TW201350124A - Pharmaceutical compositions and methods for treating, controlling, ameliorating, or reversing conditions of dry eye - Google Patents

Abstract

A pharmaceutical composition comprises a polyethylene glycol having a molecular weight in the range from about 1, 000 to about 10, 000, and a water-soluble cellulose derivative having a molecular weight in the range from about 50, 000 to 120, 000. The composition can further comprise boric acid and/or phosphate, a non-ionic surfactant, and/or an ophthalmic therapeutic agent. The composition is effective in treating, controlling, ameliorating, or reversing one or more conditions or symptoms of dry eye.

Description

Pharmaceutical composition and method for treating, controlling, ameliorating or reversing the condition of dry eye Cross reference

This application is a partial succession application and claims the right to apply for patent application No. 13/116,100 dated May 26, 2011. The latter patent application claims the provisional patent application No. 61/ filed on June 25, 2010 358,463. Both of these applications are incorporated herein by reference.

The present invention relates to compositions and methods for making the eyes comfortable. In particular, the present invention relates to compositions and methods for treating, managing, ameliorating or reversing the condition or symptoms of the eye of a patient suffering from a dry eye condition.

Dry eye or dry keratoconjunctivitis ("KCS") usually causes most discomfort in ophthalmic patients. Untreated eye disease causes corrosive and abrasion of corneal epithelial cells and increases susceptibility. The development of the disease can lead to corneal ulcers and even blindness.

A variety of irritants, injuries, and medical conditions can cause an individual to provoke an event that ultimately leads to a lack of tear film that protects and nourishes the surface of the eye. There are environmental factors that can form and/or enhance the amount and quality of tears (such as high altitude, drought and windy climates), air pollution, dry air from central heating and central air conditioning, and exposure to cigarette smoke. Even the widespread use of computers can be a contributing factor, as research has shown that users are paying attention to them. Focusing on the computer screen, the blink rate is significantly reduced. Some advances in eye care have been linked to the increase in the number of individuals who have started with contact lenses and are currently using LASIK for vision correction and the number of individuals with recent dry eye syndrome. The use of contact lenses causes the tear film to be absorbed through the lens, creating physical stimulation of the conjunctiva in the eyelids. LASIK can have a secondary effect on eye damage because nerves are usually severed or excised during laser refractive surgery, which can result in at least temporary dry eye syndrome for several months.

Some diseases and some physical conditions may also cause individuals to suffer from dry eye syndrome. Such diseases or conditions include allergy, diabetes, lupus, Parkinson's disease, Sjogren's syndrome, rheumatoid arthritis, rosacea and other diseases or conditions. Drugs used in other diseases, including diuretics, antidepressants, allergy drugs, birth control pills, decongestants and other drugs, can cause or aggravate dry eye syndrome.

Age-related changes can also induce or aggravate dry eye syndrome. After menopause, women experience changes in hormone levels, which can trigger dry eye or worsen dry eye, and thyroid imbalance can cause similar changes. Eventually, aging itself can lead to a decrease in lipid production leading to dry eye syndrome.

In the human eye, the tear film covering the surface of the eye is composed of three layers from the outermost to the innermost; the lipid layer, the water layer and the mucosa layer. The mucosal layer in contact with the surface of the eye contains mucin, a high molecular weight glycoprotein that is used to coat and lubricate the cornea. Mucin is secreted by goblet cells present in the conjunctiva. The intermediate aqueous layer comprising the tear film body and promoting tear film spreading, infection control, and weight osmolality adjustment is produced by lacrimal glands located outside the eyelids. The outermost layer is a thin (less than 250 nm) layer containing a variety of lipids called "lipid" or "sebum". The rouge is secreted by the meibomian glands located in the upper and lower eyelids to form a lipid layer of the tear film for slowing the evaporation of the water layer. Impaired production of materials necessary to form any of these layers results in a lack of tear film and ultimately a dry eye condition.

Until recently, therapeutic interventions were still limited to mitigating measures to increase the moisture content of the eye. This is most often achieved by instillation of a fluid in the form of a solution, gel or ointment that acts as an artificial tear. However, these interventions only provide short-term relief of symptoms of dry eye, and none of them The underlying cause of this condition is targeted to promote natural tear film remodeling.

Therefore, there is an urgent need to develop advantageous compositions and methods that are effective in promoting the natural production of tear film and rebuilding or improving the surface of the eye damaged by dry eye patients. It is also desirable to have such compositions and methods that minimize side effects.

In general, the present invention provides improved pharmaceutical compositions and methods that are effective in promoting the natural production of tear film and reconstituting or improving the surface of damaged eye in patients with dry eye.

In one aspect, the invention provides such compositions and methods with minimal side effects.

In another aspect, the invention provides a pharmaceutical composition comprising one or more compounds that promote the production of one or more components of the tear film or the repair or amelioration of the damaged ocular surface of a dry eye patient.

In another aspect, the present invention provides a pharmaceutical composition comprising polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da. In one embodiment, the cellulose derivative is a nonionic water soluble cellulose derivative.

In another aspect, the present invention provides an aqueous pharmaceutical composition comprising polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da. In one embodiment, the composition is an aqueous solution.

In another aspect, the present invention provides an aqueous pharmaceutical composition comprising polyethylene glycol having a molecular weight in the range of from 2000 to 8,000 Da and a nonionic water-soluble cellulose derivative having a molecular weight in the range of from 60,000 to 100,000 Da. In one embodiment, the composition is an aqueous solution.

In another aspect, the invention provides a method of treating, managing, ameliorating or reversing the condition of dry eye. The method comprises administering to the affected eye a dose comprising a molecular weight in the range of 1,000 to 10,000 Da in an amount or frequency effective to treat, control, ameliorate or reverse the dry eye condition A pharmaceutical composition of polyethylene glycol and a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da. In one embodiment, the composition is an aqueous solution.

In another aspect, the invention provides a method of treating, managing, ameliorating or reversing the condition of dry eye. The method comprises administering to a diseased eye a pharmaceutical composition comprising a polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da in an amount and at a frequency such that This administration promotes wound healing, improves the protective ability of the diseased cornea or increases the production of mucin in the affected eye. In one embodiment, the composition is an aqueous solution.

Other features and advantages of the invention will be apparent from the description and appended claims.

Figure 1 shows the effect of the composition of the present invention on corneal epithelial regeneration in a Riken transformed human corneal epithelial cell. A single level of abrasion is formed on the HCEpiC monolayer. The cells are then incubated with minimal medium containing PEG 3350 or HPMC 2910. Wound healing was calculated from the difference between the baseline measurements and the gap width after 16 hours of incubation and expressed as a percentage of gap closure. The bars represent the difference in gap width between the baseline measurements and the treatment group (n=3) after 16 hours of incubation; the line on the bars represents the mean standard error. The raw data were analyzed successively using the one-way ANOVA and Dunnett's Method tests. * indicates statistical significance relative to vehicle control; p < 0.05.

Figure 2 shows the effect of PEG 3350 and hydroxypropyl methylcellulose on the death of HCEpiC cells induced by dryness. The cells were cultured in complete (containing HCGS) medium until confluent. Cells were pretreated with minimal medium containing PEG 3350 or HPMC 2910 for 10 minutes followed by drying for 0 to 45 minutes. Live cells (top panel) were labeled with calcein using a live/dead assay kit (Invitrogen) and dead cells were labeled with ethidium homodimer (bottom panel). N=8,* vs. medium control at the same time point; p<0.05.

Figure 3 shows the effect of NaCl high volume osmolality and PEG 3350 on the cell monolayer integral resistance of the Institute of Physical Chemistry of Japan.

Figure 4 shows the effect of the high volume molar permeation concentration of NaCl on the normalized resistance of the cell monolayer of the Institute of Physical and Chemical Research, Japan over a period of 3 hours.

Figure 5 shows the effect of the high volume molar permeation concentration of NaCl on the original resistance of the cell monolayer of the Institute of Physical and Chemical Research, Japan over a period of 3 hours.

Figure 6 shows the effect of sucrose high volume molar permeation concentration and PEG 3350 on the cell monolayer integral resistance of the Institute of Physical Chemistry of Japan.

Figure 7 shows the effect of a 24-hour time course sucrose high volume molar permeation concentration on the monolayer normalized resistance of the Japanese Institute of Physical Chemistry.

Figure 8 shows the effect of a 24-hour time course sucrose high volume molar permeation concentration on the cell monolayer primary resistance of the Institute of Physical and Chemical Research, Japan.

Figure 9 shows the effect of PEG-3350 on HCEpiC MUC1 and MUC16 mRNA levels. The cells were cultured in complete (containing HCGS) medium until confluent. Cells were treated with 3% or 10% PEG-3350 for 4, 8, 18 or 24 hours; or treated with 10% PEG-3350 for 2 hours followed by 2, 6, 16 or 22 hours. Total RNA was extracted from the cells and QPCR was performed using Taqman MUC1 or MUC16 primer/probe sets. (A) MUC1 mRNA; (B) MUC16. N = 3, * indicates the control relative to the same time point; p < 0.05.

Figure 10 shows the effect of 10% PEG-3350 on the activation of pAkt, pERK, pEGFR and pPI3K as shown by Western blotting. Human corneal epithelial cells (HCEpiC) were treated with a serum-free medium containing 10% PEG-3350 over a 16 hour time period in an attempt to understand the molecular mechanisms behind the positive effects of the observed corneal epithelial regeneration. Cell lysates were collected and protein activation was assessed by Western blot using antibodies that target key phosphorylation sites.

Figure 11 is a graphical representation of the time points of peak phosphorylation of pAkt, pERK and pEGFR. Treatment of human corneal epithelial cells (HCEpiC) with a serum-free medium containing 10% PEG-3350 over a 16-hour period, attempting to understand the positive effects of the observed corneal epithelial regeneration Molecular mechanism. Cell lysates were collected and protein activation was assessed by Western blot using antibodies that target key phosphorylation sites. The peak protein phosphorylation time point for each protein is shown. The ratio of phosphorylated protein to total non-phosphorylated protein is quantified by densitometry.

Figure 12 shows the distribution of ZO-1 and actin in RT-HCEpiC after 2 hours of PEG-3350 pretreatment and incubation with basic or hypertonic medium for 2 hours.

Figure 13 shows the distribution of ZO-1 and actin in RT-HCEpiC after 2 hours of 3% PEG-3350 pretreatment and incubation with basic or hypertonic medium for 2 hours.

Figure 14 shows the distribution of ZO-1 and actin in RT-HCEpiC after pretreatment with 2% 10% PEG-3350 and incubation with basic or hypertonic medium for 2 hours.

Figure 15 shows a comparison of ZO-1 and actin in RT-HCEpiC after pretreatment with 10% PEG-3350 for 2 hours and incubation with minimal medium for 2 hours.

Figure 16 shows a comparison of ZO-1 and actin in RT-HCEpiC after pretreatment with 2% 10% PEG-3350 and incubation with hypertonic medium for 2 hours.

Figure 17 shows a comparison of ZO-1 and actin in RT-HCEpiC after 2 hours of pretreatment with 10% PEG-3350 and incubation with hypertonic medium for 2 hours.

Figure 18 shows the effect of sucrose high volume osmolality and 3% PEG-3350 on the cell monolayer integral resistance of the Institute of Physical Chemistry of Japan.

Figure 19 shows the effect of a 24-hour time course sucrose high volume osmolality and a 3% PEG 3350 on the normalized resistance of the cell monolayer of the Institute of Physical and Chemical Research, Japan.

Figure 20 shows the effect of 24-hour time course sucrose high volume osmolality and 3% PEG 3350 on the original resistance of the cell monolayer of the Institute of Physical and Chemical Research, Japan.

Throughout the present invention, when the numerical range is recited, it is understood that the range includes the lower and upper limits.

In general, the present invention provides for effectively promoting the natural production and reconstruction or improvement of the tear film. Improved pharmaceutical compositions and methods for the surface of damaged eye in patients with dry eye.

In one aspect, the invention provides such compositions and methods with minimal side effects.

In another aspect, the invention provides a pharmaceutical composition comprising one or more compounds that promote the production of one or more components of the tear film or the repair or amelioration of the damaged ocular surface of a dry eye patient.

In another aspect, the present invention provides a pharmaceutical composition comprising polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da.

In another aspect, the present invention provides an aqueous pharmaceutical composition comprising polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da. In one embodiment, the composition is an aqueous solution.

In another aspect, the present invention provides an aqueous pharmaceutical composition comprising polyethylene glycol having a molecular weight in the range of from 2000 to 8,000 Da and a nonionic water-soluble cellulose derivative having a molecular weight in the range of from 60,000 to 100,000 Da. In one embodiment, the composition is an aqueous solution.

In another aspect, the invention provides a method of treating, managing, ameliorating or reversing one or more conditions of dry eye. The method comprises administering to a diseased eye a polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da and a molecular weight in the range of 50,000 to 120,000 Da in an amount and frequency effective to treat, control, ameliorate or reverse the dry eye condition. A pharmaceutical composition of a water-soluble cellulose derivative. In one embodiment, the condition comprises ocular surface discomfort, such as feeling dry, grit, stinging or lack of water layer, lipid or mucin production. In another embodiment, the composition is an aqueous solution.

