure 6 is an image depicting the HPLC/UV chromatograms of dexamethasone sodium phosphate in PVP-I for Day 0.

Figure 7 is an image depicting the HPLC/UV chromatograms of dexamethasone sodium phosphate.

Figure 8 is an image depicting the HPLC/UV chromatograms of dexamethasone sodium phosphate in PVP-I for two weeks.

Figure 9 is an image depicting the HPLC/UV chromatograms of dexamethasone sodium phosphate in PVP-I for two weeks.

Figure 10 is an image depicting the HPLC/UV chromatograms of dexamethasone sodium phosphate in PVP-I for one month.

Figure 11 is an image depicting the HPLC/UV chromatograms of dexamethasone sodium phosphate in PVP-I for one month.

Figure 12 is an image depicting the HPLC/UV chromatograms (expanded) of dexamethasone sodium phosphate in PVP-I for one month.

Figure 13 is an image depicting the HPLC/UV chromatograms (expanded) of dexamethasone sodium phosphate in PVP-I for one month.

Figure 14 is an image depicting the mass ion chromatograms (MRM Mode) of dexamethasone sodium phosphate in reference standard samples.

Figure 15 is an image depicting the mass ion chromatograms (MRM Mode) of dexamethasone sodium phosphate in one month room temperature stability sample in the presence of PVP-I.

Figure 16 is an image depicting the mass ion chromatograms (MRM Mode) of dexamethasone sodium phosphate in one month 40°C stability sample in the presence of PVP-I.

Figure 17 is an image depicting the HPLC/UV chromatograms of PVP-I at the concentration of 20 µg/mL for prednisolone acetate.

Figure 18 is an image depicting the HPLC/UV chromatograms of prednisolone acetate in PVP-I for Day 0.

Figure 19 is an image depicting the HPLC/UV chromatograms of prednisolone acetate in PVP-I for Day 0.

Figure 20 is an image depicting the HPLC/UV chromatograms of prednisolone acetate in PVP-I for two weeks.

Figure 21 is an image depicting the HPLC/UV chromatograms of prednisolone acetate in PVP-I for two weeks.

Figure 22 is an image depicting the HPLC/UV chromatograms of prednisolone acetate in PVP-I for one month.

Figure 23 is an image depicting the HPLC/UV chromatograms of prednisolone acetate in PVP-I for one month.

Figure 24 is an image depicting the mass ion chromatograms (MRM Mode) of prednisolone acetate in reference standard samples.

Figure 25 is an image depicting the mass ion chromatograms (MRM Mode) of prednisolone acetate in one month room temperature stability sample in the presence of PVP-I.

Figure 26 is an image depicting the mass ion chromatograms (MRM Mode) of prednisolone acetate in one month 40°C stability sample in the presence of PVP-I.

Figure 27 is an image depicting the HPLC/UV chromatograms of PVP-I at the concentration of 40 µg/mL for loteprednol etabonate.

Figure 28 is an image depicting the HPLC/UV chromatograms of loteprednol etabonate in PVP-I for Day 0.

Figure 29 is an image depicting the HPLC/UV chromatograms of loteprednol etabonate in PVP-I for Day 0.

Figure 30 is an image depicting the HPLC/UV chromatograms of loteprednol etabonate in PVP-I for two weeks.

Figure 31 is an image depicting the HPLC/UV chromatograms of loteprednol etabonate in PVP-I for two weeks.

Figure 32 is an image depicting the HPLC/UV chromatograms of loteprednol etabonate in PVP-I for one month.

Figure 33 is an image depicting the HPLC/UV chromatograms of loteprednol etabonate in PVP-I for one month.

Figure 34 is an image depicting the mass ion chromatograms (MRM Mode) of loteprednol etabonate in reference standard samples.

Figure 35 is an image depicting the mass ion chromatograms (MRM Mode) of loteprednol etabonate in one month room temperature stability sample in the presence of PVP-I.

Figure 36 is an image depicting the mass ion chromatograms (MRM Mode) of loteprednol etabonate in one month 40°C stability sample in the presence of PVP-I.

Figure 37 is an image depicting the HPLC/UV chromatograms of PVP-I at the concentration of 400 µg/mL for difluprednate.

Figure 38 is an image depicting the HPLC/UV chromatograms of difluprednate in PVP-I for Day 0.

Figure 39 is an image depicting the HPLC/UV chromatograms of difluprednate in PVP-I for Day 0.

Figure 40 is an image depicting the HPLC/UV chromatograms of difluprednate in PVP-I for two weeks.

Figure 41 is an image depicting the HPLC/UV chromatograms of difluprednate in PVP-I for two weeks.

Figure 42 is an image depicting the HPLC/UV chromatograms of difluprednate in PVP-I for one month.

Figure 43 is an image depicting the HPLC/UV chromatograms of difluprednate in PVP-I for one month.

Figure 44 is an image depicting the mass ion chromatograms (MRM Mode) of difluprednate in reference standard samples.

Figure 45 is an image depicting the mass ion chromatograms (MRM Mode) of difluprednate in one month room temperature stability sample in the presence of PVP-I.

Figure 46 is an image depicting the mass ion chromatograms (MRM Mode) of difluprednate in one month 40°C stability sample in the presence of PVP-I.

DETAILED DESCRIPTION It is known that iodine, including preparations of PVP-I, reacts chemically with various steroids when combined with a steroid, resulting in an unstable composition, due in part to reactivity of the iodine with the steroid. U.S. Patent 3,886,268 demonstrates the well-known instability of steroid-iodine combinations. It is also known that certain non-steroidal anti- inflammatory compounds (“NSAIDS”) also react with iodine. However, U.S. Patent 7,767,217, incorporated herein by reference in its entirety, illustrates that under certain specific conditions, dexamethasone, for example, can be combined with PVP-I to form an effective antimicrobial- steroid pharmaceutical composition. U.S. Provisional Patent Application No. 61/485,475, to which the present application claims priority, is also incorporated herein by reference in its entirety.

Compositions In an embodiment, compositions disclosed herein comprise PVP-I and a steroid. In an embodiment, compositions disclosed herein comprise PVP-I and an NSAID. In another embodiment, a composition disclosed herein is a pharmaceutical composition. In another embodiment, a composition disclosed herein is an ophthalmic composition.

Disclosed herein are compositions comprising PVP-I in the range of about 0.01% to about 10% (weight/weight or weight/volume) and a steroid at a concentration of about 0.001% to about 10%. Also disclosed herein are ophthalmic compositions comprising povidone-iodine in the range of about 0.01% to about 10% (weight/weight or weight/volume) and a therapeutically effective amount of a steroid at a concentration of about 0.001% to about 10%. Disclosed herein are compositions comprising PVP-I in the range of about 0.01% to about 10% (weight/weight or weight/volume) and an NSAID at a concentration of about 0.001% to about 10%. Also disclosed herein are ophthalmic compositions comprising povidone-iodine in the range of about 0.01% to about 10% (weight/weight or weight/volume) and a therapeutically effective amount of an NSAID at a concentration of about 0.001% to about 10%.

The affinity of free iodine for reaction with --OH, --SH and --NH functional groups is well described in the literature and forms the basis for the anti-microbial activity of iodine- containing solutions (Rackur H. J. Hosp. Infect., 1985; 6: 13-23, and references therein).

Dexamethasone, (9-Fluoro-11.beta., 17, 21-trihydroxy-16.alpha.-methylpregna-1, 4-diene-3, 20- dione) for example, contains three such moieties (--OH) at the 11, 17 and 21 positions. The skilled artisan would conclude that these hydroxyl groups would be prone to covalent substitution reactions by the free iodine generated in the solution equilibrium reaction described above for PVP-I.

In preparing the present compositions, experiments of combinations of various steroids and PVP-I, as well as combinations of various NSAIDS and PVP-I, were performed. It was observed that many formulations were unsuccessful because of the rapid reaction between PVP-I and the added steroid. It was surprising to discover that separate solutions of PVP-I and prednisolone acetate, PVP-I and loteprednol etabonate, PVP-I and hydrocortisone acetate, and PVP-I and difluprednate demonstrate unexpected stability, based on what was previously known in the art. It was also surprising to discover that solutions of PVP-I and bromfenac demonstrate unexpected stability, based on what was previously known in the art. In an embodiment, a combination of PVP-I and one of the steroids or NSAIDS identified above each remains stable for a month or longer.

In an embodiment, a composition comprises PVP-I and prednisolone acetate. In another embodiment, a composition is a pharmaceutical composition comprising PVP-I and prednisolone acetate. In another embodiment, a composition is an ophthalmic preparation comprising PVP-I and prednisolone acetate.

In an embodiment, a composition comprises PVP-I and loteprednol etabonate. In another embodiment, a composition is a pharmaceutical composition comprising PVP-I and loteprednol etabonate. In another embodiment, a composition is an ophthalmic preparation comprising PVP- I and loteprednol etabonate.

In an embodiment, a composition comprises PVP-I and hydrocortisone acetate. In another embodiment, a composition is a pharmaceutical composition comprising PVP-I and hydrocortisone acetate. In another embodiment, a composition is an ophthalmic preparation comprising PVP-I and hydrocortisone acetate.

