RU2792627C2 - Compositions and methods for treating eye disorders - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is intended for treatment of ophthalmic diseases. A method for treating a patient with a reduced or low level of production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea includes the step of locally administering to the patient's eye a composition containing: i) a safe and effective amount of one or more extracts, or sources of extracts, yeast of the genus Pichia to achieve a concentration of yeast extract of the genus Pichia in the corneal fluid in the tissues of the cornea of the eye of at least 0.3 mg/ml, in which the yeast extract of the genus Pichia is Pichia anomala; and ii) an ophthalmologically acceptable carrier.

EFFECT: using the invention allows to induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea.

15 cl, 2 dwg, 2 tbl, 4 ex

Description

FIELD OF THE INVENTION

The present invention relates to compositions containing one or more compounds and/or extracts that induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid from the cornea and/or to the cornea, as well as methods of using these compositions for treatment eye.

BACKGROUND OF THE INVENTION

"Dry eye is a multifactorial disease of the ocular surface characterized by loss of tear film homeostasis and accompanied by ocular symptoms in which tear film instability and hyperosmolarity, inflammation and damage to the ocular surface, and neurosensory abnormalities play an etiological role." Craig, J.P. et al. TFOS DEWS II definition and classification report. Ocul Surf. 2017; 15:276-283. Dry eye syndrome can be the result of abnormal or insufficient tear fluid production and a deficiency in mucin secretion (i.e., keratoconjunctivitis sicca). The symptoms of dry eye syndrome can manifest as a result of various underlying disorders, such as autoimmune diseases that affect the lacrimal (i.e., tear-producing) glands, such as rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, systemic sclerosis, and sarcoidosis. Dry eye syndrome can also occur after eye surgery such as Lasik® surgery. It is estimated that more than 13 million people in the United States suffer from dry eye syndrome.

Regardless of the underlying pathological conditions, dry eye syndrome usually involves rapid destruction of the preocular tear film, resulting in dehydration of the exposed outer surface. Normal tear fluid production is necessary to keep the cornea and conjunctiva moist, which in turn helps prevent ulceration and also maintains the transparency of the cornea. In addition, tears facilitate the movement of the eyelid over the surface of the eye (for example, blinking) and the removal of foreign matter from the eye. Tears also usually contain lysozyme, which helps prevent eye infections. Dry eye syndrome can cause both mild discomfort and severe eye pain. When this occurs for extended periods of time, blurred vision, graininess and/or a burning and itching sensation may occur. If left untreated, the disease can also lead to corneal ulcers and/or scarring.

Symptoms of dry eye syndrome include eye pain or fatigue, increased blinking, and inflamed eyes. In addition, bacteria can enter through scratches and cause infection, and if the scratch is deep enough, it can even affect vision. In addition to eye strain, dry eye causes include Sjögren's syndrome, Stevens-Johnson syndrome, eye burns and injuries, as well as side effects of antihypertensive drugs, tranquilizers, glaucoma eye drops, and other similar drugs.

Eye drops are an effective way to treat dry eye syndrome. Such eye drops usually contain active substances for the treatment of dry eye syndrome, the most common active substance in such drops is hyaluronic acid. Hyaluronic acid is a macromolecular substance of biological origin, has extremely high water retention and characteristic properties such as high viscoelasticity, good thickening performance and high threading ability, and is used as a humectant in topical preparations for the treatment of various kinds of skin problems, etc. n. In the case of dry eye syndrome caused by Sjögren's syndrome, in which dryness is observed throughout the body, eye drops containing hyaluronic acid are effectively used. However, when administered as eye drops, hyaluronic acid has a relatively short residence time on the cornea, so the effect of hyaluronic acid eye drops lasts only about 2-3 hours, which means that the patient must use the drops more frequently (for example, 5 to 10 times a day). day).

Hyaluronic acid (HA) is produced by the epithelial cells of the cornea of the eye. Notably, significantly higher concentrations of hyaluronic acid have been found in the corneas of young people (compared to older people). ( See Pacella, E., Pascella, F., De Paolis, G., et al. Glycosaminoglycans in the human cornea: age-related changes . Ophthalmol. Eye Dis. 7:1-5, 2015).

Hyaluronic acid can also be used for normal wound healing and is essential for maintaining overall eye health.

Thus, there is a need for an ophthalmic pharmaceutical composition that stimulates and/or improves the production and/or release of hyaluronic acid from or into the cornea.

The present inventors have found extracts, or sources of extracts, of yeasts of the genus Pichia that, when sufficiently concentrated in the corneal fluid, can induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea.