In another aspect, the invention provides a method for treating, managing, ameliorating or reversing one or more conditions of dry eye. The method comprises administering to a diseased eye a polyethylene glycol comprising a molecular weight in the range of 1,000 to 10,000 Da and a molecular weight of 50,000 to A pharmaceutical composition of a water-soluble cellulose derivative in the range of 120,000 Da; wherein the administration promotes wound healing, improves the protective ability of the diseased cornea, or increases the production of mucin in the affected eye. In one embodiment, the composition is an aqueous solution.

In one embodiment, any of the pharmaceutical compositions of the present invention disclosed herein further comprise one or more ophthalmically acceptable ingredients that provide benefits to the patient, such as buffers, antioxidants, vitamins, viscosity modifying materials, tonicity adjustments. Materials, preservatives, demulcents, surfactants, pH adjusting materials, etc.

In another embodiment, the pharmaceutical composition of the present invention comprises: (a) a polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da; And (c) buffer. In one embodiment, the buffer comprises boric acid and/or phosphate buffer.

In another embodiment, the pharmaceutical composition of the present invention comprises: (a) a polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da; (c) a buffer selected from the group consisting of boric acid, phosphate buffer, and mixtures thereof; and (d) a pharmaceutically acceptable preservative.

In another embodiment, the pharmaceutical composition of the present invention comprises: (a) a polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da; And (c) a buffer selected from the group consisting of boric acid, a phosphate buffer, and mixtures thereof; (d) a pharmaceutically acceptable preservative; and (e) a preservative enhancing material selected from the group consisting of: D-glucose, sucrose, maltose, D-mannose, trehalose, glutamic acid, a mixture thereof, wherein the preservative efficacy enhancing material provides the pharmaceutical composition with an enhanced composition compared to a composition without the preservative enhancing material The antiseptic effect of spore-forming microorganisms.

In another aspect, the polyethylene glycol included in any of the compositions of the invention disclosed herein is selected from the group consisting of polyethylene glycol having a molecular weight in the range of from about 1,000 to about 10,000 Da. Alternatively, the polyethylene glycol is selected from a molecular weight of from about 2,000 to about 10,000 Da Within the range; or a group of polyethylene glycols ranging from about 3,000 to about 8,000 Da. Non-limiting examples of such polyethylene glycols are known by the common names PEG-1000, PEG-2000, PEG-3350, PEG-4000, PEG-6000, PEG-8000, and PEG-10000. Suitable polyethylene glycols having a molecular weight within this range are referred to as PEG-20, PEG-32, PEG-75, PEG-100 according to the CTFA (Cosmetic, Toiletry and Fragrance Association) nomenclature. PEG-150 has molecular weights of 1000, 1450, 3350, 4500 and 8000 Da, respectively. Particularly suitable polyethylene glycols are polyethylene glycols having a molecular weight in the range of from about 2,000 to about 8,000 Da.

The amount of polyethylene glycol in the compositions of the present invention is in the range of from about 2 to about 25 weight percent. Alternatively, the amount of polyethylene glycol in the composition of the invention is from about 2 to about 20% by weight, or from about 3 to about 20% by weight, or from about 3 to about 15% by weight, or from about 3 to about 12% by weight, or From about 3 to about 10% by weight, or from about 5 to about 15% by weight, or from about 5 to about 12% by weight, from about 5 to about 10% by weight, or from about 7 to about 25% by weight, or from about 7 to about 15 weight %, or from about 7 to about 12% by weight, or from about 7 to about 10% by weight. In one aspect, the amount of polymer included in the composition varies inversely with its molecular weight.

In another aspect, the water soluble cellulose derivative included in any of the compositions of the invention disclosed herein is selected from the group consisting of hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), A group consisting of hydroxyethyl cellulose (HEC), methyl cellulose, carboxymethyl cellulose (CMC), hydroxypropyl guar, and mixtures thereof. In a preferred embodiment, the water soluble cellulose derivative included in any of the compositions of the invention disclosed herein is HPMC. Various HPMC grades having different viscosities are commercially available, for example, from the Dow Chemical Company. Generally, the viscosity of these cellulose derivatives is specified as the apparent viscosity of a 2% by weight aqueous solution at 20 °C. The apparent viscosity of commercially available cellulose derivatives ranges from about 80 to about 14,000 cp.

The amount of the water-soluble cellulose derivative in the composition of the present invention is in the range of from about 0.1% by weight to about 10% by weight. Alternatively, the amount of water-soluble cellulose derivative in the composition of the invention is From about 0.1 to about 7% by weight, or from about 0.1 to about 5% by weight, or from about 0.1 to about 3% by weight, or from about 0.1 to about 2% by weight, or from about 0.1 to about 1% by weight, or from about 0.3 to about 3 % by weight, from about 0.3 to about 2% by weight, or from about 0.3 to about 1% by weight, or from about 0.4 to about 1% by weight, or from about 0.5 to about 1% by weight, or from about 1 to about 3% by weight, or about 1 Up to about 4% by weight, or from about 1 to about 5% by weight.

In another embodiment, the aqueous pharmaceutical composition of the present invention comprises: (a) a polyethylene glycol having a concentration in the range of from about 2 to about 25% by weight of the total composition in the range of from 1,000 to 10,000 Da; a water-soluble cellulose derivative having a concentration of from about 0.1 to about 10% by weight of the total composition in the range of from 50,000 to 120,000 Da; and (c) selected from the group consisting of boric acid, phosphate buffer, and mixtures thereof Group of buffers.

In another embodiment, any of the pharmaceutical compositions of the present invention further comprise a pharmaceutically active ingredient.

Any of the pharmaceutical compositions or formulations of the present invention disclosed herein may comprise: (a) polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da; (b) water-soluble cellulose having a molecular weight in the range of 50,000 to 120,000 Da a derivative; and (c) a buffer selected from the group consisting of boric acid, a phosphate buffer, and mixtures thereof; (d) a pharmaceutically acceptable preservative; and (e) an antioxidant. In one embodiment, the antioxidant is selected from the group consisting of BHT (butylated hydroxytoluene), thiosulfate (such as sodium, potassium, calcium or magnesium salts) and mixtures thereof.

Embodiments of the pharmaceutical compositions or formulations of the invention disclosed herein may comprise, consist of, or consist essentially of: (a) a concentration of from about 2 to about 25% by weight of the total composition of from 1,000 to 10,000 a polyethylene glycol in the range of Da; (b) a water-soluble cellulose derivative having a concentration of from about 0.1 to about 10% by weight of the total composition in the range of from 50,000 to 120,000 Da; and (c) selected from a buffer of a group consisting of boric acid, a phosphate buffer, and mixtures thereof; (d) a pharmaceutically acceptable preservative; (e) an antioxidant; and (f) water. In one embodiment, the antioxidant is selected from the group consisting of BHT (butylated hydroxytoluene), A group of thiosulfate (such as sodium, potassium, calcium or magnesium salts) and mixtures thereof.

The pharmaceutical compositions or formulations of the invention disclosed herein may comprise, consist of, or consist essentially of (a) polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da; (b) a molecular weight in the range of 50,000 to 120,000 Da. a water-soluble cellulose derivative; and (c) a buffer selected from the group consisting of boric acid, a phosphate buffer, and mixtures thereof; (d) a pharmaceutically acceptable preservative; (e) selected from the group consisting of BHT, An antioxidant comprising a group of thiosulfate (such as sodium, potassium, calcium or magnesium salts) and mixtures thereof; (f) a preservative enhancing material selected from the group consisting of D-glucose, sucrose, maltose, D- Mannose, trehalose, glutamic acid, a mixture thereof; and (g) water; wherein the pharmaceutical composition has enhanced preservative efficacy against spore-forming microorganisms.

In one aspect, a preservative included in any of the pharmaceutical compositions or formulations disclosed herein comprises, consists of, or consists essentially of one or more pharmaceutically acceptable alcohols, amines, and An ammonium compound, hydrogen peroxide, and a compound which produces hydrogen peroxide in the composition (such as carbenamine peroxide, carboguanamine hydroperoxide, carboguanamine or perborate), oxychloro compound ( Such as chlorine dioxide), zinc compounds or mixtures thereof. In one embodiment, the pharmaceutically acceptable preservative is selected from the group consisting of polytetra-ammonium-1, polytetra-ammonium-2, poly-quaternary ammonium-4, polytetra-ammonium-5, polytetra-ammonium- 6. Polytetra-ammonium-7, poly-quaternary ammonium-8, polytetra-ammonium-9, poly-quaternary ammonium-45, polytetra-ammonium-54, polytetra-ammonium-71 and polytetra-ammonium-72 a group of people. The chemical formulas of these compounds are known in the medical literature.

In a preferred embodiment, the pharmaceutically acceptable preservative is polytetraammonium-1 having the formula below.

Polytetraamethylene-1

In another embodiment, the pharmaceutically acceptable preservative is selected from the group consisting of hydrogen peroxide sources (such as perborate, peracetate or carbamide peroxide), hydrogen peroxide, and stabilized oxygen chloride complexes. And their mixtures.

In one aspect, when the composition of the present invention comprises an anti-corrosion enhancing material as disclosed above, the material provides the composition with enhanced preservative efficacy against spore forming microorganisms, otherwise the composition is within the comfort of the user. It cannot be achieved with low concentration of preservatives. In one embodiment, the spore forming microorganism is a mold or a yeast. In another embodiment, the preservative efficacy needs to comply with the European Pharmacopoeia A ("EP-A") standard.

In another embodiment, when the composition of the present invention comprises a preservative and a preservative enhancing material as disclosed above, the antiseptic enhancing material provides the composition with enhanced preservative efficacy against spore forming microorganisms, wherein the concentration of the preservative is separate The composition cannot be made to meet the EP-A preservative efficacy standard.

In another aspect, the spore forming microorganism is spore-forming Aspergillus oryzae (A. brasiliensis ).

Procedure for assessing the antiseptic efficacy ("PE") of a pharmaceutical formulation of the invention against microorganisms

The microorganisms against which the PE of the pharmaceutical formulation of the present invention is evaluated are S. aureus , E. coli , P. aeruginosa , C. albicans , and Brazil. Aspergillus. This procedure applies to the US FDA Pre-Sale Notice (510(k)) guidance document and the USP/ISO/DIS 14730 standard preservative efficacy test with a 14-day re-excitation test. Three individual batches of each test solution were evaluated for each microorganism. Each batch was tested with different formulations of each microorganism.

The bacterial cells are grown on a tryptic soy agar ("TSA") slant in a incubator at a temperature ranging from 30 ° C to 35 ° C for a period of 18 to 24 hours. The fungal cells are grown on a Sabouraud Dextrose Agar ("SDA") slope in a incubator at a temperature ranging from 20 ° C to 25 ° C for a period of 2 to 7 days. The cells were collected in saline solution (5-10 ml, USP, 0.9% saline, with or without 0.1% Tween 80 surfactant), and the saline solution was added to each agar slants, followed by slow agitation with a sterile cotton swab. The cell suspension was aseptically dispensed into separate sterile polypropylene centrifuge tubes. The cells were harvested by centrifugation at 3000 rpm for 10 minutes, washing once and suspended in saline TS to a concentration of 2 x 10 ⁸ cells per ml.

The test solution was diluted with 20ml of cell suspension (0.1ml) reaches 1.0 × 10 ⁵ to 1.0 × 10 ⁶ communities were forming units ( "CFU") of the final concentration. Phosphate buffered saline ("PBS") was used as a control solution. The inoculated test and control solutions were incubated in static culture at temperatures ranging from 20 °C to 25 °C. At time zero, 1 ml PBS (USP, pH 7.2) from the control solution was diluted with 9 ml PBS and serially diluted cells were applied in triplicate to TSA (for bacteria) and SDA (for fungi). The bacterial culture plate is incubated at a temperature ranging from 30 ° C to 35 ° C for a period of 2 to 4 days. The fungal culture plates are incubated at temperatures ranging from 20 ° C to 25 ° C for a period of 2 to 7 days.

Similarly, on day 7 and day 14, 1 ml volume from the test solution was added to 9 ml Dey-Engley Neutralization Medium ("DEB") and serially diluted in DEB and applied in triplicate to TSA (for bacteria) and SDA (for fungi). The bacterial culture plate is incubated at a temperature ranging from 30 ° C to 35 ° C for a period of 2 to 4 days. The fungal culture plates are incubated at temperatures ranging from 20 ° C to 25 ° C for a period of 2 to 7 days. Count the developing communities.

The test solution was inoculated again immediately after sampling on the 14th day so that the final concentration of each microorganism was 1.0 × 10 ⁴ to 1.0 × 10 ⁵ . At time zero, 1 ml from the inoculum control was added to 9 ml PBS and subsequent serial dilutions were applied in triplicate to TSA (for bacteria) and SDA (for fungi). The bacterial culture plate is incubated at a temperature ranging from 30 ° C to 35 ° C for a period of 2 to 4 days. The fungal culture plates are incubated at temperatures ranging from 20 ° C to 25 ° C for a period of 2 to 7 days.