In an embodiment, a composition comprises PVP-I and difluprednate. In another embodiment, a composition is a pharmaceutical composition comprising PVP-I and difluprednate. In another embodiment, a composition is an ophthalmic preparation comprising PVP-I and difluprednate.

In an embodiment, a composition comprises PVP-I and bromfenac. In another embodiment, a composition is a pharmaceutical composition comprising PVP-I and bromfenac.

In another embodiment, a composition is an ophthalmic preparation comprising PVP-I and bromfenac.

Percentages for components of compositions are provided herein as weight/weight (w/w), unless otherwise indicated. For example, 0.6% PVP-I indicates 0.6% PVP-I by weight, with respect to the total weight of 100% for a composition.

In an embodiment, a composition comprises povidone-iodine (PVP-I) at a concentration in the range of about 0.1% to about 2.5%. In another embodiment, a composition comprises povidone-iodine (PVP-I) at a concentration in the range between 0.2 and 1.5%, and in yet another embodiment, between 0.3% and 1.0%. In an embodiment, a composition comprises PVP-I at a concentration in the range of about 0.2 to about 2.0%, about 0.3% to about 1.5%, about 0.36% to about 1.0%, and about 0.4% to about 0.75%. In an embodiment, a composition comprises PVP-I at a concentration of about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% or about 1.0%. In an embodiment, a composition comprises povidone-iodine PVP-I at a concentration of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, or 1.0%. In another embodiment, a composition comprises PVP-I at a concentration of about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10%. In another embodiment, a composition comprises PVP-I at a concentration of about 2% or less, about 3% or less, about 4% or less, about 5% or less, about 6% or less, about 7% or less, about 8% or less, about 9% or less or about 10% or less. In another embodiment, a composition comprises PVP-I at a concentration of about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more or about 10% or more. In another embodiment, a composition comprises PVP-I at a concentration of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

Compositions disclosed herein may comprise one or more steroids. Steroids include, but are not limited to, dexamethasone, dexamethasone alcohol, dexamethasone sodium phosphate, fluorometholone acetate, fluorometholone acetate, fluorometholone alcohol, loteprednol etabonate, medrysone, prednisolone acetate, prednisolone sodium phosphate, difluprednate, rimexolone, hydrocortisone, hydrocortisone acetate, lodoxamide tromethamine, and any combinations thereof. In an embodiment, a steroid is present in the composition at a level of about 0.001% to about 10%. In an embodiment, a steroid is present in the composition or preparation at a level of 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%. In an embodiment, a steroid is present in the composition or preparation at a level of about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%. In an embodiment, a steroid is present in the composition or preparation at a level of about 0.001% or less, about 0.002% or less, about 0.003% or less, about 0.004% or less, about 0.005% or less, about 0.006% or less, about 0.007% or less, about 0.008% or less, about 0.009% or less, about 0.01% or less, about 0.02% or less, about 0.03% or less, about 0.04% or less, about 0.05% or less, about 0.06% or less, about 0.07% or less, about 0.08% or less, about 0.09% or less, about 0.1% or less, about 0.2% or less, about 0.3% or less, about 0.4% or less, about 0.5% or less, about 0.6% or less, about 0.7% or less, about 0.8% or less, about 0.9% or less, about 1.0% or less, about 1.1% or less, about 1.2% or less, about 1.3% or less, about 1.4% or less, about 1.5% or less, about 1.6% or less, about 1.7% or less, about 1.8% or less, about 1.9% or less, or about 2.0% or less. In an embodiment, a steroid is present in the composition or preparation at a level of about 0.001% or more, about 0.002% or more, about 0.003% or more, about 0.004% or more, about 0.005% or more, about 0.006% or more, about 0.007% or more, about 0.008% or more, about 0.009% or more, about 0.01% or more, about 0.02% or more, about 0.03% or more, about 0.04% or more, about 0.05% or more, about 0.06% or more, about 0.07% or more, about 0.08% or more, about 0.09% or more, about 0.1% or more, about 0.2% or more, about 0.3% or more, about 0.4% or more, about 0.5% or more, about 0.6% or more, about 0.7% or more, about 0.8% or more, about 0.9% or more, about 1.0% or more, about 1.1% or more, about 1.2% or more, about 1.3% or more, about 1.4% or more, about 1.5% or more, about 1.6% or more, about 1.7% or more, about 1.8% or more, about 1.9% or more, or about 2.0% or more.

Compositions disclosed herein may comprise one or more NSAIDS. NSAIDS include, but are not limited to, bromfenac, ketorolac, nepafenac, ketotifen fumarate, diclofenac sodium, flurbiprofen sodium, ketorlac tromethamine, suprofen, celecoxib, naproxen, rofecoxib, and any combinations thereof. In an embodiment, an NSAID is present in the composition at a level of about 0.001% to about 10%. In an embodiment, an NSAID is present in the composition or preparation at a level of 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%. In an embodiment, an NSAID is present in the composition or preparation at a level of about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%. In an embodiment, an NSAID is present in the composition or preparation at a level of about 0.001% or less, about 0.002% or less, about 0.003% or less, about 0.004% or less, about 0.005% or less, about 0.006% or less, about 0.007% or less, about 0.008% or less, about 0.009% or less, about 0.01% or less, about 0.02% or less, about 0.03% or less, about 0.04% or less, about 0.05% or less, about 0.06% or less, about 0.07% or less, about 0.08% or less, about 0.09% or less, about 0.1% or less, about 0.2% or less, about 0.3% or less, about 0.4% or less, about 0.5% or less, about 0.6% or less, about 0.7% or less, about 0.8% or less, about 0.9% or less, about 1.0% or less, about 1.1% or less, about 1.2% or less, about 1.3% or less, about 1.4% or less, about 1.5% or less, about 1.6% or less, about 1.7% or less, about 1.8% or less, about 1.9% or less, or about 2.0% or less. In an embodiment, an NSAID is present in the composition or preparation at a level of about 0.001% or more, about 0.002% or more, about 0.003% or more, about 0.004% or more, about 0.005% or more, about 0.006% or more, about 0.007% or more, about 0.008% or more, about 0.009% or more, about 0.01% or more, about 0.02% or more, about 0.03% or more, about 0.04% or more, about 0.05% or more, about 0.06% or more, about 0.07% or more, about 0.08% or more, about 0.09% or more, about 0.1% or more, about 0.2% or more, about 0.3% or more, about 0.4% or more, about 0.5% or more, about 0.6% or more, about 0.7% or more, about 0.8% or more, about 0.9% or more, about 1.0% or more, about 1.1% or more, about 1.2% or more, about 1.3% or more, about 1.4% or more, about 1.5% or more, about 1.6% or more, about 1.7% or more, about 1.8% or more, about 1.9% or more, or about 2.0% or more.

The compositions disclosed herein can be administered as solutions, suspensions, emulsions (dispersions), gels, creams, or ointments in a suitable ophthalmic vehicle. In any of the compositions of this disclosure for topical administration, such as topical administration to the eye, the mixtures are preferably formulated as aqueous solutions at a pH of 3.5 to 6.5.

Preferentially the pH is adjusted to between 4 and 5. This pH range may be achieved by the addition of acids/bases to the solution.

In an embodiment, an ophthalmic composition may comprise an optional co-solvent. In another embodiment, the solubility of the components of the present compositions may be enhanced by a surfactant or other appropriate co-solvent in the composition. Such co-solvents or surfactants include polysorbate -20, -60, and -80, a polyoxyethylene/polyoxypropylene surfactant (e.g. Pluronic F-68, F-84 and P-103), cyclodextrin, tyloxapol, PEG 35 Castor oil (Cremophor EL), polyoxyl 40 Stearate (Myrj 52), other agents known to those skilled in the art, or a combination thereof. Typically, such co-solvents are present at a level of from about 0.01% to about 2% by weight. In an embodiment, a co-solvent is present at a level of about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%.

In an embodiment, a composition may comprise an optional agent that can increase viscosity. As will be understood by the skilled artisan when armed with the present disclosure, it may be desirable to increase viscosity above that of a simple aqueous solution in order to increase ocular absorption of the active compound, to decrease variability in dispensing the formulation, to decrease physical separation of components of a suspension or emulsion of the formulation and/or to otherwise improve the ophthalmic formulation. Such viscosity-enhancing agents include, but are not limited to, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, other agents known to those skilled in the art, or any combination thereof. Such agents are typically employed at a level of from about 0.01% to about 2% by weight. In an embodiment, such optional agents are present at about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%.

In another aspect, bioadhesive agents may comprise the compositions, in order to increase the retention time of the drug gradient over a biological substrate. The bioadhesive agents include, but are not limited to, polyvinylpyrrolidone (PVP), xanthan gum, locust bean gum, acacia gum, hydroxypropyl methylcellulose (HPMC), sodium alginate, pectin, gelatin, carbomer, polyvinylalcohol, gellan gum, tragacanth, acacia, and sodium carboxymethyl cellulose, as well as other agents known to those skilled in the art, or any combination thereof.

In yet another embodiment, compositions of the invention may comprise viscoelastic agents such as methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, polyvinyl alcohol, dextran, chondroitin sulfate and salts thereof, and hyaluronic acid and salts thereof.