On days 21 and 28, 1 ml from the test article was added to 9 ml DEB and serial dilutions were applied to TSA in triplicate. At temperatures between 30 ° C and 35 ° C The plates were incubated for 2 to 4 days and the developing communities were counted.

Based on the United States Pharmacopoeia ("USP") bacterial acceptance criteria, if the concentration of live bacteria recovered per ml is reduced by at least 1 log on day 7 (base 10 log or log ₁₀ ), reduce at least 3 log on day 14. And after re-excitation on day 14, the bacterial concentration was reduced by at least 3 log on day 28, and the solution was acceptable. In addition, if the concentration of live yeast and mold recovered per ml of solution on day 14 is maintained at or below the initial concentration (within experimental uncertainty of ±0.5 log) and lived on day 28 after re-excitation on day 14 The concentration of yeast and mold is maintained at or below the initial concentration (within experimental uncertainty of ±0.5 log) and the solution is acceptable.

It is worth noting that the acceptance criteria for products sold in Europe are stricter than the above guidelines. Pharmaceutical compositions that meet this more stringent criteria may be referred to as "enhanced anti-corrosion efficacy against microorganisms."

Based on a more stringent target acceptance criteria for bacteria ("EP-A" or European target guidelines), if the concentration of live bacteria recovered per ml is reduced by at least 2 log (log ₁₀ ) at the end of 6 hours, at 24 hours At the end of the reduction of at least 3 log, and after re-excitation on the 14th day, on the 28th day no bacteria for the recovery concentration ("no recovery" is considered to be equal to or greater than 4 log), the solution is acceptable. In addition, if the concentration of live yeast and mold recovered per ml of solution on day 7 is reduced by at least 2 log and re-excited on day 14, the concentration of live yeast and mold remains at or below the initial concentration on day 28 (in The solution is acceptable within ±0.5 log of experimental uncertainty.

Based on another set of more stringent bacterial acceptance criteria ("EP-B" or Europeanly accepted guidelines), if the concentration of live bacteria recovered per ml is reduced by at least 1 log (log ₁₀ ) at the end of 24 hours, at 7th The day is reduced by at least 3 log, and after re-excitation on day 14, the bacterial concentration is maintained at or below the initial concentration on day 28 (within experimental uncertainty of ±0.5 log), then the solution is acceptable. In addition, if the concentration of live yeast and mold recovered per ml of solution on day 14 is reduced by at least 1 log and re-excited on day 14, the concentration of live yeast and mold remains at or below the initial concentration on day 28 (in The solution is acceptable within ±0.5 log of experimental uncertainty.

The previously accepted criteria are summarized in Table 1.

"--" means "no requirement"

Any of the pharmaceutical compositions or formulations of the invention may be in the form of a solution, suspension, emulsion, dispersion, ointment or cream.

Any of the pharmaceutical compositions or formulations of the invention may be in the form of a solution or suspension or may comprise a solution or suspension.

Any pharmaceutical composition or formulation may be in the form of an aqueous solution or may comprise an aqueous solution.

Furthermore, the ophthalmic solution of the present invention may comprise an active pharmaceutical ingredient (or therapeutic agent) such as an anti-inflammatory agent, an antibiotic, an immunosuppressive agent, an antiviral agent, an antifungal agent, an antiprotozoal agent, a combination thereof, or a mixture thereof. Non-limiting examples of anti-inflammatory agents include glucocorticol (eg, for short-term treatment) and non-steroidal anti-inflammatory drugs ("NSAID").

Non-limiting examples of glucocorticols are: 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, times Betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprenol Cloprednol), corticosterone, cortisone, cortivazol, deflazacort, desonide, deoxymetazone (desoximetasone), dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort , flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluorocodone Fluocortolone), fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate ), formocortal, hacinionide, halobetasol propionate, halometasone, halopedone acetate, hydrocortannate ), hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone Methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamine Base-acetate, prednisolone sodium phosphate, prednisone, predival, prednylidene, rimexolone, teicosone (tixocortol), triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, its physiologically acceptable salt, its derivatives , combinations thereof, and mixtures thereof. In one embodiment, the therapeutic agent is selected from the group consisting of difluprednate, loteprednol carbonate, prednisolone, combinations thereof, and mixtures thereof.

Non-limiting examples of NSAIDs are: amine aryl carboxylic acid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid (flufenamic) Acid), isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, tirofenac ester (terofenamate), tolfenamic acid, aryl acetic acid derivatives (eg aceclofenac, acemetacin, alclofenac, aminfenic acid (amfenac) ), amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodo Acid (etodolac), fenbinac (fenbinac), fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin , isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pyrella Pyrazolac, proglumetacin, sulindac, tiaramide, tolmetin, tropa (tropesin), zomepirac, aryl butyric acid derivatives (eg, bumadizon, butibufen, fenbufen, xenbucin) , aryl carboxylic acids (such as clidanac, ketorolac, tinoridine), aryl propionic acid derivatives (such as alminprofen, phenoxazole) Benoxaprofen, bermofolfen, bucloxic acid, carprofen, fenoprofen, flonoxaprofen, flurbiprofen ), ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin (oxaprozin), piketoprofen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, Ximoprofen, zaltoprofen, pyrazole (eg difenamizole, eptazole) (epirizole)), pyrazolone (eg, apazone, benzpiperylon, feprazone, mofebutazone, morazone, hydroxybine) Oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, thiazolidine (thiazolinobutazone)), a salicylic acid derivative (such as acetaminosalol, aspirin, benorilate, bromosaligenin, acetamidine salicylic acid) Calcium, diflunisal, etersalate, fendosal, gentisic acid, diol salicylate, imidazolium salicylate, acetaminosalicylic acid Amino acid, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, acetaminophen phenyl salicylate Ester, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylic acid sulfate, double Salicylate, sulfasalazine, thiazide carboxamide (eg, ampiroxicam, droxicam, isoxicam, lornoxicam) , piroxicam, tenoxicam, ε-acetamidohexanoic acid, S-(5'-adenosyl)-L-methionine, 3-amino-4 - hydroxybutyric acid, amixetrine, bentazac, benzydamine, bisabolol, bucolome, difenpiramide, Ditazol, emorfazone, fepradinol, guaiazulene, nabumetone, nimesulide, oxa Oxaloprol, paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, zileuton, Physiologically acceptable salts, combinations thereof, and mixtures thereof.

Non-limiting examples of antibiotics include cranberries; aminosides (eg, amikacin) (amikacin), apramycin, arbekacin, bambermycin, butirosin, dibekacin, dihydrostreptomycin (dihydrostreptomycin), fortimicin, gentamicin, isepamicin, kanamycin, micronomicin, neomycin, Neodecanoic acid neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin Streptomycin), tobramycin, trospectomycin, valine alcohols (eg, azidamfenicol, chloramphenicol, florfenicol, Thiamphenicol, ansamycins (eg rifamide, rifampin, rifamycin SV, rifapentine) , rifaximin, β-indoleamines (eg, carbacephems (eg, loracarbef), carbapenems) Carbapenem (eg, biapenem, imipenem, meropenem, panipenem), cephalosporins (eg cefaclor (eg cefaclor) Cefaclor), cefadroxil, cefmandole, cefazozine, cefazedone, cefazolin, cefcapene pivoxil, cefixime (cefclidin), cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefinenoxime, cefodizime (cefclidin) Cefodizime), cefonicid, cefoperazone, ceforamide, cefotaxime, cefotiam, cefozopran, cefpimizole , cefpiramide, cefpirome, cefpodoxime proxetil, cetprozil, cefoxadine, cefsulodin, ceftazidime, cephalosporin special Cefteram, ceftezole, ceftibuten, ceftizoxime, ceftrixone, cefuroxime, cefazolam, cefsai Cephacetrile sodium, cephalexin, cephaloglycin, cephaloridine, cephalosporin, cephalothin, cephapirin sodium, cephalosporin Cephradine, pivcefalexin, cephamycins (eg cefbuperazone, cefinetazole, cefininox, cefotetan) ), cefoxitin, monocyclic beta-lactams (eg, aztreonam, carumonam, tigemonam), oxycephalosporin (oxacephems) (flomoxef, moxalactam), penicillins (eg, amdinocillin, amdinocillin pivoxil, amoxicillin, ampicillin) (ampicillin), apalcillin (apalcillin) ), apoxicillin, azidocillin, azlocillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, carboxybenzyl Carbenicillin, carindacillin, clometacillin, cloxacillin, cyclacillin, dicloxacillin, epicillin, fenbylillin (fenbenicillin), floxacillin, hetacillin, lenampicillin, metampicillin, methicillin sodium, mezlocillin, naf Nafcillin sodium, oxacillin, penamecillin, penehamate hydriodide, penicillin G benethamine, penicillin G benzathine ), penicillin G benzhydrylamine, penicillin G calcium (penicillin G Calcium, penicillin G hydrabamine, penicillin G potassium, procaine penicillin G (penicillin G procaine), penicillin N, penicillin O, penicillin V, benzathine penicillin V, baibamin penicillin V, Penimepicycline, phenethicillin potassium, piperacillin, pivampicillin, propicillin, quinacillin, sulfacillin (sulbenicillin), sultamicillin, talampicillin, temocillin, ticarcillin, lincosamides (eg clindamycin, Lincomycin, macrolides (eg azithromycin, carbomycin, clarithromycin, dihithromycin, erythromycin, vinegar) Erythromycin acistrate, erythromycin estolate, erythromycin glucoheptonate, erythromycin lactobionate, C Erythromycin propionate, erythromycin stearate, josamycin, leucomycins, midecamycins, miokamycin ), oleandomycin, primycin, rokitamycin, rosaramicin, roxithromycin, spiramycin, vinegar (troleandomycin), polypeptides (eg, amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviromycin) (enviomycin), fusafungine, gramicidin S, gramicidin (class), micotiin, polymyxin, pristinamycin, Ristomycin, teicoplanin, thiostrepton, tuberactinomycin, tyrocidine, tyrothricin, wangkuo Vancomycin (viomycin), virginiamycin, zinc bacitracin, tetracyclines (eg apicycline, chlortetracycline, clomocycline, dexamethasone) Demeclocycline, doxycycline, guamecycline, lymecycline, meclocycline, methacycline, minocycline, Oxytetracycline, penimepicycline, pipacycline, rolitetracycline, sancycline, tetracycline, and other antibiotics (eg cycloserine) , mupirocin, tuberin.

Other examples of antibiotics are synthetic antibacterial agents, such as 2,4-diaminopyrimidines (e.g., brodimoprim, tetroxoprim, trimethoprim), nitrofuran. Classes (eg furtaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol) , nifurprazine, nifurtoinol, nitrofurantoin, quinolones and analogues (eg cinoxacin, ciprofloxacin, gram) Clinafloxacin, difloxacin, enoxacin, fleroxacin, flumequine, grepafloxacin, lomefloxacin, Miloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, Pazufloxacin, pefloxacin, pipemidic acid, pyridin Puromidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin Star (trovafloxacin), sulfonamides (such as acetyl sulfamethoxypyrazine, benzylsulfamide, chloramine-B, chloramine-T, dichloramine-T, n ² - A cable pyrimidin-acyl sulfonamide ^{(n 2 -formylsulfisomidine), n 4} -β-D- glucosyl amine benzenesulphonamide Amides ^{(n 4 -β-D-glucosylsulfanilamide} ), mafenide (mafenide), 4 '- ( Methyl sulfonyl sulfanilanilide, noprylsulfamide, phthalylsulfacetamide, phthalylsulfathiazole, salazosulfadimidine ), succinylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachrysoidine, sulfacytine, sulfadiazine (sulfadiazine), sulfadicram Ide), sulfadimethoxine, sulfadoxine, sulfaethidole, sulfaguanidine, sulfaguanol, sulfalene, sulfa Sulfaxic acid, sulfamerazine, sulfameter, sulfamethazine, sulfamethizole, sulfamethomidine, sulfamethamine Sulfamethoxazole, sulfamethoxypyridazine, sulfametrole, sulfamidochrysoidine, sulfamoxole, sulfanilamide, 4- amine benzenesulfonamide Amides acid (4-sulfanilamidosalicylic acid), n 4 - amine benzenesulfonamide acyl amine benzenesulphonamide Amides (n ⁴ -sulfanilylsulfanilamide), benzyl amine sulfonylurea (sulfanilylurea), N-sulfanilyl-3,4-xylamide, sulfanitran, sulfaperine, sulfaphenazole, Sulfaproxyl Sulfapyrazine, sulfapyridine, sulfasomizole, sulfasymazine, sulfathiazole, sulfathiourea, sulfatolamide, sulfonamide Sulsopidine, sulfisoxazole, terpenoids (eg acedapsone, acediasulfone, acetosulfone sodium, dapsone) , diathymosulfone, glucosulfone sodium, solasulfone, succisulfone, sulfanilic acid, p-aminobenzenesulfonate P-sulfanilylbenzylamine, sulfoxone sodium, thiazolsulfone, and other antibiotics (eg clofoctol, hexedine, hexamethylenetetrazine) Methenamine, methenamine anhydromethylene citrate, methenamine hippurate, methenamine mandelate, sulfonate Methenamine sulfosalicylate, nitroxoline, taurolidine, xibomol.