In an embodiment, an ophthalmic composition may further comprise one or more of (1) a penetration enhancer which enhances the penetration of povidone-iodine into the tissues of the eye (this may be a topical anesthetic) (2) a co-solvent or a nonionic surface agent - surfactant, which, for example, may be about 0.01% to 2% by weight; (3) a viscosity increasing agent, which, for example, may be about 0.01% to 2% by weight; and (4) a suitable ophthalmic vehicle.

The ophthalmic composition may be in the form of a solution, a suspension, an emulsion, a preparation, an ointment, a cream, a gel, or a controlled-release/sustain-release vehicle. By way of a non-limiting example, the composition may be in the form of a contact lens solution, eyewash, eyedrop, and the like.

Methods In an embodiment, compositions disclosed herein are useful for preparation of and use as pharmaceutical compositions. In another embodiment, compositions disclosed herein are useful for preparation of and use as compositions other than pharmaceutical compositions.

In an embodiment, compositions disclosed herein are useful for preparation of and use as ophthalmic compositions. In an aspect, a composition of the invention is useful in the treatment of infections of the conjunctiva and cornea. In another aspect, the broad spectrum antimicrobial activity of povidone-iodine enables a composition of the invention to be used to treat ocular conjunctival or corneal infection caused by mycobacteria, viruses, fungi, and amoeba.

Additionally the composition is useful in the infectious prophylaxis of patients recovering from ophthalmic surgery.

In an embodiment, an ophthalmic composition is provided that is suitable for topical administration to an eye, effective for treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye. Prophylaxis may be, for example, prophylaxis from infection following surgery, prophylaxis from infection after birth for the newborn, or prophylaxis from accidental contact with contaminating material. Accidental contact with contaminating material may occur, for example, during surgery or during food processing.

In an aspect, the ophthalmic composition may be used for treatment and/or prophylaxis of a microorganism infection. The microorganism may be a bacterium, a virus, a fungus, or an amoeba, a parasite, or a combination thereof. In an embodiment, the bacteria may be a mycobacterium.

In an aspect, an ophthalmic composition may be used to treat a disorder such as, but not limited to, conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis, herpesvirus-related keratitis, ocular surface irregularity, tear deficiency, dry syndrome, meibomian gland dysfunction, blepharitis and uveitis. In another aspect, an ophthalmic composition may be used for prophylaxis of disorders such as conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis, herpesvirus-related keratitis, ocular surface irregularity, tear deficiency, dry syndrome, meibomian gland dysfunction, blepharitis and uveitis.

Disclosed herein is a method for treating and/or prophylaxis of an eye disorder or a microorganism infection of at least one tissue of the eye comprising the step of administering one of more doses of an ophthalmic composition, discussed above, to the eye. The eye disorder may be, for example, a microorganism infection of at least one tissue of the eye, conjunctivitis, corneal abrasion, ulcerative infectious keratitis, epithelial keratitis, stromal keratitis, herpes virus-related keratitis, ocular surface irregularity, tear deficiency, dry syndrome, meibomian gland dysfunction, and blepharitis. The microorganism may be bacteria (e.g., mycobacteria), virus, fungi, or amoebae.

In an embodiment, the dose volume administered to a subject may be between about 10 microliters and about 200 microliters, in another embodiment, between about 20 microliters and 100 microliters, and in another embodiment, between about 50 microliters and about 80 microliters, or about one drop per eye. Two or more drops may be added to an eye. Treatment of an eye may be effected by adding a single drop of composition disclosed herein, or by adding two or more drops, as required to achieve the desired result.

In an embodiment, administration frequency may be between 1 and 24 times a day. In an embodiment, administration frequency may be between 1 and 48 times a day. In another embodiment, administration frequency may be between 2 and 24 times a day. In another embodiment, administration frequency may be between 2 and 4 times a day. In another embodiment, administration frequency may be twice a day. In another embodiment, administration frequency may be once a day. In another embodiment, administration frequency may be less frequent than once a day. In another embodiment, administration frequency may be on demand, as therapeutic treatment is required or desired. In another embodiment, administration frequency may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, or 96 times a day.

In an embodiment, a composition disclosed herein is used for prophylaxis and/or treatment of a non-ophthalmic tissue by contacting the tissue with the composition.

The invention is further described by the following examples. It should be recognized that variations based on the inventive features are within the skill of the ordinary artisan, and that the scope of the invention should not be limited by the examples. To properly determine the scope of the present disclosure, an interested party should consider the claims herein, and any equivalent thereof. All patents, patent applications, and references cited herein are hereby incorporated by reference in their entirety.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

EXAMPLES The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Example 1: Stability Testing For Steroids Combined With Povidone Iodine The objective of this study was to determine whether povidone iodine (PVP-I) at the concentration of 4 mg/mL (0.4%) reacts with any of four different steroids (dexamethasone sodium phosphate, prednisolone acetate, loteprednol etabonate, and difluprednate), the active ingredients, in pharmaceutical formulations under both room temperature and 40 ºC for a time period of one month.

Dexamethasone sodium phosphate ophthalmic solution (USP, 0.1%) from Alcon Laboratories, prednisolone acetate ophthalmic suspension (USP, 1%) from Alcon Laboratories, loteprednol etabonate ophthalmic suspension (0.5%) from Bausch & Lomb, and difluprednate ophthalmic emulsion (0.05%) from Sirion Therapeutics were used for this study. PVP-I was prepared in water at the concentration of 100 mg/mL (10%). One milliliter of the solution, suspension, or emulsion was mixed with 40 µL of 10% PVP-I in 1.5 mL amber glass vials, followed by storage under both room temperature and 40 ºC for 2 weeks and one month. The resultant samples in the presence of PVP-I were analyzed using HPLC. The four steroid levels were measured against the reference standard samples stored under room temperature in the absence of PVP-I (0.4%). The One Month stability test samples were analyzed with the reference standard sample using LC-MS/MS Method in MRM mode with three characteristic ion transitions to confirm the identity of four steroids in stability testing samples. The presence of each of the four steroids in the respective pharmaceutical formulations tested was confirmed by LC/UV-MS and MS/MS. Thus, the four pharmaceutical formulations can be used in the study.

After storage under room temperature and 40 C at the presence of PVP-I (0.4%), the levels of dexamethasone phosphate in two week samples were only 83.04% and 84.57% of those in room temperature and 40 C Day 0 samples, respectively. The respective data are 84.24% and 84.09% for one month testing, indicating that dexamethasone phosphate was not stable in the presence of PVP-I (0.4%) under the current testing conditions. Three degradation products (D1, D2, and D3) were observed.

After storage under room temperature and 40 C in the presence of PVP-I (0.4%), the levels of prednisolone acetate in two week testing samples were 99.24% and 96.60% of those in room temperature and 40 C Day 0 samples, respectively. The respective data are 95.66% and 96.79% for one month testing. Identical mass ion chromatograms and same intensities of mass ion response were observed in the reference standard and one month stability testing samples.

The results from both HPLC/UV and LC-MS/MS analysis indicate that prednisolone acetate was stable in the presence of PVP-I (0.4%) under the current testing conditions.

After storage under room temperature and 40 C in the presence of PVP-I (0.4%), the levels of loteprednol etabonate in two week testing samples were 101.43% and 100.07% of those in room temperature and 40 C Day 0 samples, respectively. The respective data are 100.72% and 96.02% for one month testing. Identical mass ion chromatograms and same intensities of mass ion response were observed in the reference standard and one month stability testing samples. The results from both HPLC/UV and LC-MS/MS analysis indicate that loteprednol etabonate was stable in the presence of PVP-I (0.4%) under the current testing conditions.

After storage under room temperature and 40 C in the presence of PVP-I (0.4%), the levels of difluprednate in two week testing samples were 103.23% and 99.30% of those in room temperature and 40 C Day 0 samples, respectively. The respective data are 104.47% and 100.24% for one month testing. Identical mass ion chromatograms and same intensities of mass ion response were observed in the reference standard and one month stability testing samples.

The results from both HPLC/UV and LC-MS/MS analysis indicate that difluprednate was stable in the presence of PVP-I (0.4%) under the current testing conditions. 1. MATERIALS 1.1 Test Pharmaceutical Formulations The four steroids and their related pharmaceutical formulations are listed in Table I and Table II. 1.2 Povidone Iodine Povidone iodine (USP) was obtained from Spectrum Chemicals. Lot No. and expiration date are YQ0429 and January 31, 2011, respectively. 1.3 Solvents, Reagents, and Supplies OmniSolv Water was obtained from EM Science. Acetonitrile, methanol, and ammonium acetate were purchased from Sigma-Aldrich. 1.4 Suppliers and Equipment 1.4.1 Supplies • Serological Pipettes, Kimble Glass Inc • Wiretrol Micropipettes, Drummond Scientific Company • Autosampler Vials, Sun International • Automatic Pipettes, Gilson 1.4.2 Equipment • Sartorius Balances, BP301S, Sartorius Corporation 2. METHODS 2.1 Preparation of Stability Test Samples 2.1.1 Preparation of PVP-I Solution (10%, 100mg/mL) Weigh 1 g of PVP-I and dissolve in 10 mL of water. 2.1.2 Preparation of Stability Test Samples 2.1.2.1 Preparation of Dexamethasone Sodium Phosphate Stability Test Samples Aliquot 1 mL of ophthalmic solution (USP, 0.1%) into eight amber HPLC vials to give the following samples: Dexamethasone Sodium Phosphate-1, 2, 3, 4, 5, 6, 7, 8, and 9.

Added 40 µL of PVP-I stock solution (10%) into Dexamethasone Sodium Phosphate-3, 4, 5, and 6, and mixed well to give the following samples: Dexamethasone Sodium Phosphate+ PVP-I-3, 4, 5, and 6 Store Dexamethasone Sodium Phosphate+PVP-I-3 and 4 on the lab bench at room temperature and store Dexamethasone Sodium Phosphate+PVP-I -5 and 6 in a stability test chamber at 40 ºC.

Added 40 µL of water into Dexamethasone Sodium Phosphate-7, 8, and 9, and mixed well to give the following samples: Dexamethasone Sodium Phosphate+H O-7, 8, and 9 Stored Dexamethasone Sodium Phosphate+H O-9 on the lab bench at room temperature and store Dexamethasone Sodium Phosphate+H O-7 and 8 in a stability test chamber at 40 ºC.

Used Dexamethasone Sodium Phosphate+ PVP-I-3 and -5 and Dexamethasone Sodium Phosphate+H O-7 for two week stability test. Used Dexamethasone Sodium Phosphate+ PVP-I- 4 and -6 and Dexamethasone Sodium Phosphate+H O-8 for one month stability test. Used Dexamethasone Sodium Phosphate+H O-9 to prepare reference standard.

Stored Dexamethasone Sodium Phosphate-1 and 2 on the lab bench at room temperature.

On Week 4, added 40 µL of PVP-I (10%, freshly prepared) and mix well to give Dexamethasone Sodium Phosphate+PVP-I-1 and 2. Used the resultant samples as time zero samples for HPLC analysis. 2.1.2.2 Preparation of Prednisolone Acetate Stability Test Samples Aliquot ted1 mL of ophthalmic suspension (USP, 1%) into eight amber HPLC vials to give the following samples: Prednisolone Acetate-1, 2, 3, 4, 5, 6, 7, 8, and 9 Added 40 µL of PVP-I stock solution (10%) into Prednisolone Acetate -3, 4, 5, and 6, and mixed well to give the following samples: Prednisolone Acetate + PVP-I-3, 4, 5, and 6 Stored Prednisolone Acetate +PVP-I-3 and 4 on the lab bench at room temperature and stored Prednisolone Acetate +PVP-I -5 and 6 in a stability test chamber at 40 ºC.

Added 40 µL of water into Prednisolone Acetate -7, 8, and 9, and mixed well to give the following samples: Prednisolone Acetate +H O-7, 8, and 9 Stored Prednisolone Acetate +H O-9 on the lab bench at room temperature and stored Prednisolone Acetate +H O-7 and 8 in a stability test chamber at 40 ºC.

Used Prednisolone Acetate + PVP-I-3 and -5 and Prednisolone Acetate +H O-7 for two week stability test. Used Prednisolone Acetate + PVP-I-4 and -6 and Prednisolone Acetate +H O-8 for one month stability test. Used Prednisolone Acetate +H O-9 to prepare reference standard.

Stored Prednisolone Acetate -1 and 2 on the lab bench at room temperature. On Week 4, added 40 µL of PVP-I (10%, freshly prepared) and mixed well to give Prednisolone Acetate +PVP-I-1 and 2. Used the resultant samples as time zero samples for HPLC analysis. 2.1.2.3 Preparation of Difluprednate Stability Test Samples Aliquotted 1 mL of Ophthalmic emulsion (0.05%) into eight amber HPLC vials to give the following samples: Difluprednate-1, 2, 3, 4, 5, 6, 7, 8, and 9 Added 40 µL of PVP-I stock solution (10%) into Difluprednate-3, 4, 5, and 6, and mixed well to give the following samples: Difluprednate+ PVP-I-3, 4, 5, and 6 Stored Difluprednate+PVP-I-3 and 4 on the lab bench at room temperature and stored Difluprednate+PVP-I -5 and 6 in a stability test chamber at 40 ºC.

Added 40 µL of water into Difluprednate-7, 8, and 9, and mixed well to give the following samples: Difluprednate+H O-7, 8, and 9 Stored Difluprednate+H O-9 on the lab bench at room temperature and stored Difluprednate+H O-7 and 8 in a stability test chamber at 40 ºC.

Used Difluprednate+ PVP-I-3 and -5 and Difluprednate+H2O-7 for two week stability test. Used Difluprednate+ PVP-I-4 and -6 and Difluprednate+H O-8 for one month stability test.

Used Difluprednate+H O-9 to prepare reference standard.

Stored Difluprednate-1 and 2 on the lab bench at room temperature. On Week 4, added 40 µL of PVP-I (10%, freshly prepared) and mix well to give Difluprednate+PVP-I-1 and 2.

Used the resultant samples as time zero samples for HPLC analysis. 2.1.2.4 Preparation of Loteprednol Etabonate Stability Test Samples Aliquotted 1 mL of ophthalmic solution (USP, 0.1%) into eight amber HPLC vials to give the following samples: Loteprednol Etabonate-1, 2, 3, 4, 5, 6, 7, 8, and 9 Added 40 µL of PVP-I stock solution (10%) into Loteprednol Etabonate-3, 4, 5, and 6, and mix well to give the following samples: Loteprednol Etabonate+ PVP-I-3, 4, 5, and 6 Stored Loteprednol Etabonate+PVP-I-3 and 4 on the lab bench at room temperature and stored Loteprednol Etabonate+PVP-I -5 and 6 in a stability test chamber at 40 ºC.

Added 40 µL of water into Loteprednol Etabonate-7, 8, and 9, and mixed well to give the following samples: Loteprednol Etabonate+H O-7, 8, and 9 Stored Loteprednol Etabonate+H O-9 on the lab bench at room temperature and stored Loteprednol Etabonate+H O-7 and 8 in a stability test chamber at 40 ºC.

Used Loteprednol Etabonate+ PVP-I-3 and -5 and Loteprednol Etabonate+H O-7 for two week stability test. Used Loteprednol Etabonate+ PVP-I-4 and -6 and Loteprednol Etabonate+H2O-8 for one month stability test. Used Loteprednol Etabonate+H2O-9 to prepare reference standard.

Stored Loteprednol Etabonate-1 and 2 on the lab bench at room temperature. On Week 4, added 40 µL of PVP-I (10%, freshly prepared) and mix well to give Loteprednol Etabonate+PVP-I-1 and 2. Used the resultant samples as time zero samples for HPLC analysis. 2.2 Preparation of Stability Test Samples for HPLC/UV Analysis 2.2.1 Preparation of PVP-I Solution for HPLC/UV Analysis 2.2.1.1 Preparation of PVP-I-4 mg/mL Mixed 40 µL of PVP-I (10%) with 1 mL of water to give PVP-I-4 mg/mL. 2.2.1.2 Preparation of PVP-I Solution for Dexamethasone Sodium Phosphate Testing Mixed 100 µL of PVP-I-4 mg/mL with 1.9 mL of water to give PVP-I-200 µg/L for HPLC analysis. 2.2.1.3 Preparation of PVP-I Solution for Prednisolone Acetate Testing Mixed 100 µL of PVP-I-4 mg/mL with 9.9 mL of acetonitrile:water (1:1) to give PVP-I- 40 µg/L.

Mixed 750 µL of PVP-I-40 µg/L with 750 µL of acetonitrile:water (1:1) to give PVP-I-20 µg/L for HPLC analysis. 2.2.1.4 Preparation of PVP-I Solution for Difluprednate Testing Mixed 100 µL of PVP-I-4 mg/mL with 0.9 mL of methanol to give PVP-I-400 µg/L for HPLC analysis. 2.2.1.5 Preparation of PVP-I Solution for Loteprednol Etabonate Testing Mixed 100 µL of PVP-I-4 mg/mL with 9.9 mL of acetonitrile:water (1:1) to give PVP-I- 40 µg/L for HPLC analysis. 2.2.2 Preparation of Dexamethasone Sodium Phosphate for HPLC/UV Analysis 2.2.2.1 Preparation of Dexamethasone Sodium Phosphate Standard Mixed 100 µL of Dexamethasone Sodium Phosphate +H O-9 with 1.9 mL of H O in an HPLC vial to give Dexamethasone Sodium Phosphate+ H O50 µg/mL. 2.2.2.2 Preparation of Dexamethasone Sodium Phosphate Stability Test Samples Mixed 100 µL of Dexamethasone Sodium Phosphate+PVP-1, 2, 3, 4, 5, or 6 with 1.9 mL of H O in an HPLC vial to give Dexamethasone Sodium Phosphate+PVP-1, 2, 3, 4, 5, or 6- 50µg/mL for HPLC analysis. 2.2.2.3 Preparation of Control Dexamethasone Sodium Phosphate Stability Test Samples Mixed 100 µL of Dexamethasone Sodium Phosphate+H O-7, or 8 with 1.9 mL of H O in an HPLC vial to give Dexamethasone Sodium Phosphate+H O-7, or 8-50 µg/mL for HPLC analysis. 2.2.3 Preparation of Prednisolone Acetate for HPLC/UV Analysis 2.2.3.1 Preparation of Prednisolone Acetate Standard Mixed 100 µL of Prednisolone Acetate+H2O-9 with 9.9 mL of acetonitrile:water (1:1) to give Prednisolone Acetate+H O100 µg/mL.