Non-limiting examples of immunosuppressive agents include dexamethasone, cyclosporin A, azathioprine, brequinar, gusperimus, 6-mercaptopurine, Mizoribine, rapamycin, tacrolimus (FK-506), folic acid analogues (eg, denopterin, edatrexate, methylamine) Meth (methotrexate), piritrexim, pteropterin, Tomudex®, trimetrexate, guanidine analogues (eg cladribine, fludarabine) , 6-mercaptopurine, thiamiprine, thiaguanine, pyrimidine analogs (eg, ancitabine, azacitidine, 6-azuridine, carmofur (carmofur), cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine (gemcitabine), tegafur (tegafur), fluocinolone, triamcinolone (triacinolone), anecortave acetate, fluorometholone, hydroxysodium, and prednisolone.

Non-limiting examples of antifungal agents include polyenes (e.g., amphotericin B, candicidin, dermostatin, filipin, mycophenolin) (fungichromin), hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pexi Pecilocin, perimycin, azaserine, griseofulvin, oligomycin, neodecanoate neomycin, pyrrolnitrin (pyirolnitrin), siccanin, tubercidin, viridin, allylamine (eg butenafine, naftifine, terbinaphthalene) Terbinafine, imidazoles (such as bifonazole, butoconazole, chlordantoin, chlormidazole, cloconazole, gram Clotrimazole, econazole, enilconazole, fenticonazole, flurmazo Le), isoconazole, ketoconazole, lanoconazole, miconazole, omoconazole, oxiconazole nitrate, Sertaconazole, sulconazole, tioconazole, thiocarbamate (eg tolciclate, tolindate, tolnaphthalene) Tolnaftate, triazoles (such as fluconazole, itraconazole, saperconazole, terconazole), acridine (acrisorcin), Amorolfine, biphenamine, bromosalicylchloranilide, buclosamide, calcium propionate, chlorphenesin, ciclopirox , cloxyquin, coparaffinate, dexamethasone dihydrochloride, exalamide, fluoride Flucytosine, halethazole, hexetidine, loflucarban, nifuratel, potassium iodide, propionic acid, pyrithione ), salicylanilide, sodium propionate, sulbentine, tenonitrozole, triacetin, benzylthiodiazine acetate (ujothion), undecene Acid and zinc propionate.

Non-limiting examples of antiviral agents include acyclovir, carbovir, famciclovir, ganciclovir, penciclovir, and zidovudine. ).

Non-limiting examples of antiprotozoal agents include pentamidine isethionate, quinine, chloroquine, and mefloquine.

In one aspect, the amount of therapeutic agent is from 0.001 to 10% (or 0.005 to 5%, or 0.01 to 2%, or 0.01 to 1%, or 0.01 to 0.5%, or 0.1 to the weight of the pharmaceutical composition) Within the range of 0.5%, or 0.1 to 1%, or 0.1 to 2%, or 0.5 to 2%, or 0.5 to 5%).

In one embodiment, the pharmaceutical component comprises a fluoroquinolone of formula I (a new generation of fluoroquinolone antibacterial agent, disclosed in U.S. Patent No. 5,447,926, incorporated herein by reference).

Wherein R ¹ is selected from the group consisting of hydrogen, unsubstituted C ₁ -C ₅ alkyl, substituted C ₁ -C ₅ alkyl, C ₃ -C ₇ cycloalkyl, unsubstituted C ₅ -C ₂₄ aryl group, the substituted C ₅ -C ₂₄ aryl group, the unsubstituted C ₅ -C ₂₄ heteroaryl and substituted heteroaryl of C ₅ -C ₂₄ aryl group; R ² selected from the group consisting of a group of: hydrogen, an unsubstituted amine group, and an amine group substituted with one or two C ₁ -C ₅ alkyl groups; R ³ is selected from the group consisting of hydrogen, unsubstituted C ₁ -C ₅ Alkyl, substituted C ₁ -C ₅ alkyl, C ₃ -C ₇ cycloalkyl, unsubstituted C ₁ -C ₅ alkoxy, substituted C ₁ -C ₅ alkoxy, unsubstituted Substituted C ₅ -C ₂₄ aryl, substituted C ₅ -C ₂₄ aryl, unsubstituted C ₅ -C ₂₄ heteroaryl, substituted C ₅ -C ₂₄ heteroaryl, unsubstituted C ₅ -C ₂₄ aryloxy, substituted C ₅ -C ₂₄ aryloxy, unsubstituted C ₅ -C ₂₄ heteroaryloxy and substituted C ₅ -C ₂₄ heteroaryloxy; X-system is selected from the group consisting of a halogen atom; the Y selected from the group consisting of the group consisting _{of: CH 2, O, S,} SO, SO 2 and NR ^4, wherein R ⁴ is selected from the group consisting of lines consisting of: hydrogen, The substituted C ₁ -C ₅ alkyl, the substituted C ₁ -C ₅ alkyl and C ₃ -C ₇ cycloalkyl; and Z is selected from the group consisting of oxygen, and the group consisting of two hydrogen atoms; and wherein when the group When the group is substituted, the substituent is selected from the group consisting of hydroxyl, amine, halogen, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ halogenated alkyl, SO ₂ And sulfhydryl groups.

In another embodiment, the pharmaceutical component comprises a fluoroquinolone having the formula H.

((R)-(+)-7-(3-Amino-2,3,4,5,6,7-hexahydro-1H-azain-1-yl)-8-chloro-1-cyclopropane -6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid).

In another embodiment, the pharmaceutical component comprises a glucocorticoid receptor agonist having Formula III or IV as disclosed in U.S. Patent Application Publication No. 2006/0116396, which is incorporated herein by reference. .

Wherein R ⁴ and R ^{5 are} independently selected from the group consisting of hydrogen, halogen, cyano, hydroxy, C ₁ -C ₁₀ (or C ₁ -C ₅ or C ₁ -C ₃ )alkoxy, unsubstituted a C ₁ -C ₁₀ (or C ₁ -C ₅ or C ₁ -C ₃ ) linear or branched alkyl group, substituted C ₁ -C ₁₀ (or C ₁ -C ₅ or C ₁ -C ₃ ) a linear or branched alkyl group, an unsubstituted C ₃ -C ₁₀ (or C ₃ -C ₆ or C ₃ -C ₅ ) cyclic alkyl group, and a substituted C ₃ -C ₁₀ (or C ₃ -C) _{a 6} or C ₃ -C ₅ ) cyclic alkyl group, wherein when the group is substituted, the substituent is selected from the group consisting of hydroxyl, amine, halogen, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ halogenated alkyl and sulfhydryl.

In another embodiment, the pharmaceutical component comprises a glucocorticoid receptor agonist of formula V (a compound of formula III).

In another embodiment, the therapeutic agent is loteprednol carbonate of formula VI, an anti-inflammatory agent.

The pharmaceutical composition of the present invention may further comprise a material selected from the group consisting of a buffer, a tonicity adjuster, a viscosity modifier, a pH adjuster, an antioxidant, a chelating agent, a surfactant, and other pharmaceutically acceptable materials, as needed. Accepted reagents.

The ophthalmic solutions of the present invention can be formulated in physiologically acceptable buffers to adjust pH and tonicity to a range compatible with ophthalmic use and with any active ingredients present therein. A physiologically acceptable non-limiting examples of buffers include phosphate buffer; Tris-HCl buffer (containing a reference (hydroxymethyl) aminomethane and HCI); based on the pK _a at 25 deg.] C and a pH value of 7.5 a buffer of HEPES (N-{2-hydroxyethyl}piperazine-N'-{2-ethanesulfonic acid}) having a value in the range of about 6.8-8.2; based on _a pK _a value of 7.1 at 25 ° C and a pH value Buffer of BES (N,N-bis{2-hydroxyethyl}2-aminoethanesulfonic acid) in the range of about 6.4-7.8; based on _a pK _a value of 7.2 at 25 ° C and a pH of about 6.5- MOPS (3- {N- morpholino} propanesulfonic acid) within the range of 7.9 buffer; 25 deg.] C based on the pK _a value of 7.4 and the pH TES (N- {parameter in the range of about 6.8-8.2 hydroxyalkyl methyl} - methyl-2-amino-ethanesulfonic acid) buffer was; 25 based on the pK _a value of 7.6 deg.] C and the pH in the range of about 6.9-8.3 MOBS of (4- {N- morpholinyl} Buffer of butanesulfonic acid); based on DIPSO (3-(N,N-bis{2-hydroxyethyl}amino)-2 with _a pKa value of 7.52 at 25 ° C and a pH in the range of about 7-8.2 - dihydroxypropane) of the buffer; 25 based on the pK _a 7.61 deg.] C and the pH value in the range of about 7-8.2 TAPSO (2- hydroxy-3 {parameter (hydroxymethyl) methylamino} -1-propanesulfonic acid) of the buffer; based at 25 ℃ pK _a value of 8.4 and a pH value TAPS ({(2- hydroxy-1,1-bis (hydroxymethyl) ethyl) amino} -1-propanesulfonic acid) within the range of about 7.7-9.1 of the buffer; deg.] C based on the pK _a value of 25 a buffer of TABS (N-paraxyl (hydroxymethyl)methyl-4-aminobutyric acid) having a pH in the range of about 8.2-9.6; based on _a pK _a value of 9.0 at 25 ° C and a pH at Buffer of AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid) in the range of about 8.3-9.7; based on pK _a value at 25 ° C a buffer of CHES (2-(cyclohexylamino)ethanesulfonic acid) having _a pH of about 8.6 to 10.0; based on _a pK _a value of 9.6 at 25 ° C and a pH in the range of about 8.9 to 10.3 a buffer of CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid); or _a CAPS based on _a pKa value of 10.4 at 25 ° C and a pH in the range of about 9.7-11.1. - (cyclohexylamino)-1-propanesulfonic acid) buffer.

Although the buffer itself is a "tonicity adjuster" and a "pH adjuster" for maintaining an ophthalmic solution at a specific ion concentration and pH, other "tension modifiers" may be added to adjust the final tension of the solution. Non-limiting examples of tonicity adjusting agents include, but are not limited to, mannitol, sorbitol, urea, propylene glycol, and glycerol. Various salts including a halide salt of a monovalent cation such as NaCl or KCl can also be utilized.

In the presence of a tonicity adjusting agent, the concentration may range from about 0.01 to about 10, or from about 0.01 to about 7, or from about 0.01 to about 5, or from about 0.1 to about 2, or from about 0.1 to about 1% by weight. In some embodiments in which a tonicity modifier is present, the solution may contain a single tonicity modifier or a combination of different tonicity modifiers. Typically, the tension of the formulations of the invention is in the range of from about 200 to 400 mOsm/kg. Alternatively, the tension of the formulation of the invention is from about 220 to 400 mOsm/kg, or from about 220 to 350 mOsm/kg, or from about 220 to 300 mOsm/kg, or from about 250 to 350 mOsm/kg, or from about 290 to 350 mOsm. /kg, or in the range of about 240 to 290 mOsm/kg. For certain applications, such as alleviating symptoms of dry eye or treating inflammation of the eye, the ophthalmic formulations of the present invention may desirably be hypotonic, such as a tension of from about 200 to about 270 mOsm/kg or from about 250 to about 270 mOsm/kg. Within the scope.

Non-limiting examples of viscosity modifiers include synthetic and natural polymers such as poly(acrylic acid) (eg, lightly crosslinked poly(acrylic acid) known as Carbopol®, carbomer, or polycarbophil )), polysaccharides (such as alginic acid, gellan gum, β-glucan, guar gum, gum arabic (arabinogalactan oligosaccharides, polysaccharides and glycoproteins) Mixture), locust bean gum, pectin, xanthan gum, hyaluronic acid, carboxymethyl starch, carboxymethyl polydextrose, polyglucose sulfate, carboxymethyl poly Amino sugar or chondroitin sulfate (such as chondroitin sulfate A, chondroitin sulfate B or chondroitin sulfate C), carrageenan or curdlan gum, cellulose derivatives (such as carboxymethyl) Cellulose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose or hydroxyethyl Methylcellulose) or a salt thereof. It will be appreciated that some of the polysaccharides listed above may not have naturally occurring salts. Various polyethylene glycols (such as PEG-1000, PEG-3350, PEG-4000, PEG-8000, PEG-10000) can also be considered as viscosity modifiers.

The amount of viscosity modifier is selected to produce a viscosity of from about 2 to about as measured by a Brookfield viscometer (RVDV Type III) at 25 ° C and a shear rate of 1-7 sec ^-1 using a CPE-40 spindle. A pharmaceutical composition in the range of 2,000 centipoise (or mPa.s) (or from about 2 to about 1,000, or from about 2 to about 500, or from about 2 to about 100 centipoise). The amount of viscosity modifier added to achieve a certain viscosity can be readily determined experimentally.

Non-limiting examples of antioxidants include ascorbic acid (vitamin C) and its salts and esters; tocopherols (such as alpha-tocopherol) and tocotrienols (vitamin E) and their salts and esters (such as vitamin E TGPS (succinic acid) D-α-tocopherol polyethylene glycol 1000)); glutathione; lipoic acid; uric acid; butylated hydroxyanisole ("BHA"); butylated hydroxytoluene ("BHT"); Butylhydroquinone ("TBHQ"); and polyphenolic antioxidants (such as gallic acid, cinnanmic acid, flavonoids and their salts, esters and derivatives). In some embodiments, the antioxidant comprises ascorbic acid (vitamin C) and salts and esters thereof; tocopherols (such as alpha-tocopherol) and tocotrienols (vitamin E) and salts and esters thereof; BHT; or BHA.