Mixed 750 µL of Prednisolone Acetate+H O100 µg/mL with 750 µL of acetonitrile:H2O (1:1) in HPLC vial to give Prednisolone Acetate+H O50 µg/mL for HPLC analysis. 2.2.3.2 Preparation of Prednisolone Acetate Stability Test Samples Mixed 100 µL of Prednisolone Acetate+PVP-I-1, 2, 3, 4, 5, or 6 with 9.9 mL of acetonitrile:water (1:1) to give Prednisolone Acetate+PVP-I-1, 2, 3, 4, 5, or 6-100 µg/mL.

Mixed 750 µL of give Prednisolone Acetate+PVP-I-1, 2, 3, 4, 5, or 6-100 µg/mL with 750 µL of acetonitrile:H2O (1:1) in HPLC vial to give Prednisolone Acetate+PVP-I-1, 2, 3, 4, 5, or 6-50 µg/mL for HPLC analysis. 2.2.3.3 Preparation of Control Prednisolone Acetate Stability Test Samples Mixed 100 µL of Prednisolone Acetate+H O-7, or 8 with 9.9 mL of acetonitrile:water (1:1) to give Prednisolone Acetate+H O-7, or 8-100 µg/mL.

Mixed 750 µL of give Prednisolone Acetate+H O-7, or 8-100 µg/mL with 750 µL of acetonitrile:H2O (1:1) in HPLC vial to give Prednisolone Acetate+H O-7, or 8-50 µg/mL for HPLC analysis. 2.2.4 Preparation of Loteprednol Etabonate for HPLC/UV Analysis 2.2.4.1 Preparation of Loteprednol Etabonate Standard Mixed 100 µL of Loteprednol Etabonate+H O-9 with 9.9 mL of acetonitrile:water (1:1) to give Loteprednol Etabonate+H O50 µg/mL. 2.2.4.2 Preparation of Loteprednol Etabonate Stability Test Samples Mixed 100 µL of Loteprednol Etabonate+PVP-I-1, 2, 3, 4, 5, or 6 with 9.9 mL of acetonitrile:water (1:1) to give Loteprednol Etabonate+PVP-I-1, 2, 3, 4, 5, or 6-50 µg/mL. 2.2.4.3 Preparation of Control Loteprednol Etabonate Stability Test Samples Mixed 100 µL of Loteprednol Etabonate+H O-7, or 8 with 9.9 mL of acetonitrile:water (1:1) to give Loteprednol Etabonate+H O-7, or 8-50 µg/mL. 2.2.5 Preparation of Difluprednate for HPLC/UV Analysis 2.2.5.1 Preparation of Difluprednate Standard Mixed 100 µL of Difluprednate +H O-9 with 0.9 mL of methanol in an HPLC vial to give Difluprednate+ H O50 µg/mL. 2.2.5.2 Preparation of Difluprednate Stability Test Samples Mixed 100 µL of Difluprednate+PVP-1, 2, 3, 4, 5, or 6 with 0.9 mL of methanol in an HPLC vial to give Difluprednate+PVP-1, 2, 3, 4, 5, or 6-50µg/mL for HPLC analysis. 2.2.5.3 Preparation of Control Difluprednate Stability Test Samples Mixed 100 µL of Difluprednate+H O-7, or 8 with 0.9 mL of methanol in an HPLC vial to give Difluprednate+H O-7, or 8-50 µg/mL for HPLC analysis. 2.3 Preparation of Stability Test Samples for LC-MS/MS Analysis 2.3.1 Preparation of Dexamethasone Sodium Phosphate for LC-MS/MS Analysis 2.3.1.1 Preparation of Dexamethasone Sodium Phosphate Standard Mixed 100 µL of Dexamethasone Sodium Phosphate+ H O50 µg/mL with 0.9 mL of water in an HPLC vial. 2.3.1.2 Preparation of Dexamethasone Sodium Phosphate Stability Test Samples Mixed 100 µL of Dexamethasone Sodium Phosphate+PVP-4, or 6-50µg/mL with 0.9 mL of water in an HPLC vial. 2.3.2 Preparation of Prednisolone Acetate for HPLC Analysis 2.3.2.1 Preparation of Prednisolone Acetate Standard Mixed 100 µL of Prednisolone Acetate+H O50 µg/mL with 0.9 mL of acetonitrile:water (1:1) in an HPLC vial. 2.3.2.2 Preparation of Prednisolone Acetate Stability Test Samples Mixed 100 µL of Prednisolone Acetate+PVP-I-4, or 6-50 µg/ with 0.9 mL of acetonitrile:water (1:1) in an HPLC vial. 2.3.3 Preparation of Loteprednol Etabonate for HPLC Analysis 2.3.3.1 Preparation of Loteprednol Etabonate Standard Mixed 100 µL of Loteprednol Etabonate+H O50 µg/mL with 0.9 mL of acetonitrile:water (1:1) in an HPLC vial. 2.3.3.2 Preparation of Loteprednol Etabonate Stability Test Samples Mixed 100 µL of Loteprednol Etabonate+PVP-I-4, or 6-50 µg/mL with 0.9 mL of acetonitrile:water (1:1) in an HPLC vial. 2.3.4 Preparation of Difluprednate for HPLC Analysis 2.3.4.1 Preparation of Difluprednate Standard Mixed 100 µL of Difluprednate+ H O50 µg/mL with 0.9 mL of methanol in an HPLC vial. 2.3.4.2 Preparation of Difluprednate Stability Test Samples Mixed 100 µL of Difluprednate+PVP-4, or 6-50µg/mL for HPLC analysis with 0.9 mL of methanol in an HPLC vial. 2.4 HPLC/UV Chromatography 2.4.1 HPLC Method 1 (for Dexamethasone Sodium Phosphate) HPLC System: SHIMADZU HPLC system (Pump: LC-10ADVP; Autosampler: SIL-HTC) UV: SPD-10AVvp @239 and 210nm Column: Waters XTerra MS C18 3.5µm, 2.1x150mm, S/N 019435216117 Column Temperature: Room Temperature Autosampler Temperature: Room Temperature Injection Vol.: 10 µL Mobile Phase A: 0.01M NH4OAc in H2O Mobile Phase B: ACN Gradient: Time (min) Flow (mL/min) A B Initial 0.2 100 0 40 0.2 40 60 45 0.2 2 98 50 0.2 2 98 51 0.2 100 100 70 0.2 Stop 2.4.2 HPLC Method 2 (for Prednisolone Acetate) The same as Method 1 except the gradient was changed as follows: Time (min) Flow (mL/min) A B Initial 0.2 100 0 40 0.2 30 70 45 0.2 2 98 50 0.2 2 98 51 0.2 100 100 70 0.2 Stop 2.4.3 HPLC Method 3 (for Loteprednol Etabonate and Difluprednate) The same as Method 1 except the gradient was changed as follows: Time (min) Flow (mL/min) A B Initial 0.2 100 0 40 0.2 20 80 45 0.2 2 98 50 0.2 2 98 51 0.2 100 100 70 0.2 Stop 2.4.4 Date Integration and Calculation The software provided with the HPLC system (LCSolution software, version 1.23, installed by SHIMADZU) was used to integrate the peak area.

The measured peak area was converted into concentrations (µg/mL) using the following equation: C =A Cs A x x x where, C = Concentration (µg/mL) of analyte in stability samples A = Peak area from analyte in stability samples Cs = Concentration (µg/mL) of analyte in standard samples A = Peak area from analyte in standard samples 2.5 Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) HPLC Methods: The same as HPLC Method 1,2, and 3 under Section 2.4.

MS Conditions: Mass Spectrometer: API 3000 LC/MS/MS System Ionization Mode: ESI in Positive mode ESI: 5,000 V Temperature: 350 C Nebulizer Gas Flow (NEB): 12 psi Curtain Gas Flow (CUR): 12 units Turbo-Ion Spray Gas Flow: 7,000-8,000 mL/min Collision Gas (CAD): 6 units DP: 30 FP: 80 EP: 8 CXP: 10 Precursor Ion, Product Ion, Collision Energy, and HPLC Retention Time Compound Precursor ion Product ion Collision Retention (m/z) (m/z) Energy (eV) Time (min) 473.3 355.2 20 ~21.82 Dexamethasone 473.3 337.2 20 ~21.82 Phosphate 473.3 237.2 35 ~21.82 403.1 325.2 20 ~27.62 307.2 20 ~27.62 Prednisolone Acetate 403.1 403.1 147.1 30 ~27.62 467.3 359.2 20 ~33.15 Loteprednol 467.3 265.2 30 ~33.15 Etabonate 467.3 147.1 35 ~33.15 509.3 303.2 20 ~31.85 Difluprednate 509.3 279.2 20 ~31.85 509.3 101.1 30 ~31.85 3. RESULTS 3.1 LC/MS and MS/MS Analyses of Four Formulations The four formulations used in this study were analyzed by HPLC –UV and MS and MS/MS. The HPLC-UV chromatograms and ESI-MS and MS/MS spectral data were presented in Figure 1 to Figure 4.

The presence of four steroids in the pharmaceutical formulations was confirmed by LC/UV-MS and MS/MS. Thus, the four pharmaceutical formulations can be used for this study. 3.2 HPLC System Suitability Testing The four standard samples at the concentration of 50 µg/mL were analyzed using HPLC/UV methods developed at PharmaOn. The data are summarized in Table III.

As shown in Table III, the system used in this study was suitable to determine the levels of four steroids in the stability test samples. 3.3 HPLC/UV and LC-MS/MS Analysis of Stability Testing Samples 3.3.1 Dexamethasone Sodium Phosphate 3.3.1.1 PVP-I Sample PVP-I in solvent at the same concentration as in stability test samples of dexamethasone sodium phosphate was analyzed using HPLC Method 1. The HPLC/UV chromatograms are depicted in Figure 5.

No dexamethasone phosphate was observed in PVP-I sample. 3.3.1.2 Dexamethasone Sodium Phosphate Stability Samples The Day 0, Two Week, and One Month stability test samples were analyzed with reference standard samples (stored at room temperature in the absence of PVP I) using HPLC Method 1. The sample in the absence of PVP-I with the same concentration of dexamethasone phosphate as those stability samples at the presence of PVP-I was stored in the same stability chamber at 40°C for one month as control sample. The control sample was analyzed under the same conditions. The concentrations of dexamethasone phosphate in the stability samples were calculated. The data were summarized in Table IV. The HPLC/UV chromatograms of all reference standards and stability testing samples are depicted in Figure 6 to Figure 13.

The One Month stability test samples were analyzed with the reference standard sample using LC-MS/MS Method in MRM mode with three characteristic ion transitions to confirm the identity of dexamethasone phosphate in stability testing samples. The mass ion chromatograms are presented in Figure 14 to Figure 16.

Identity of dexamethasone phosphate in reference standard sample and two One month stability test samples was confirmed by LC-MS/MS.

After storage at room temperature and 40°C in the presence of PVP-I (0.4%), the levels of dexamethasone phosphate in two weeks samples were only 83.04% and 84.57% of those in room temperature and 40°C Day 0 samples, respectively (Table IV). The respective data are 84.24% and 84.09% for one month testing (Table IV), indicating that dexamethasone phosphate was not stable in the presence of PVP-I (0.4%) under the current testing conditions.

As shown in Figure 6 to Figure 13, three additional peaks, Degradation Product 1, 2, and 3 (D1, D2, and D2), were observed in both Two Week and/or One Month stability testing samples at the presence of PVP I. 3.3.2 Prednisolone Acetate 3.3.2.1 PVP-I Sample PVP-I in solvent at the same concentration as in stability test samples of prednisolone acetate was analyzed using HPLC Method 2. The HPLC/UV chromatograms are depicted in Figure 17.

No prednisolone acetate was observed in PVP-I sample. 3.3.2.2 Prednisolone Acetate Stability Samples The Day 0, Two Week, and One Month stability test samples were analyzed with reference standard samples (stored at room temperature in the absence of PVP I) using HPLC Method 2. The sample in the absence of PVP-I with the same concentration of prednisolone acetate as those stability samples at the presence of PVP-I was stored in the same stability chamber at 40 oC for two week and one month as control samples. The control samples were analyzed under the same conditions. The concentrations of prednisolone acetate in the stability samples were calculated. The data were summarized in Table V. The HPLC/UV chromatograms of all reference standards and stability testing samples are depicted in Figure 18 to Figure 23.

The One Month stability test samples were analyzed with the reference standard sample using LC-MS/MS Method in MRM mode with three characteristic ion transitions to confirm the identity of prednisolone acetate in stability testing samples. The mass ion chromatograms are presented in Figure 24 to Figure 26.

After storage at room temperature and 40°C in the presence of PVP-I (0.4%), the levels of prednisolone acetate in two week testing samples were 99.24% and 96.60% of those in room temperature and 40°C Day 0 samples, respectively (Table V). The respective data are 95.66% and 96.79% for one month testing (Table V). Identical mass ion chromatograms and same intensities of mass ion response were observed in the reference standard and one month stability testing samples. The results from both HPLC/UV and LC-MS/MS analysis indicate that prednisolone acetate was stable in the presence of PVP-I (0.4%) under the current testing conditions. 3.3.3 Loteprednol Etabonate 3.3.3.1 PVP-I Sample PVP-I in solvent at the same concentration as in stability test samples of loteprednol etabonate was analyzed using HPLC Method 3. The HPLC/UV chromatograms are depicted in Figure 27.

No loteprednol etabonate was observed in PVP-I sample. 3.3.3.2 Loteprednol Etabonate Stability Samples The Day 0, Two Week, and One Month stability test samples were analyzed with reference standard samples (stored at room temperature in the absence of PVP I) using HPLC Method 3. The sample in the absence of PVP-I with the same concentration of loteprednol etabonate as those stability samples at the presence of PVP-I was stored in the same stability chamber at 40°C for two week and one month as control samples. The control samples were analyzed under the same conditions. The concentrations of loteprednol etabonate in the stability samples were calculated. The data were summarized in Table VI. The HPLC/UV chromatograms of all reference standards and stability testing samples are depicted in Figure 28 to Figure 33.

The One Month stability test samples were analyzed with the reference standard sample using LC-MS/MS Method in MRM mode with three characteristic ion transitions to confirm the identity of loteprednol etabonate in stability testing samples. The mass ion chromatograms are presented in Figure 34 to Figure 36.

After storage at room temperature and 40°C in the presence of PVP-I (0.4%), the levels of loteprednol etabonate in two week testing samples were 101.43% and 100.07% of those in room temperature and 40°C Day 0 samples, respectively (Table VI). The respective data are 100.72% and 96.02% for one month testing (Table VI). Identical mass ion chromatograms and same intensities of mass ion response were observed in the reference standard and one month stability testing samples. The results from both HPLC/UV and LC-MS/MS analysis indicate that loteprednol etabonate was stable in the presence of PVP-I (0.4%) under the current testing conditions. 3.3.4 Difluprednate 3.3.4.1 PVP-I Sample PVP-I in solvent at the same concentration as in stability test samples of difluprednate was analyzed using HPLC Method 3. The HPLC-UV chromatograms are depicted in Figure 37.

No difluprednate was observed in PVP-I sample. 3.3.4.2 Difluprednate Stability Samples The Day 0, Two Week, and One Month stability test samples were analyzed with reference standard samples (stored at room temperature in the absence of PVP I) using HPLC Method 3. The sample in the absence of PVP-I with the same concentration of difluprednate as those stability samples at the presence of PVP-I was stored in the same stability chamber at 40°C for two week and one month as control samples. The control samples were analyzed under the same conditions. The concentrations of difluprednate in the stability samples were calculated.

The data were summarized in Table VII. The HPLC/UV chromatograms of all reference standards and stability testing samples are depicted in Figure 38 to Figure 43.

The One Month stability test samples were analyzed with the reference standard sample using LC-MS/MS Method in MRM mode with three characteristic ion transitions to confirm the identity of difluprednate in stability testing samples. The mass ion chromatograms are presented in Figure 44 to Figure 46.

After storage at room temperature and 40°C in the presence of PVP-I (0.4%), the levels of difluprednate in two week testing samples were 103.23% and 99.30% of those in room temperature and 40°C Day 0 samples, respectively (Table VII). The respective data are 104.47% and 100.24% for one month testing (Table VII). Identical mass ion chromatograms and same intensities of mass ion response were observed in the reference standard and one month stability testing samples. The results from both HPLC/UV and LC-MS/MS analysis indicate that difluprednate was stable in the presence of PVP-I (0.4%) under the current testing conditions.

TABLES Table I Four Pharmaceutical Formulations Steroids Name Formulation/Product Manufacture/Vendor Lot No.