In another embodiment, the amount of antioxidant in the pharmaceutical formulation of the invention is in the range of from about 0.0001 to about 5% by weight of the formulation. Alternatively, the amount of antioxidant is from about 0.001 to about 3%, or from about 0.001 to about 1%, or from greater than about 0.01 to about 2%, or greater than about 0.01 to about 1%, or greater than about 0.01 to the weight of the solution. About 0.7%, or greater than about 0.01 to about 0.5%, or greater than about 0.01 to about 0.2%, or greater than about 0.01 to about 0.1%, or greater than about 0.01 to about 0.07%, or greater than about 0.01 to about 0.05%, or Greater than about 0.05 to about 0.15%, or greater than about 0.03 to about 0.15%, or greater than about 0.1 to about 1%, or greater than about 0.1 to about 0.7%, or greater than about 0.1 to about 0.5%, or greater than about 0.1 to about 0.2%, or greater than about 0.1 to about 0.15%.

Non-limiting chelating agents include compounds of formula VII, VIII or IX.

Wherein n ₁ , n ₂ , n ₃ , n ₄ , n ₅ , n ₆ and n ₇ are integers independently ranging from 1 to 4 (including 1 and 4); m is 1 to 3 (including 1 and 3) An integer in the range; p ₁ , p ₂ , p ₃ and p _{4 are} independently selected from integers in the range of 0 and 1 to 4 inclusive.

In some embodiments, the chelating agent comprises a salt selected from the group consisting of ethylenediaminetetraacetic acid ("EDTA"), diethylenetriamine pentas(methylphosphonic acid), hydroxyethylphosphoric acid, pharmaceutically acceptable salts thereof, and mixtures thereof a group of compounds.

In some other embodiments, the chelating agent comprises a tetrasodium salt of isethionate (also known as "HAP", which is available in a 30% solution).

In some other embodiments, the chelating agent comprises a sodium salt of EDTA.

The ophthalmic solution of the present invention may also comprise one or more surfactants. Suitable surfactants can include cationic, anionic, nonionic or amphoteric surfactants. Preferred surfactants are neutral or nonionic surfactants. Non-limiting examples of surfactants suitable for use in the formulations of the present invention include polyethylene glycol ("PEG" such as PEG-400, PEG-800, PEG-1000, PEG-3350, PEG-4000, PEG-8000, PEG-10000), polysorbate (such as polysorbate 80 (polyoxyethylene sorbitan monooleate), polysorbate 60 (polyoxyethylene sorbitan monostearate) Polysorbate 20 (polyoxyethylene sorbitan monolaurate), usually known under the trade names Tween ^® 80, Tween ^® 60, Tween ^® 20), poloxamers (ethylene oxide and propylene oxide block polymers of synthetic, such as generally employed in its synthesis block polymers known under the trade name of Pluronic ^®; e.g. Pluronic ^® F127 or Pluronic ^® F108)), poloxamer amines (poloxamines ) (ethylene oxide and propylene oxide attached to the synthetic block polymers of ethylene diamine, such as generally employed in its synthesis tradename Tetronic ^® block polymer of known; e.g. Tetronic ^® 1508 or Tetronic ^® 908, etc.), other Nonionic surfactants such as Brij ^® , Myrj ^® and carbon chains have about 12 or more Long chain fatty alcohols having a carbon atom (e.g., from about 12 to about 24 carbon atoms) (i.e., oleyl alcohol, stearyl alcohol, tetradecanol, docosahexanol, etc.). Such compounds are depicted in Martindale, 34th edition, pages 1411-1416 (Martindale, "The Complete Drug Reference", SCSweetman (ed.), Pharmaceutical Press, London, 2005) and Remington, "The Science and Practice of Pharmacy", 21st edition, pages 291 and 22, Lippincott Williams & Wilkins, New York, 2006. The concentration of the nonionic surfactant (if present) in the compositions of the present invention may range from about 0.001 to about 5% by weight (or from about 0.01 to about 4, or from about 0.01 to about 2, or from about 0.01 to about 1% by weight). Within the scope of).

In addition to the ingredients of the classes disclosed above, the pharmaceutical formulations of the invention (such as ophthalmic solutions) may further comprise one or more additional ingredients, such as vitamins (other than the vitamins disclosed above) or provided to the user. Other ingredients for additional health benefits.

The compositions of the invention may also be used as contact lens care. In this case, it can contain It is generally used to clean and maintain other known components of contact lenses as long as they are compatible with the other ingredients in the formulation. In one embodiment, the contact lens care solution can comprise a microabrasive (eg, polymeric microbeads).

In one embodiment, the pharmaceutical composition of the invention comprises, consists of, or consists essentially of: PEG-3350, polysorbate 80, HPMC (hydroxypropyl methylcellulose) 2910 or HPMC E15LV, boric acid, phosphate , glycerol, sodium thiosulfate, EDTA salt (such as disodium salt), BHT, polytetraamethylene-1 and water. HPMC 2910 or HPMC E15LV is available from the Dow Chemical Company.

In one aspect, the pharmaceutical composition of the present invention comprises, consists of, or consists essentially of: PEG-3350, polysorbate 80, HPMC (hydroxypropyl methylcellulose) 2910, boric acid, phosphate, C3 Alcohol, sodium thiosulfate, EDTA salt (such as disodium salt), BHT, polytetraammonium-1 and water; wherein the viscosity of the composition is in the range of 5 to 30 mPa.s (or cp) and the pH is 6 to 8 (or 6.5 to 7.7, or 6.5 to 7.5, or 7 to 7.5).

Exemplary concentrations of the components of the composition are shown in Tables 2 and 3.

In another aspect, the present invention provides a method for the manufacture of an ophthalmic pharmaceutical formulation for treating, controlling, ameliorating or reversing a condition of a dry eye patient (such as eye irritation, discomfort, dryness, Gravel or stinging, or lack of water, lipid layer or sticky Film layer). The method comprises combining: (a) a polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da; and (c) an ophthalmically acceptable Carrier. In one embodiment, the ophthalmically acceptable carrier comprises water and the pharmaceutical formulation is an aqueous solution.

In another aspect, the present invention provides a method for the manufacture of an ophthalmic pharmaceutical formulation for treating, controlling, ameliorating or reversing a condition of a dry eye patient (such as eye irritation, discomfort, dryness, There is grit or tingling, or lack of water, lipid or mucous layers). The method comprises combining: (a) a polyethylene glycol having a concentration ranging from about 5 to about 15% of the total composition in the range of 1,000 to 10,000 Da; (b) a concentration of from about 0.5 to about 2% of the total composition. a water-soluble cellulose derivative having a molecular weight in the range of 50,000 to 120,000 Da; and (c) an ophthalmically acceptable carrier. In one embodiment, the ophthalmically acceptable carrier comprises water and the pharmaceutical formulation is an aqueous solution.

In another aspect, the invention provides a method for making an ophthalmic pharmaceutical formulation for treating, managing or ameliorating a condition (such as eye irritation or discomfort) in a dry eye patient. The method comprises combining the ingredients listed in Tables 2 and 3 of the respective concentrations to produce an ophthalmic formulation.

In another aspect, the method further comprises the step of mixing the combined components to achieve substantial homogeneity.

In another aspect, the method further comprises the steps of sterilizing the formulation to produce a sterilized formulation and encapsulating the sterilized formulation in a suitable container.

In one embodiment, the method may also include: (1) adding some material and mixing together to produce a first mixture; and (2) adding the remaining material to the first mixture while continuing to mix to produce the composition.

In another embodiment, the method may further comprise: (1) adding some materials and mixing together to produce a first mixture; (2) adding remaining materials and mixing together to produce a second mixture; and (3) combining the first Mixing the mixture with the second mixture while continuing to mix to produce a combination Things.

Other non-limiting examples of the invention are shown in the table below.

Example 3: Ophthalmic Formulation with NSAID Anti-Inflammatory Drug

The formulation is produced by combining the following ingredients.

Example 4: Ophthalmic Formulation for Treatment or Control of High Intraocular Pressure

The following ingredients are combined to produce an exemplary formulation for treating or controlling high intraocular pressure.

Example 5: Ophthalmic Formulations for Treatment or Control of Eye Infections

The formulation is produced by combining the following ingredients.

Example 6: Ophthalmic Formulations for Treatment or Control of Eye Infections

The formulation is produced by combining the following ingredients.

Example 7: Ophthalmic Formulations for Treatment or Control of Eye Infections

The formulation is produced by combining the following ingredients.

Example 8: Ophthalmic Formulations for Treatment or Control of Eye Allergy

The following ingredients are combined to produce an exemplary formulation for treating or controlling eye irritation.

Example 9: Ophthalmic Formulations for Treatment or Control of Eye Allergy

The following ingredients are combined to produce an exemplary formulation for treating or controlling eye irritation.

Example 10: Ophthalmic Formulations for Treatment or Control of Eye Infections

The following ingredients are combined to produce an exemplary formulation for treating or controlling an eye infection.

Example 11: Ophthalmic Formulations for Treatment or Control of Eye Infections

The following ingredients are combined to produce an exemplary formulation for treating or controlling an eye infection.

Example 12: Ophthalmic Formulation for Treatment or Control of Inflammation of the Eye

The following ingredients are combined to produce an exemplary formulation for treating or controlling inflammation of the eye.

Example 13: Ophthalmic Formulation for Treatment or Control of Inflammation of the Eye

The following ingredients are combined to produce an exemplary formulation for treating or controlling inflammation of the eye.

Example 14: Ophthalmic Formulation for Treatment or Control of Inflammation of the Eye

The following ingredients are combined to produce an exemplary formulation for treating or controlling inflammation of the eye.

Example 15: Ophthalmic Formulations for Treatment or Control of Intraocular Pressure

The following ingredients are combined to produce an exemplary formulation for treating or controlling intraocular pressure. The following ingredients are combined to produce an exemplary formulation for treating or controlling inflammation of the eye.

Example 16: Formulation comprising a second preservative

The following ingredients are combined to produce an exemplary formulation. This formulation can be used as a vehicle for ophthalmic active agents or as a contact lens for treating, cleaning, wetting or storing solutions.

The following ingredients are combined to produce an exemplary formulation for treating or controlling inflammation of the eye.

In another aspect, the ophthalmic solution of the invention as described in Table 2 can be used to treat, control or ameliorate conditions or symptoms associated with dry eye, eye irritation or allergy.

In another aspect, the ophthalmic solution of the present invention as described in Table 2 can be used to promote healing of a damaged eye surface, wherein the damage is caused by dryness, trauma or irritation.

In another aspect, the invention provides methods for making and using the pharmaceutical formulations of the invention. Any of the materials, compounds and ingredients disclosed herein may be suitable for use in the present invention. Any method is used together or included in any of the methods of the present invention.

In one embodiment, the method comprises: (a) combining (i) a pharmaceutically acceptable carrier; (ii) polyethylene glycol having a molecular weight in the range of 1,000 to 10,000 Da; and (iii) having a molecular weight of 50,000 a water soluble cellulose derivative in the range of 120,000 Da; and (b) mixing the ingredients (i), (ii) and (iii) together for a time sufficient to produce a substantially uniform pharmaceutical composition.

In another embodiment, the method further comprises adding one or more ingredients selected from the group consisting of a therapeutic agent, a buffer, a tonicity modifier, a surfactant, a viscosity modifier, and other pharmaceuticals to the pharmaceutical composition. Acceptable reagents. The therapeutic agent can be selected from the group consisting of anti-inflammatory agents, intraocular pressure reducing agents, ocular neuroprotective agents, antibiotics, immunosuppressants, anti-allergic agents, antiviral agents, antifungal agents, antiprotozoal agents, and mixtures thereof. Non-limiting examples of each of the classes of agents, compounds, and ingredients are disclosed throughout the specification.

In another aspect, the pharmaceutically acceptable carrier comprises boric acid and a phosphate buffer.

Test 1: The compositions of the present invention show statistically significant improvements over baseline for all major and minor endpoints of dry eye syndrome

A randomized multicenter study of 73 patients was performed for 12 weeks to assess the effectiveness of the compositions of the invention in improving the condition or symptoms of dry eye. The composition for the study is shown in Table T-1-1 below. Two eyes per subject received one drop of the composition twice a day.

Common primary end

‧ Total corneal staining ("TCS") score (sum of 5 designated areas in the cornea, week 12)

‧Day 12 worst baseline dry eye symptoms (eye discomfort, dryness, grit and tingling) visual analog scale ("VAS") score secondary efficacy endpoint

‧The percentage of individuals (study eyes) whose central corneal staining completely subsided

‧Changes in conjunctival staining compared to baseline values as determined by the sum of the Lissamine green conjunctival staining scores in each of the 6 conjunctival regions

‧Use the Schirmer test (without anesthesia) Percentage of individuals weighed 10mm

‧Changes in tear secretion in mm as measured by the Hillmer test (not anesthetized) compared to baseline values

‧ Percentage of safety endpoints for individuals with complete corneal staining (study eyes)

‧ Individual ratio of eye treatment sudden adverse events ("TEAE"): 15/71 individuals (or 21.1%) experienced at least one TEAE. However, no TEAE is serious enough to require an early stop. Three patients (or 4.5%) showed an increase in intraocular pressure compared to baseline ("IOP") 5mm Hg, but less than 10mm Hg. No patient shows an increased IOP 10mm Hg. Therefore, it was concluded that the composition was safe to use.

The results of the 12th week study are shown in Table T-1-2.

in conclusion

The composition of the invention resulted in a significant reduction in total corneal staining, conjunctival staining, and worst VAS scores and a significant increase in tear production (as indicated by the Hillmer test) after the diseased eyes were administered twice a day for 12 weeks. Thus, it is demonstrated that compositions such as the compositions of the present invention are effective in treating, controlling, ameliorating or reversing the condition or symptoms of dry eye. In addition, it has also proved that the lack of tears can be reversed.

Test 2: Composition of the invention promotes corneal epithelial regeneration Introduction

Dry eye syndrome is an ocular surface disorder attributed to lack of tears, excessive tear evaporation, or incorrect tear formation. Dryness of the resulting ocular surface causes eye irritation and discomfort.

An in vitro wound healing model using a single layer of scaffolded transformed human corneal epithelial cells has been developed and monitored for growth in the resulting acellular gap. In vivo and in vitro models have been used in the literature to show that heparin-binding endothelial growth factor ("HB-EGF") stimulates corneal wound healing and that HB-EGF expression is observed during corneal wound healing (see, for example, DM Foreman et al., A Simple Organ Culture Model for Assessing the Effects of Growth Factors on Corneal Re-epithelization", Exp.Eye Res., Vol. 62, 555-64 (1996); A. Wells, "EGF Receptor", IJBCB , Volume 31 , 637-43 (1999)). In the scratch analysis model system presented here, it has been found that HB-EGF provides consistent results in a human corneal epithelial cell line ("RT-HCEpiC") of the Japan Institute of Physical Chemistry as a positive analysis control.

Current studies have determined the ability of PEG 3350 and HPMC 2910 to contribute to corneal epithelial regeneration following RT-HCEpiC injury.

method Single layer abrasion analysis

A vertical line was drawn with a marker at the bottom of each well of the 24-well culture plate at the bottom of the plate, and an image of the intersection of the line and the cell-free gap was taken for measurement. RT-HCEpiC was prepared in a suspension of 2.5 x 10 ⁵ cells per ml in complete medium, and 500 μl of cell suspension was added to each well. The plates were incubated at 37 ° C, 5% CO ₂ and 95% humidity until a complete monolayer was formed. When the cells reached confluence, the medium was removed from each well and replaced with a minimal medium without growth factors. The wells were serum starved for 18 hours in the incubator. After this incubation, 500 μl of HBSS was added to each well. The monolayer was disrupted by a single horizontal abrasion with a sterile P200 pipette tip. The HBSS was aspirated and the wells were washed once again with HBSS. Finally, the treatment solution in the minimal medium is applied to the appropriate wells. A baseline image of the single layer gap was taken and the cells were returned to the incubator for regeneration of the intercellular epithelium for 16 hours. At this point, the cells were examined using an optical microscope and photographed to record a single layer gap closure.

Experimental design and progress overview

data analysis

Light microscopy after 16 hours of incubation and after treatment Cells were photographed to monitor the closure of the acellular gap. The percentage of closure was determined using the formula below.

((gap width _baseline - gap width _{16 hours} ) / gap width _baseline ) × 100 = closure percentage

A comparison was made between the treatment group and the vehicle control to determine the effect of treatment on epithelial regeneration. Statistical analysis of the net decrease in gap width was performed by one-way ANOVA and Dunnett's method, and P < 0.05 was considered significant. All data were analyzed using statistical analysis software JMP (SAS Institute, Cary, NC).

result

Corneal epithelial regeneration was significantly increased by 10 ng/mL HB-EGF and PEG 3350 (10%) 16 hours after wounding (Figure 1).

10% strength of PEG 3350 promotes wound healing. This effect appeared to be dose dependent, however no significant effect was observed at 1% or 3% PEG 3350.

HPMC 2910 (1%) did not contribute to corneal epithelial regeneration (Figure 1).

In the preliminary experiment, 0.1% and 0.3% HPMC 2910 were also tested. However, neither a significant effect on corneal epithelial regeneration nor a dose-dependent response was observed.

Overview of research results

PEG 3350 (10%) significantly increased corneal epithelial regeneration of HCEpiC, while 1% and 3% PEG 3350 and HPMC 2910 (1%) did not work.

Test 3: Compositions of the invention reduce dry-induced cell death in transformed human corneal epithelial cells Introduction

Several published studies have used in vitro drying models to determine the effects of polymers, lubricants, and protectants on the ocular surface (see, for example, JLUbels et al., "Preclinical Investigation of the Efficacy of an Artificial Tear Solution Containing Hydroxypropyl Guar as a Gelling Agent, Curr.Eye Res., Vol. 28, 437-44 (2004); T. Matsuo, "Trehalose Protects Corneal Epithelial Cells From Death by Drying", Br. J. Ophthalmol., Vol. 85, 610-12 (2001); K. Paulsen et al., "Lubricating Agents Differ in Their Protection of Cultured Human Epithelial Cells Against Desiccation", Med. Sci. Monit., Vol. 14, PI 12-16 (2008)). In this model, the corneal epithelial cells are exposed to the test agent, followed by drying after removal of the test agent. Cell viability was then measured. An in vitro drying model has been established in transforming human corneal epithelial cells ("HCEpiC"). This study consists in determining the ability of the compositions of the invention to protect cells from drying-induced cell death.

method design

Transforming human corneal epithelial cells from ATCC (T-HCEpiC) in EpiLife medium + 1% human corneal growth supplement ("HCGS"; containing bovine pituitary extract, bovine insulin, hydrocortisone, bovine transferrin and small Murine epidermal growth factor) was seeded at 1.25 x 10 ⁴ cells/well in 4 black-walled 96-well culture plates and cultured until confluent. The medium was removed from the cells and pretreated with minimal medium or minimal medium containing HPMC 2910 (0.1-1%) or PEG 3350 (1-10%) for 10 minutes (Table T-3-1). The plates were then placed in a tissue culture hood without air flow for 0, 15, 30 and 45 minutes. Cell viability was assessed using a live/dead viability/cytotoxic kit (Invitrogen).

* indicates 8 holes per group.

data analysis

Substrate fluorescence from the separate medium was subtracted. Changes in live and dead cell content are expressed in RFU.

Data are expressed as mean ± SEM. Statistical analysis was performed using a two-way ANOVA-Tukey Kramer test (factor 1 for drying time; factor 2 for OTC dry eye drops) using JMP 8 software (SAS Institute, Cary, NC). p < 0.05 was considered statistically significant. Data was analyzed directly or after Box-Cox transformations.

result

Compared to the medium control, the cells showed a significant increase in calcein fluorescence (indicating an increase in viable cells) after exposure to 0.3% or 1% HPMC 2910 after 15, 30 or 45 minutes of drying. Ethidium fluorescein was significantly smaller (indicating a decrease in cell death) in cells exposed to 0.3% or 1% HPMC 2910 after 15, 30 or 45 minutes (Figure 2).

There was no significant difference in calcein or ethidium fluorescence after exposure to 0.1% HPMC 2910 or 1%, 3% or 10% PEG 3350 after 15 to 45 minutes of drying (Figure 2).

Overview of research results

At all time points (15, 30, and 45 minutes) measured, HPMC 2910 (0.3% and 1%) reduced dry-induced HCEpiC death. Therefore, the composition of the present invention comprising a nonionic cellulose derivative such as HPMC can improve the anti-drying activity of the corneal surface.

0.1% HPMC 2910 or 1%, 3% or 10% PEG 3350 had no significant effect on dry-induced HCEpiC cell death.

Test 4: Effect of high volume molar permeation concentration and 10% PEG-3350 on monolayer resistance in transformed human corneal epithelial cells of the Institute of Physical Chemistry, Japan Introduction

In this study, effective monitoring using electronic cell-matrix impedance sensing ("ECIS") The Institute of Physical Chemistry of Japan transformed the single layer resistance of human epithelial cells ("RT-HCEpiC"), which is an indication of barrier function. This novel ECIS system has recently been designed to determine the effect of the molar osmolality of sodium chloride (NaCl) on the monolayer resistance of human epithelial conjunctival cells, thereby indicating a change in monolayer permeability. First, try to demonstrate that ECIS can be used to monitor changes in cell monolayer resistance induced by NaCl or sucrose hypertonic medium. Second, these experiments examined the ability of PEG-3350 to increase the single layer barrier function for hypertonic induced single layer resistance changes.

method

RT-HCEpiC cells were seeded on ECIS 8-well slides at a density of 1 or 2×10 ⁵ per ml in DMEM/F12 medium (DMEM/F12 complete HCGS medium) (0.25 ml/well) containing 15% FBS and HCGS. Incubate in an incubator at 37 ° C, 5% CO ₂ and 95% humidity until it reaches confluence (about 2 to 3 days after inoculation). The medium was removed by aspiration and the cells were incubated in minimal medium containing the test components of the concentrations listed in Table 1. Cells were cultured under these conditions and the change in resistance was monitored by ECIS at 20 minute intervals. Each slide was tested with a single well medium as a negative control and cells of one well were tested with 33 ppm Benzododecinium bromide (BOB) as a positive control to measure the change in electrical resistance.

data analysis

The integral response of the impedance change is analyzed by calculating the area under the curve of each test hole over the time course using the trapezoidal rule defined by the following equation: Σ (time (hours) n) - (n-1)) × (impedance (ohms) n+n-1)/2=ohm*hour

The integral reaction was analyzed by two-way ANOVA and Taki Kremer test. Before the statistical analysis, the equivalent concentration and variance homogeneity of the data were evaluated, and if necessary, the results were subjected to Box-Cox conversion. Any conversion of the data is listed in the schema description.

Overview of research results

Treatment of cells with 460 mOsm/kg NaCl medium with or without PEG 3350 did not significantly reduce the integral monolayer resistance as compared to the minimal medium control as shown by ECIS (Figure 3).

Treatment of cells with 535 mOsm/kg and 610 mOsm/kg NaCl medium with or without PEG 3350 significantly reduced the integral monolayer resistance compared to the minimal medium control (Figure 3).

Treatment of cells with 535 mOsm/kg NaCl without PEG 3350 significantly reduced the integral monolayer resistance compared to cells treated with 535 mOsm/kg NaCl and pretreated with PEG 3350 (Figure 3).

The normalization (to 0 o'clock) after treatment with PEG 3350 and basic and NaCl high volume molar permeate media and the time course of the original monolayer resistance at 180 minutes are shown in Figures 4 and 5, respectively.

Treatment of cells with 465 and 525 mOsm/kg sucrose medium with or without PEG 3350 did not significantly reduce the integral monolayer resistance as measured by ECIS compared to the minimal medium control (Figure 6).

Treatment of cells with 585 mOsm/kg and 645 mOsm/kg sucrose medium with or without PEG 3350 significantly reduced the integral monolayer resistance compared to the minimal medium control. (Figure 6).

Treatment of cells with 585 mOsm/kg sucrose without PEG 3350 pretreatment significantly reduced the integral monolayer resistance compared to cells treated with 585 mOsm/kg sucrose and PEG 3350. (Figure 6).

The normalization (to 0 o'clock) after treatment with PEG 3350 and basic and sucrose high volume molar permeation media and the time course of the original monolayer resistance at 180 minutes are shown in Figures 7 and 8, respectively.

Test 5: Effect of PEG-3350 on the mRNA content of mucin (MUC1 and MUC16) in human corneal epithelial cells of the Institute of Physical Chemistry, Japan Introduction

The DEWS Definition and Classification Subcommittee interprets dry eye as a multifactorial disease that causes discomfort, visual impairment, and tear instability, as well as potential ocular surface damage, accompanied by an increase in the osmotic concentration of the tear film and The surface of the eye is inflamed.