Dexamethasone Ophthalmic solution Alcon Laboratories 153643F Sodium Phosphate USP, 0.1% Ophthalmic Prednisolone Suspension USP, Alcon Laboratories 148757F Acetate Loteprednol Ophthalmic Bausch & Lomb 437291 Etabonate Suspension, 0.5% Ophthalmic Difluprednate Sirion Therapeutics SIR9F001 emulsion, 0.05% Table II Four Steroids Name Structure MW Rt (min) O P ONa Dexamethasone 516.41 ~21.13 Sodium Phosphate Prednisolone Acetate 402.49 ~26.51 Loteprednol O 466.96 ~32.15 Etabonate Difluprednate 508.56 ~31.04 Table III Summary of System Suitability Testing Dexamethasone Sodium Phosphate Replicate HPLC Run No. Rt (min) Peak Area 1 09701005_002 21.16 4860116 2 09701005_003 21.12 4887168 3 09701005_004 21.16 4845056 4 09701005_005 21.12 4841633 09701005_006 21.11 4815314 Mean 21.13 4849857 SD 0.024 26369 CV (%) 0.11 0.54 Prednisolone Acetate Replicate HPLC Run No. Rt (min) Peak Area 1 09701005_012 26.53 5275846 2 09701005_013 26.52 5280425 3 09701005_014 26.54 5197617 4 09701005_015 26.39 5262924 09701005_016 26.55 5237854 Mean 26.51 5250933 SD 0.066 34088 CV (%) 0.25 0.65 Loteprednol Etabonate Replicate HPLC Run No. Rt (min) Peak Area 1 09701005_017 32.19 4352552 2 09701005_018 32.27 4272956 3 09701005_019 32.11 4368753 4 09701005_020 32.11 4281766 09701005_021 32.08 4292832 Mean 32.15 4313772 SD 0.078 43748 CV (%) 0.24 1.01 Difluprednate Replicate HPLC Run No. Rt (min) Peak Area 1 09701005_007 31.02 4746034 2 09701005_008 31.02 4715228 3 09701005_009 31.04 4761819 4 09701005_010 31.06 4715455 09701005_011 31.07 4728211 Mean 31.04 4733349 SD 0.023 20288 CV (%) 0.07 0.43 Table IV Analytical Data Summary of Dexamethasone Sodium Phosphate Stability Testing in PVP-I (0.4 %) Nominal Conc. Calc Conc. Calc Conc. a b c d Samples HPLC Run No. Rt (min) Peak Area (µg/mL) (µg/mL) DF (mg/mL) % of Std % of Day 0 Day 0 Std1 09701006_002 20.96 4964292 Std2 09701006_003 21.23 4873676 Mean 4918984 50 — 20 1.0000 — — Room Temp 1 09701006_004 21.17 5084959 51.69 20 1.0337 103.37 — Room Temp 2 09701006_005 21.20 5093624 51.78 20 1.0355 103.55 — Mean 5089292 51.73 20 1.0346 103.46 — 2 Weeks Std1 09701004_001 23.91 5019426 Std2 09701004_002 23.07 5004047 Mean 5011737 50 — 20 1.0000 — — Room Temp 09701004_003 23.08 4305845 42.96 20 0.8592 85.92 83.04 40 C 09701004_004 23.08 4385137 43.75 20 0.8750 87.50 84.57 One Month Std1 09701007_023 20.99 4845855 Std2 09701007_024 21.03 4810095 Mean 4827975 50 — 20 1.0000 — — Room Temp 09701007_025 21.06 4207982 43.58 20 0.8716 87.16 84.24 40 C 09701007_026 21.08 4216932 43.67 20 0.8734 87.34 84.42 40 C 09701007_027 21.07 4184100 43.33 20 0.8666 86.66 83.76 Mean 4200516 43.50 20 0.8700 87.00 84.09 40 C Control 09701007_028 21.11 4471624 46.31 20 0.9262 92.62 92.62 a b c d e : Nominal concentration in HPLC samples; : Calculated concentration in HPLC samples; : Dilution factor; : Calculated concentration in stability samples; : Stored at 40 C without PVP-I.

Table V Analytical Data Summary of Prednisolone Acetate Stability Testing in PVP-I (0.4 %) Nominal Conc. Calc Conc. Calc Conc. a b c d Samples HPLC Run No. Rt (min) Peak Area (µg/mL) (µg/mL) DF (mg/mL) % of Std % of Day 0 Day 0 Std1 09701006_010 26.74 5112497 Std2 09701006_011 26.75 5081143 Mean 5096820 50 — 200 10.000 — — Room Temp 1 09701006_012 26.76 5342803 52.41 200 10.483 104.83 Room Temp 1 09701006_013 26.77 5323574 52.22 200 10.445 104.45 Mean 5333189 52.32 20 10.464 104.64 2 Weeks Std1 09701004_012 27.70 5305927 Std2 09701004_013 27.73 5317386 Mean 5311657 50 — 200 10.000 — — Room Temp 09701004_014 27.74 5515685 51.92 200 10.384 103.84 99.24 40 C 09701004_015 27.71 5369264 50.54 200 10.108 101.08 96.60 40 C Control 09701004_016 27.61 5351149 50.37 200 10.074 100.74 100.74 One Month Std1 09701007_012 26.78 5181293 Std2 09701007_013 26.79 5127543 Mean 5154418 50 — 200 10.000 — — Room Temp 09701007_014 26.81 5159554 50.05 200 10.010 100.10 95.66 40 C 09701007_015 26.78 5220242 50.64 200 10.128 101.28 96.79 40 C Control 09701007_016 26.80 5169543 50.15 200 10.029 100.29 100.29 a b c d e : Nominal concentration in HPLC samples; : Calculated concentration in HPLC samples; : Dilution factor; : Calculated concentration in stability samples; : Stored at 40 C without PVP-I.

Table VI Analytical Data Summary of Loteprednol Etabonate Testing in PVP-I (0.4 %) Nominal Conc. Calc Conc. Calc Conc. a b c d Samples HPLC Run No. Rt (min) Peak Area (µg/mL) (µg/mL) DF (mg/mL) % of Std % of Day 0 Day 0 Std1 09701006_014 32.41 4172610 Std2 09701006_015 32.41 4193226 Mean 4182918 50 — 100 5.0000 — — Room Temp 1 09701006_016 32.45 4224688 50.50 100 5.0499 101.00 Room Temp 2 09701006_017 32.27 4180845 49.98 100 4.9975 99.95 Mean 09701006_017 32.27 4202767 50.24 20 5.0237 100.48 2 Weeks Std1 09701004_017 32.87 4460467 Std2 09701004_018 33.02 4431159 Mean 4445813 50 — 100 5.0000 — — Room Temp 09701004_019 33.03 4530572 50.95 100 5.0953 101.91 101.43 40 C 09701004_020 32.99 4470012 50.27 100 5.0272 100.54 100.07 40 C Control 09701004_021 32.98 4521010 50.85 100 5.0846 101.69 101.69 One Month Std1 09701007_017 32.45 4074874 Std2 09701007_018 32.30 4068504 Mean 4071689 50 — 100 5.0000 — — Room Temp 09701007_019 32.34 4120353 50.60 100 5.0598 101.20 100.72 40 C 09701007_020 32.48 3928248 48.24 100 4.8239 96.48 96.02 40 C Control 09701007_021 32.46 3975565 48.82 100 4.8820 97.64 97.64 a b c d e : Nominal concentration in HPLC samples; : Calculated concentration in HPLC samples; : Dilution factor; : Calculated concentration in stability samples; : Stored at 40 C without PVP-I.

Table VII Analytical Data Summary of Difluprednate Stability Testing in PVP-I (0.4 %) Nominal Conc. Calc Conc. Calc Conc. a b c d Samples HPLC Run No. Rt (min) Peak Area (µg/mL) (µg/mL) DF (mg/mL) % of Std % of Day 0 Day 0 Std1 09701006_006 31.17 4647615 Std2 09701006_007 31.10 4757011 Mean 4702313 50 — 10 0.5000 — — Room Temp 1 09701006_008 31.17 4503933 47.89 10 0.4789 95.78 — Room Temp 2 09701006_009 31.16 4548076 48.36 10 0.4836 96.72 — Mean 09701006_009 31.16 4526005 48.13 20 0.4813 96.25 — 2 Weeks Std1 09701004_007 31.76 4849758 Std2 09701004_008 31.76 4871971 Mean 4860865 50 — 10 0.5000 — — Room Temp 09701004_009 31.75 4829559 49.68 10 0.4968 99.36 103.23 40 C 09701004_010 31.74 4645691 47.79 10 0.4779 95.57 99.30 40 C Control 09701004_011 31.85 4350242 44.75 10 0.4475 89.50 89.50 One Month Std1 09701007_007 31.26 4519656 Std2 09701007_008 31.21 4538123 Mean 4528890 50 — 10 0.5000 — — Room Temp 09701007_009 31.20 4554140 50.28 10 0.5028 100.56 104.47 40 C 09701007_010 31.21 4369678 48.24 10 0.4824 96.48 100.24 40 C Control 09701007_011 31.24 4432171 48.93 10 0.4893 97.86 97.86 a b c d e : Nominal concentration in HPLC samples; : Calculated concentration in HPLC samples; : Dilution factor; : Calculated concentration in stability samples; : Stored at 40 C without PVP-I.

Example 2: Stability Testing For Steroids and NSAIDS Combined With 0.6% Povidone Iodine Steroids and NSAIDS were mixed with PVP-I at the concentration of 0.6% w/w on Day 1. The resultant mixtures will be split to glass vials and stored at room temperature. fluorometholone alcohol, medrysone, prednisone sodium phosphate, rimexolone, hydrocortisone, hydrocortisone acetate, lodoxamide tromethamine, nepafenac, bromfenac, and ketorolac. Testing timepoints included day 0 (Time Zero), and week 4. Tests were conducted at room temperature. The testing samples were analyzed using liquid chromatography and tandem mass spectrometry (LC/MS/MS) methods at Day 0, and Week 4. The steroids and NSAIDS standards were also analyzed and steroids and NSAIDS levels in testing samples were determined.

Rimexolone, hydrocortisone acetate, lodoxamide, and bromfenac samples appeared to be stable. Nepafenac was generally stable, but to a lesser degree. Prednisone sodium phosphate was stable to a lesser degree than nepafenac. In an embodiment, a result wherein about 10% or greater reduction in concentration of a compound of interest is observed is an indication that the compound is not stable. In an embodiment, a result wherein a reduction in the concentration of a compound of interest is observed, but about less than 10% reduction in concentration of a compound of interest is observed, is an indication that the compound is semi-stable. In an embodiment, a result wherein there is substantially no reduction in concentration of a compound of interest observed is an indication that the compound is stable.