Ocular surface mucin is critical for maintaining tear film stability, providing lubrication, and maintaining corneal and conjunctival epithelial cell barrier function. Three membrane-associated mucins MUC1, MUC4, and MUC16 have been shown to be expressed in the corneal epithelium, with MUC1 and MUC16 performing higher (see IK Gipson, "Distribution of Mucins at the Ocular Surface", Exp. Eye Res., Vol. 78, 379 -88 (2004)). Several papers have shown that MUC1 and MUC16 levels have changed in patients with dry eye. Recent studies have shown that MUC1 is significantly lower in conjunctival epithelium in patients with water-deficient dry eye (RM Corrales et al., "Ocular Mucin Gene Expression Levels as Biomarkers for the Diagnosis of Dry Eye Syndrome", Invest. Ophthalmol. Vis . Sci, 52nd Volume, 8263-69 (2011)). Another study has shown that there are fewer MUC1/A splice variants in patients with dry eye than in the normal control group (Y. Imbert et al., "MUC1 Splice Variants in Human Ocular Surface Tissues: Possible Differences Between Dry Eye Patients and Normal Controls", Exp.Eye Res., Vol. 83, 493-501 (2006)). In patients with dry eyes with Hugh's syndrome, there are increased levels of MUC1 and MUC16 mRNA and protein in conjunctival and tear samples (B. Caffery et al., "MUC1 Expression in Sjogren's Syndrome, KCS, and Control Subjects", Mol. Vis., vol. 16, 1720-27 (2010); B. Caffery et al., "MUC16 Expression in Sjogren's Syndrome, KCS, and Control Subjects", Mol. Vis., Vol. 14, 2547-55 (2008) . The aim of this study was to determine the effect of PEG-3350 on MUC1 and MUC16 mRNA levels in human corneal epithelial cells ("RT-HCEpiC") transformed by the Japanese Institute of Physical Chemistry (SV40) at different time points.

method design

Human corneal epithelial cells ("RT-HCEpiC") were seeded in four medium well plates (DMEM/F12 + 10% fetal bovine serum ("FBS")) in four 12-well culture plates. The cells were cultured for 1 week (about 3 days after confluence). The medium was replaced with DMEM/F12 + 10% charcoal adsorbed FBS 18 hours prior to treatment. Then with or without 3% or 10% PEG-3350 DMEM/F12 minimal medium was treated with HCEpiC for 0 to 24 hours; or DMEM/F12 minimal medium containing 10% PEG-3350 for 2 hours, followed by incubation in DMEM/F12 minimal medium for 4 to 24 hours (Table T-5-1) . Total RNA was isolated from cells using the RNeasy Plus Mini Kit, followed by quantification using the Quant-It RNA kit from Invitrogen. The assembly was performed using the affinity script qPCR cDNA to prepare cDNA. QPCR was performed to determine mRNA expression of MUC1 and MUC16. Glucuronidase β (GUSB) is used as a housekeeping gene.

data analysis

The amplification curves were examined to verify that each consisted of a linear baseline region, an amplified log phase, and a subsequent plateau phase. To confirm the linearity and efficiency of the reaction and the efficiency of MUC1 or MUC16 DNA synthesis is similar to GUSB, by using a serial dilution of the selected sample to amplify and then using the Mx3005P software to target the relative amount to the measured cycle limit (Ct The value is plotted to produce a correlation curve. The R ² values for all correlation curves were <0.99, indicating the linearity of the reaction. Efficiency is typically greater than 80% and is equal for MUC and GUSB.

To quantify the fold increase in MUC1 and MUC16 mRNA performance compared to the control under various treatments, the Mx3005P software calculates relative quantitative data in which the degree of performance of the PEG-3350 sample was compared to the control sample after correction for endogenous control GUSB. Data are expressed as the difference in gene performance (at 4 hours) compared to the control.

Data are expressed as mean ± SEM (mean standard error). Statistical analysis was performed using a two-way ANOVA-Taki Kremer test (factor 1 is time; factor 2 is treatment; JMP 8 software), p < 0.05 was considered statistically significant. Analyze data directly or after the Box-Cox conversion.

result

HCEpiC was treated with 10% PEG-3350 for 8, 18 or 24 hours; 3% PEG-3350 for 18 or 24 hours; or treated with 10% PEG for 2 hours, followed by treatment with control minimal medium for 6 hours, MUC1 mRNA content increased significantly (Fig. 9A).

HCEpiC exposure to 10% PEG 3350 4, 18 or 24 hours MUC16 mRNA content was significantly reduced (Figure 9B).

Overview of research results

10% PEG-3350 increased MUC1 mRNA content at 8, 18 or 24 hours. At 18 or 24 hours, 3% PEG-3350 increased MUC1 mRNA levels. In addition, treatment with PEG-3350 for 2 hours followed by incubation with control minimal medium for 6 hours resulted in a significant increase in MUCl mRNA. Thus, compositions of the invention comprising polyethylene glycol (such as PEG-3350) can stimulate the production of mucin in the eye.

At 4, 18 or 24 hours, the MUC16 mRNA content was significantly reduced in the case of 10% PEG-3350.

Test 6: PEG-3350 (10%) for the activation of the Japanese Institute of Physical Chemistry to transform human cornea Role of cell signaling pathways in dermal cells Introduction

Successful wound healing involves many cellular processes, particularly cell migration and proliferation (see, for example, FSXYu et al., "Growth Factor and Corneal Epithelial Wound Healing", Brain Res. Bull., Vol. 81, Nos. 2-3, 229- 35 (2010)). These processes are initiated and coordinated by most of the growth factors produced by the cornea in response to the wound. Epidermal growth factor receptor ("EGFR") is a transmembrane tyrosine kinase receptor that is activated during corneal trauma and is required for initiation of migration and healing (see JSLozano et al., "Activation of the Epidermal Growth Factor Receptor by Hydrogels in Artificial Tears, Exp.Eye Res., Vol. 86, 500-05 (2008)). EGFR is active in the phosphorylated state and provides binding sites for many signaling molecules including extracellular signal-regulated kinases 1 and 2 ("ERK1/2"). ERK1/2 has been shown to contribute to corneal wound healing by promoting cell proliferation and migration (see FSXYu et al., "ERK1/2 Mediate Wounding and G-Protein Coupled Receptor Ligands Induced EGFR Activation via Regulating ADAM 17 and HB-EGF Shedding , Invest. Ophthalmol. Vis . Sci., Vol. 50, No. 1, 132-39 (2009).

In this study, we sought to elucidate the molecular mechanism behind the positive effects of 10% PEG-3350 observed on corneal epithelial regeneration. In particular, the goal of this study was to evaluate the phosphorylation status of EGFR, ERK, Akt, and PI3K after exposure to 10% PEG-3350 over a 16 hour time course.

method Western ink point method

RT-HCEpiC cell suspension (2.5×10 ⁵ cells/ml) was prepared in complete medium with 10% fetal bovine serum (“FBS”) and added to each well of a 6-well culture plate with 2.5 mL suspension per well. . When the cells reached confluence, they were serum starved overnight in basic DMEM/F12. Treatment in minimal medium; treatment including control 10 ng/mL HB-EGF (heparin-binding EGF-like growth factor) for 10 minutes; or 10% PEG-3350 for 10, 15, 20, 30 minutes, 1, 2, 4 , 6 or 16 hours. The protein concentration of the cell lysate was analyzed and activated by western blots for Akt, ERK, EGFR and PI3K activation.

The medium was aspirated from each well and the cells were washed twice with cold non-sterile PBS. The cells were incubated with 1 x SDS lysis buffer, then scraped to the bottom of each well and transferred to a microcentrifuge tube. The cell lysate was sonicated to homogenize the sample, followed by centrifugation at 10 minutes x 13,000 RPM. The supernatant containing the cell lysate was transferred to a fresh microcentrifuge tube and stored at -70 °C. The protein concentration of the cell lysate was analyzed and analyzed by Western blots. After detecting the phosphorylated protein, the dots are removed and the corresponding total protein is detected. Western ink dots were imaged by chemiluminescence detection using a Bio Rad Versa Doc 4000 MP imager.

data analysis

Protein mass measurement: The protein concentration in cell lysates was determined based on the absorbance (OD) at 570 nm based on a standard curve generated using albumin. Follow using linear regression LP06017 analysis data.

Western dot method: Use the Quantity One software to analyze the density of Western dot dots in digital images captured and stored by the VersaDod MP 4000. The tape density is quantified. The results are reported as the ratio of the phosphorylated protein to the total protein.

result

Treatment with 10 ng/mL HB-EGF (positive control) for 10 minutes substantially increased phosphorylation of EGFR, Akt and ERK, but no effect was observed for pP13K (Figure 10).

10% PEG-3350 increased phosphorylation of EGFR as early as 10 minutes and lasted for 6 hours, and was less effective after 16 hours (Figure 10).

Phosphorylation of Akt was observed after incubation with 10% PEG-3350 and continued from 10 minutes to 20 minutes. Phosphorylation decreased slightly at 30 and 60 minutes, followed by an increase at 120 and 240 minutes, and peak phosphorylation was seen after 6 hours (Figure 10).

10% PEG-3350 activated ERK at 10, 15 or 20 minutes as indicated by phosphorylation and decreased slightly at 30 minutes (Figure 10). At 60 minutes, ERK phosphorylation was lower than in control cells. A slight increase in the level of phosphorylation near the control at 6 hours. However, after 16 hours of treatment with 10% PEG-3350, ERK phosphorylation had fallen below the control level.

No significant changes in PI3K phosphorylation were detected at any time point treated with 10% PEG-3350 (Figure 10).

Figure 11 shows a graphical representation of the peak protein phosphorylation time points for individual proteins. The ratio of phosphorylated protein to total non-phosphorylated protein is quantified by densitometry.

Overview of research results

At various points along the 16 hour duration, 10% PEG-3350 activated phosphorylation of EGFR, Akt and ERK.

Because some protein activators are involved in wound healing signaling, 10% PEG-3350 is much less effective at 10 ng/mL HB-EGF at 10 minutes.

Thus, compositions of the invention comprising polyethylene glycol (such as PEG-3350) can activate a cellular signaling pathway involving EGFR, Akt or ERK to promote healing of the damaged corneal epithelial layer.

Test 7: Effect of high volume molar permeation concentration and 3% or 10% polyethylene glycol 3350 on ZO-1 and actin distribution in transformed human corneal epithelial cells of the Institute of Physical and Chemical Research, Japan Introduction

The epithelial barrier function is maintained by a tight junction. Tight junctions are composed of protein complexes that form intimate contact between the plasma membranes of adjacent cells (a. Nusrat et al., "Molecular Physiology and Pathophysiology of Tight Junctions. IV. Regulation of Tight Junctions by Extracellular Stimuli: Nutrients, Cytokines, And Immune Cells", Am. J. Physiol, Gatroinstest . Liver Physioly, Vol . 279, G851-857 (2000)). The actin cytoskeleton is tied to a tight junction and is critical for its integrity, organized into a peripheral actin ring in corneal epithelial cells (SPSrinivas et al., "Histamine-Induced Phosphorylation of the Regulatory Light Chain" Of Myosin II Disrupts the Barrier Integrity of Corneal Endothelial cells", Invest. Ophthalmol Vis. Sci., Vol. 47, 4011-18 (2006)). Loss of epithelial barrier function due to increased contraction and division of actin and breakdown of tightly joined proteins such as occludin, ZO-1 and ZO-2 (K. Araki-Sasaki et al., "An SV40-Immortalized Human Corneal Epithelial" Cell Line and its Characterization", Invest Ophthalmol . Vis. Sci , Vol. 36, 614-21 (1995)). In this study, confocal microscopy was used to study the effect of 10% PEG-3350 pretreatment on changes in ZO-1 and actin distribution in HCEpiC 3350 induced by sucrose high volume molar permeation.

method Cell culture

RT-HCEpiC cells were seeded at a density of 5 × 10 ⁴ per ml in DMEM/F12 medium (0.5 ml/well) containing 15% fetal bovine serum ("FBS") and human corneal growth supplement ("HCGS"). The upper 4 well chamber slides were incubated in an incubator at 37 ° C, 5% CO ₂ and 95% humidity until they reached confluence (about 2 to 3 days after inoculation). Confluent cells were recultured in 15% FBS HCGS medium for 2 to 3 days to ensure tight junction formation. The medium was removed by aspiration and the cells were incubated in DMEM/F12 serum-free medium for 16 hours before being incubated in the test treatment. The medium was removed by aspiration and the cells were incubated in a PEG 3350-containing minimal medium or high volume molar osmolality sucrose medium at concentrations as described in Tables T-7-1 and T-7-2.

* 4 images per group

Immunocytochemical analysis of ZO-1 and actin:

The treatment solution was removed by aspiration and the cells were washed with phosphate buffered saline (PBS) (PBS-CM) containing 0.5 mM magnesium chloride and 1 mM calcium chloride. The cells were fixed in 3.7% paraformaldehyde for 10 minutes, followed by washing 3 times with PBS, followed by neutralization with 20 mM glycine for 10 minutes. The cells were washed 3 times with PBS, followed by permeation with PBS/0.1% Triton X-100 (TX-100) for 10 minutes, followed by blocking with PBS-CM containing 1% BSA and 10% goat serum for 30 minutes. After blocking, cells were incubated with 500 μl of PBS containing ZO-1 antibody (1:500) for 16 hours at 4 °C on a shaker. The cells were then washed 3 times with PBS on the shaker for 10 minutes each time. The cells were incubated with 500 μl PBS/1% BSA + Alexa-fluor rabbit 488 (1:2000) + 10 μl Alexa-fluor 568 phalloidin (small molecule that specifically binds to actin filamentous fibers) for 1 hour. The cells were washed 3 times with PBS-Triton X-100 for 10 minutes each time. The walls of the chamber slide and the gasket were removed and a drop of vectashield containing propidium iodide was added to each chamber well. The glass cover slip was placed on top and the edges were sealed with nail polish. The cells were observed at a magnification of 10 or 20 using a confocal microscope.

result

¹ from Figure 15 to Figure 17

² All data were compared to no PEG-3350 pretreatment followed by treatment with minimal medium for 2 hours, ++++ indicates no change in ZO-1 or actin distribution.

Overview of research results

The distribution of ZO-1 and actin is shown in Figures 12 to 14 after pretreatment with PEG-3350 or pretreatment with 3% or 10% PEG-3350 followed by treatment with increasing hypertonic medium. For cells not subjected to PEG pretreatment, ZO-1 and actin were observed at 525 and 585 mOsm/kg sucrose-treated cells instead of 465 mOsm/kg sucrose concentration compared to cells treated only with minimal medium. Changes in distribution.

For cells pretreated with 3% PEG-3350, cells treated at 525 and 585 mOsm/kg sucrose instead of 465 mOsm/kg compared to cells pretreated with 3% PEG-3350 followed by incubation in minimal medium Changes in ZO-1 and actin distribution were observed.

For cells pretreated with 10% PEG-3350, changes in ZO-1 and actin distribution were observed only in the case of 585 mOsm/kg sucrose-treated cells. After pretreatment with 10% PEG-3350, 465 and 525 mOsm/kg sucrose treatment had no effect on ZO-1 and actin distribution.

To distinguish the subjective differences between 525 and 585 mOsm/kg sucrose treatment, a more rigorous evaluation was performed in which 4 random images were obtained from cells treated with or without 10% PEG and then treated with the two hypertonic media. The results are summarized in Table T-7-3 and shown in Figures 15-17.

Figure 15 shows a comparison of cells treated with or without 10% PEG followed by treatment with minimal medium. No difference in ZO-1 or actin distribution was observed between these groups and both were evaluated as 4 + (Table T-7-3), indicating no change in ZO-1 and actin distribution.

Figure 16 shows a comparison of cells treated with 525 or 585 mOsm/kg sucrose without PEG pretreatment. The ZO-1 and actin distribution of cells treated with 525 mOsm/kg sucrose was evaluated as 3 + indicating that some division was observed. The ZO-1 and actin distribution of cells treated with 585 mOsm/kg sucrose was evaluated as 2 +, indicating significant division of tight junctions and a large number of large gaps within the cell monolayer.

Figure 17 shows a comparison of cells pretreated with 10% PEG followed by 525 or 585 mOsm/kg sucrose. The ZO-1 and actin distribution of cells treated with 525 mOsm/kg sucrose was evaluated as 4 +, indicating no change observed. The ZO-1 and actin distribution of cells treated with 585 mOsm/kg sucrose was evaluated as 3 + indicating that some division was observed.

Test 8: Effect of high volume molar permeation concentration and 3% PEG-3350 on monolayer resistance in transformed human corneal epithelial cells of the Institute of Physical Chemistry, Japan Introduction

In this study, electronic cell-matrix impedance sensing (ECIS) was used to effectively monitor the single-layer resistance change of the transformed human epithelial cells (RT-HCEpiC) of the Japan Institute of Physical Chemistry, which is an indication of barrier function. The novel ECIS system was used to determine the effect of the molar osmolality of sodium chloride (NaCl) on the monolayer resistance of human epithelial conjunctival cells, indicating a change in monolayer permeability. See test 4 disclosed above.

method

RT-HCEpiC cells were seeded on ECIS 8-well slides at a density of 1 or 2×10 ⁵ per ml in DMEM/F12 medium (DMEM/F12 complete HCGS medium) containing 15% FBS and HCGS (0.25 ml/well). And incubated at 37 ° C, 5% CO ₂ and 95% humidity in an incubator until it reached confluence (about 2 to 3 days after inoculation). The medium was removed by aspiration and the cells were incubated in minimal medium containing the test components at the concentrations listed in Table T-8-1. Cells were cultured under these conditions and the change in resistance was monitored by ECIS at 20 minute intervals. One cell of each well was tested with a single minimal medium as a negative control and cells of one well were tested with 33 ppm benzalkonium bromide (BOB) as a positive control to measure the change in electrical resistance. Only high volume molar osmolality of sucrose was tested in these experiments because NaCl interferes with ECIS measurements.

Table T-8-1 Overview of experimental design and progress

data analysis

The integral response of the impedance change is analyzed by calculating the area under the curve of each test hole over the time course using the trapezoidal rule defined by the following equation: Σ (time (hours) n) - (n-1)) × (impedance (ohms) n+n-1)/2=ohm*hour

The integral reaction was analyzed by two-way ANOVA and Taki Kremer test. Before the statistical analysis, the equivalent concentration and variance homogeneity of the data were evaluated, and if necessary, the results were subjected to Box-Cox conversion. Any conversion of the data is listed in the schema description.

Overview of research results

Treatment with 465 mOsm/kg sucrose medium without or with 3% PEG-3350 pretreatment did not significantly reduce the integral monolayer resistance as measured by ECIS compared to the minimal medium control (Figure 18). .

Treatment of cells with 525 mOsm/kg and 585 mOsm/kg sucrose medium with or without 3% PEG-3350 pretreatment significantly reduced the integral monolayer resistance compared to the minimal medium control. (Figure 18).

The normalization (to 0 o'clock) after pretreatment with PEG-3350 and basic or hypertonic sucrose medium and the time course of the original monolayer resistance up to 24 hours are shown in Figures 19-20, respectively.

In summary, the results of the studies disclosed herein together show that a composition of the invention comprising a polyethylene glycol such as PEG-3350 or PEG and a nonionic cellulose derivative such as HPMC or a similar cellulose derivative can: (1) treat Control, improve or reverse the symptoms of dry eye; (2) promote epithelial regeneration after corneal trauma; (3) protect the surface of the eye from drying-induced cell death; (4) maintain exposure to high volume molar osmotic concentration Damage to the integrity of the corneal surface; (5) promotion of mucin production by the eye to improve lubrication of the corneal surface; and (6) activation of cellular signaling pathways involving EGFR, ERK and Akt during healing of the eye wound. All such studies have shown that compositions within the scope of the present invention are effective in treating, controlling, ameliorating or reversing the condition, symptom, damage or injury caused by dry eye.

Although a particular embodiment of the invention has been described above, it will be understood by those skilled in the art that many equivalents can be made without departing from the spirit and scope of the invention as defined in the appended claims. Modifications, substitutions and changes.

Claims (23)

An ophthalmic composition comprising: (a) a concentration of from about 5 to about 15% by weight of the total composition of polyethylene glycol having a molecular weight in the range of from about 1,000 to about 10,000 Da; (b) The total composition is present in a concentration of from about 0.1 to about 5% of a nonionic water-soluble cellulose derivative having a molecular weight in the range of from about 50,000 to about 120,000 Da. The ophthalmic composition of claim 1, wherein the polyethylene glycol has a molecular weight in the range of from about 2,000 to about 8,000 Da, and the nonionic water-soluble cellulose derivative has a molecular weight in the range of from about 60,000 to about 100,000 Da. Inside. The ophthalmic composition of claim 2, wherein the polyethylene glycol is selected from the group consisting of PEG-2000, PEG-3350, PEG-4000, PEG-6000, PEG-8000, and mixtures thereof; and the cellulose derivative The system is selected from the group consisting of hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, methyl cellulose, and mixtures thereof. The ophthalmic composition of claim 3, wherein the concentration of the polyethylene glycol is in the range of about 7 to 12% by weight of the total composition, and the concentration of the cellulose derivative is in the total composition. The weight is in the range of about 0.3 to 2%. The ophthalmic composition of claim 4, wherein the polyethylene glycol is PEG-3350 or PEG-4000, and the cellulose derivative is hydroxypropylmethylcellulose. The ophthalmic composition of claim 4, which further comprises an ophthalmically acceptable therapeutic agent. The ophthalmic composition of claim 6, wherein the ophthalmically acceptable therapeutic agent is a compound having the formula II, V or VI: An ophthalmic composition consisting essentially of polyethylene glycol having a molecular weight in the range of 2,000 to 10,000; selected from the group consisting of hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and methyl a nonionic water-soluble cellulose derivative of cellulose; a nonionic surfactant selected from the group consisting of polysorbates 20, 60 and 80; boric acid; a polyol selected from the group consisting of glycerol, propylene glycol and mixtures thereof At least a phosphate; an antioxidant; a chelating agent; a preservative; and water; wherein the composition has a weight molar concentration in the range of 260 to 350 mOsm/kg, a pH in the range of 6.5 to 8 and It has a viscosity in the range of 2 to 500 centipoise. The ophthalmic composition of claim 8, wherein the polyethylene glycol is present in a concentration of from 5 to 15% by weight of the total composition; the nonionic water-soluble cellulose derivative is based on the weight of the total composition a concentration of 0.1 to 2%; and the nonionic surfactant is present in a concentration of 0.2 to 2% by weight of the total composition; and the polyol It is present at a concentration of from 0.01 to 2% by weight of the total composition. Use of a composition for the manufacture of a medicament for treating, controlling, ameliorating or reversing the condition of dry eye, wherein the composition comprises: (a) a concentration of about 5 to about the weight of the total composition About 15% of polyethylene glycol having a molecular weight in the range of from about 1,000 to about 10,000 Da; and (b) having a concentration of from about 0.1 to about 5% by weight of the total composition having a molecular weight of from about 50,000 to about 120,000 Da A nonionic water-soluble cellulose derivative. The use of claim 10, wherein the condition is selected from the group consisting of: eye discomfort, dryness, grit, tingling, eye irritation, and insufficient production of tear film material. The use of claim 10, wherein the polyethylene glycol has a molecular weight in the range of from about 2,000 to about 8,000 Da, and the nonionic water-soluble cellulose derivative has a molecular weight in the range of from about 60,000 to about 100,000 Da. The use of claim 12, wherein the polyethylene glycol is selected from the group consisting of PEG-2000, PEG-3350, PEG-4000, PEG-6000, PEG-800, and mixtures thereof; and the cellulose derivative is selected A group consisting of free hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, methyl cellulose, and mixtures thereof. The use of claim 13, wherein the concentration of the polyethylene glycol is in the range of from about 7 to 12% by weight of the total composition, and the concentration of the cellulose derivative is based on the weight of the total composition. It is in the range of about 0.3 to 2%. The use of claim 14, wherein the polyethylene glycol is PEG-3350 or PEG-4000, and the cellulose derivative is hydroxypropyl methylcellulose. The use of claim 14, wherein the composition further comprises an ophthalmically acceptable therapeutic agent. The use of claim 16, wherein the ophthalmically acceptable therapeutic agent is a compound having Formula II, V or VI: Use of a composition for the manufacture of a medicament for the treatment, management, amelioration or reversal of the condition of dry eye, wherein the composition consists essentially of polyethylene glycol having a molecular weight in the range of 2,000 to 10,000 a nonionic water-soluble cellulose derivative selected from the group consisting of hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and methylcellulose; selected from the group consisting of polysorbates 20, 60, and 80 a nonionic surfactant; boric acid; a polyol selected from the group consisting of glycerol, propylene glycol, and mixtures thereof; at least a phosphate; an antioxidant; a chelating agent; a preservative; and water; wherein the composition has 260 to The molar osmolality in the range of 350 mOsm/kg, the pH in the range of 6.5 to 8 and the viscosity in the range of 2 to 500 centipoise. The use of claim 18, wherein the polyethylene glycol is present in a concentration of from 5 to 15% by weight of the total composition; the nonionic water-soluble cellulose derivative is in the total group The weight of the compound is present at a concentration of 0.1 to 2%; and the nonionic surfactant is present at a concentration of 0.2 to 2% by weight of the total composition; and the polyol is based on the weight of the total composition A concentration of 0.01 to 2% is present. The use of claim 19, wherein the condition is selected from the group consisting of: eye discomfort, dryness, grit, tingling, eye irritation, and insufficient production of tear film material. A use of a composition for the manufacture of a medicament for: (1) treating, controlling, ameliorating or reversing the condition of dry eye; (2) promoting epithelial regeneration after corneal trauma; (3) providing for the surface of the eye Protect against cell death induced by dryness; (4) maintain the integrity of the corneal surface exposed to high volume molar penetration; (5) promote mucin production in the eye, resulting in improved lubrication of the corneal surface; or (6) promote Activation of cellular signaling pathways involving EGFR, ERK, and Akt during healing of an eye wound, wherein the composition comprises: (a) a concentration of from about 5 to about 15% by weight of the total composition having a molecular weight of about 1,000 a polyethylene glycol in the range of up to about 10,000 Da; and (b) a nonionic water soluble fiber having a molecular weight of from about 50,000 to about 120,000 Da in a concentration of from about 0.1 to about 5% by weight of the total composition A derivative. The use of claim 21, wherein the polyethylene glycol has a molecular weight in the range of from about 2,000 to about 8,000 Da, and the nonionic water-soluble cellulose derivative has a molecular weight in the range of from about 60,000 to about 100,000 Da; The composition further comprises a nonionic surfactant and a polyol. The use of claim 22, wherein the polyethylene glycol is present in a concentration of from 5 to 15% by weight of the total composition; the nonionic water-soluble cellulose derivative is 0.1 to the weight of the total composition a concentration of 2% is present; and the nonionic surfactant is present at a concentration of from 0.2 to 2% by weight of the total composition; and the polyol is present in a concentration of from 0.01 to 2% by weight of the total composition .