Table VIII illustrates the analytical data summary of bromfenac stability testing in 0.6% PVP-I at room temperature. Table IX illustrates the analytical data summary of hydrocortisone acetate stability testing in 0.6% PVP-I at room temperature. Table X illustrates the analytical data summary of rimexolone stability testing in 0.6% PVP-I at room temperature. Table XI illustrates the analytical data summary of prednisone sodium phosphate stability testing in 0.6% PVP-I at room temperature. Table XII illustrates the analytical data summary of nepafenac stability testing in 0.6% PVP-I at room temperature.

Table XIII illustrates the analytical data summary of fluorometholone stability testing in 0.6% PVP-I at room temperature. For Tables VIII-XIII, a: Nominal concentration in HPLC samples; b: Calculated concentration in HPLC samples; c: Dilution factor; d: Calculated concentration in stability samples; e: Spiked 50µL of H O and stored at room temperature without PVP-I.

Table VIII: Bromfenac testing.

Samples Rt (min) Peak Area Nominal Calc DF Calc Conc. % of Std % of Day 0 Conc. Conc. a b d (µg/mL) (µg/mL) (µg/mL) Standard 1 24.925 11390037 90 — 10 900 — — Standard 2 25.034 11288449 90 — 10 900 — — Mean 24.980 11339243 90 — 10 900 — — Day 0 Replicate 1 24.900 11310534 90 89.77 10 897.7 99.74 — Replicate 2 24.889 11107933 90 88.16 10 881.6 97.96 — Mean 24.895 11209234 90 88.97 10 889.7 98.86 — Four Weeks Replicate 1 24.960 11211003 90 88.98 10 889.8 98.87 100.01 Replicate 2 24.963 11066657 90 87.84 10 878.4 97.6 98.73 Mean 24.962 11138830 90 88.41 10 884.1 98.23 99.37 Control 24.978 11342445 90 90.03 10 900.3 100.03 101.19 Table IX: Hydrocortisone acetate testing.

Samples Rt (min) Peak Area Nominal Calc DF Calc Conc. % of Std % of Day 0 Conc. Conc. a b d (µg/mL) (µg/mL) (µg/mL) Standard 1 29.087 9578995 100 — 50 5000 — — Standard 2 29.215 9456921 100 — 50 5000 — — Mean 29.151 9517958 100 — 50 5000 — — Day 0 Replicate 1 29.067 9672596 100 101.62 50 5081 101.62 — Replicate 2 29.107 9472035 100 99.52 50 4976 99.52 — Mean 29.087 9572316 100 100.57 50 5029 100.57 — Four Weeks Replicate 1 29.125 9627042 100 101.15 50 5058 101.15 100.58 Replicate 2 29.127 9699896 100 101.91 50 5096 101.91 101.33 Mean 29.126 9663469 100 101.53 50 5077 101.53 100.95 Control 29.178 9676282 100 101.66 50 5083 101.66 101.08 Table X: Rimexolone testing.

Samples Rt (min) Peak Area Nominal Calc DF Calc Conc. % of Std % of Day Conc. Conc. 0 a b d (µg/mL) (µg/mL) (µg/mL) Standard 1 39.98 3399891 100 — 100 10,000 — — Standard 2 39.961 3404392 100 — 100 10,000 — — Mean 39.971 3402142 100 — 100 10,000 — — Day 0 Replicate 40.004 3362494 100 98.83 100 9883 98.83 — Replicate 40.018 3418997 100 100.5 100 10050 100.5 — Mean 40.011 3390746 100 99.67 100 9967 99.67 — Four Weeks Replicate 40.035 3398853 100 99.9 100 9990 99.9 100.23 Replicate 39.948 3375059 100 99.2 100 9920 99.2 99.53 Mean 39.992 3386956 100 99.55 100 9955 99.55 99.88 Control 20.117 3303121 100 97.09 100 9709 97.09 97.41 Table XI: Prednisone sodium phosphate testing.

Samples Rt Peak Area Nominal Calc DF Calc % of Std % of (min) Conc. Conc. Conc. Day 0 a b d (µg/mL) (µg/mL) (µg/mL) Standard 1 26.61 8422981 100 — 50 5000 — — Standard 2 26.748 8470831 100 — 50 5000 — — Mean 26.679 8446906 100 — 50 5000 — — Day 0 Replicate 1 26.843 8272276 100 97.93 50 4897 97.93 — Replicate 2 26.717 8243394 100 97.59 50 4880 97.59 — Mean 26.780 8257835 100 97.76 50 4888 97.76 — Four Weeks Replicate 1 26.608 7853275 100 92.97 50 4649 92.97 95.1 Replicate 2 26.738 7946048 100 94.07 50 4704 94.07 96.23 Mean 26.673 7899661.5 100 93.52 50 4676 93.52 95.66 Control 26.477 8495335 100 100.57 50 5029 100.57 102.87 Table XII: Nepafenac testing (270nm).

Samples Rt Peak Nominal Calc DF Calc Conc. % of Std % of Day 0 (min) Area Conc. Conc. a b d (µg/mL) (µg/mL) (µg/mL) Standard 1 34.589 727 50 — 100 5,000 — — Standard 2 34.580 729 50 — 100 5,000 — — Mean 34.585 728 50 — 100 5,000 — — Day 0 Replicate 1 34.568 715 50 49.11 100 4911 98.22 — Replicate 2 34.548 722 50 49.59 100 4959 99.18 — Mean 34.558 719 50 49.35 100 4935 98.7 — Four Weeks Replicate 1 34.538 703 50 48.28 100 4828 96.56 97.83 Replicate 2 34.577 694 50 47.66 100 4766 95.32 96.58 Mean 34.558 698.5 50 47.97 100 4797 95.94 97.2 Control 34.570 719 50 49.38 100 4938 98.76 100.06 Table XIII: Fluorometholone testing.

Samples Rt (min) Peak Nominal Calc DF Calc % of Std % of Day Area Conc. Conc. Conc. 0 a b d (µg/mL) (µg/mL) (µg/mL) Standard 1 38.664 1872 50 — 20 — — 1,000 Standard 2 38.614 1877 50 — 20 — — 1,000 Mean 38.639 1875 50 — 20 — — 1,000 Day 0 Replicate 1 38.648 1901 50 50.71 20 1014 101.42 — Replicate 2 38.646 1896 50 50.57 20 1011 101.14 — Mean 38.647 1899 50 50.64 20 1013 101.28 — Four Weeks Replicate 1 38.611 1861 50 49.64 20 993 99.28 98.03 Replicate 2 38.613 1877 50 50.07 20 1001 100.14 98.87 Mean 38.612 1869 50 49.85 20 997 99.7 98.44 Control 38.602 1860 50 49.61 20 992 99.22 97.97 It is to be understood that at least some of the descriptions of the invention have been simplified to focus on elements that are relevant for a clear understanding of the invention, while eliminating, for purposes of clarity, other elements that those of ordinary skill in the art will appreciate may also comprise a portion of the invention. However, because such elements are well known in the art, and because they do not necessarily facilitate a better understanding of the invention, a description of such elements is not provided herein.

Further, to the extent that the method does not rely on the particular order of steps set forth herein, the particular order of the steps should not be construed as limitation on the claims. The claims directed to the method of the present invention should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the steps may be varied and still remain within the spirit and scope of the present invention.

Claims

WHAT WE CLAIM IS:

1. An ophthalmic composition suitable for topical administration to an eye, comprising a) povidone-iodine in a concentration between 0.01% and 10% (w/w), and b) a steroid, wherein the steroid is prednisolone acetate, loteprednol etabonate or difluprednate, and wherein after a period of one month after mixing the povidone-iodine and the steroid, the steroid concentration is at least 95% by weight of the steroid starting concentration.

2. The ophthalmic composition of claim 1, comprising: 0.3 to 1% (w/w) polyvinylpyrrolidinone-iodine complex; 0.05 to 2% (w/w) steroid; 0.005% to 0.02% (w/w) EDTA; 0.01 to 0.5% (w/w) sodium chloride; 0.02 to 0.1% (w/w) tyloxapol; 0.5% to 2% (w/w) sodium sulfate; and 0.1 to 0.5% (w/w) hydroxyethylcellulose.

3. Use of an ophthalmic composition according to claim 1 or 2 for the manufacture of a medicament for the treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye.

4. Use of povidone-iodine and a steroid, wherein the steroid is prednisolone acetate, loteprednol etabonate or difluprednate, in the manufacture of a topical medicament for treatment and/or prophylaxis of a microorganism infection or a disorder of at least one tissue of the eye, wherein the concentration of povidone-iodine in the medicament is between 0.01% and 10% (w/w), and wherein after a period of one month after mixing the povidone- iodine and the steroid, the steroid concentration in the medicament is at least 95% by weight of the steroid starting concentration.

5. The use of claim 4, wherein the medicament comprises: 0.3 to 1% (w/w) polyvinylpyrrolidinone-iodine complex; 0.05 to 2% (w/w) steroid; 0.005% to 0.02% (w/w) EDTA; 0.01 to 0.5% (w/w) sodium chloride; 0.02 to 0.1% (w/w) tyloxapol; 0.5% to 2% (w/w) sodium sulfate; and 0.1 to 0.5% (w/w) hydroxyethylcellulose.

6. An ophthalmic composition of claim 1 or 2 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.

7. Use of any one of claims 3 to 5 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.