Accordingly, an aspect of the present invention relates to methods for inducing, stimulating and/or improving the production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea, comprising the step of administering ophthalmic compositions containing a safe and effective amount of one or more extracts or sources extracts, yeast of the genus Pichia .

Another aspect of the present invention relates to methods of inducing, stimulating and/or improving the production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea, comprising the step of administering ophthalmic compositions containing a safe and effective amount of one or more extracts, or sources of extracts , yeast of the genus Pichia , to induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea, and the compositions can be administered to patients with a concentration of hyaluronic acid in the lacrimal fluid less than 10 (or about 10) nanograms , optionally less than 15 (or about 15) nanograms, optionally less than 20 (or about 20) nanograms, or optionally less than 25 (or about 25) nanograms per milligram of proteins such that the concentration of hyaluronic acid in their tear fluid exceeds or becomes equal to 10 ( or about 10), not necessarily greater than or equal to 15 (or about 15), optionally greater than or equal to 20 (or about 20), optionally greater than or equal to 25 (or about 25) nanograms, optionally greater than or equal to 30 (or about 30), optionally greater than or equal to equal to 35 (or about 35), optionally greater than or equal to 40 (or about 40) or optionally greater than or equal to 45 (or about 45) nanograms per milligram of proteins up to 100 (or about 100), optionally 90 (or about 90) , optionally 80 (or about 80), optionally 70 (or about 70), or optionally 60 (or about 60) nanograms per milligram of proteins.

In some embodiments, the above-described concentration of hyaluronic acid in the lacrimal fluid of the patient, due to the compounds and/or extracts of the present invention, is maintained for a period of up to at least about 2 hours, optionally about 4 hours, optionally about 6 hours, optionally about 8 hours, optionally about 10 hours, optionally about 12 hours, or optionally from about 12 to about 24 hours.

The hyaluronic acid concentrations detailed above are determined using the Dreyfus method (described below in the definitions).

Another aspect of the present invention relates to methods of inducing, stimulating and/or improving the production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea for the treatment (e.g., reduction) and/or prevention of dry eye or symptoms associated with dry eye , comprising the step of administering ophthalmic compositions containing a safe and effective amount of one or more extracts, or sources of extracts, of the yeast of the genus Pichia .

Another aspect of the present invention relates to methods for the prevention and/or treatment of ocular symptoms resulting from reduced or low production/release/delivery/release of hyaluronic acid from and/or into the cornea by administering compositions containing a safe and effective amount of one or more extracts, or sources of extracts, of yeast of the genus Pichia to induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea, for example, the concentration of hyaluronic acid within and/or on the surface of the cornea.

Another aspect of the present invention relates to methods of promoting healing or increasing the rate of healing of wounds inside and/or on the surface of the eye (for example, non-dry eye injuries of the eye, postoperative surgical or non-specific wounds) of a patient by administering compositions containing a safe and effective amount of one or more extracts, or sources of extracts, of yeast of the genus Pichia to increase the production/release/delivery/excretion of hyaluronic acid from the cornea and/or into the cornea. In some embodiments, the above methods increase the production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea beyond the concentration of hyaluronic acid normally produced in such a patient without administering compositions containing a safe and effective amount of one or more extracts or sources extracts, yeast of the genus Pichia .

SUMMARY OF THE INVENTION

The present invention relates to methods for treating patients with reduced or low levels of production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea, comprising the step of local administration to the eye of a patient (optionally a patient in need of an increase in such production/release/delivery/ release of hyaluronic acid) composition containing:

i) a safe and effective amount of one or more extracts, or sources of extracts, of the yeast of the genus Pichia to achieve a concentration of the extract of the yeast of the genus Pichia in corneal fluid in corneal tissues of the eye of at least about 0.3 mg/ml;

ii) optionally a safe and effective amount of a penetration enhancer; And

iii) optionally an ophthalmologically acceptable carrier.

The present invention relates to methods for promoting healing or increasing the rate of healing of wounds in the eye and/or on the surface of the eye (optionally in a patient in need of such stimulated healing of eye wounds or an increased rate of healing), comprising the step of locally administering to such a patient a composition (i.e. a composition that enhances the production/release/delivery/release of hyaluronic acid from the cornea and/or to the cornea, in some embodiments, in addition to the concentration of hyaluronic acid produced by such a patient without (or in the absence of) administration of compositions containing a safe and effective amount of one or more compounds and/or extracts, or sources of extracts, yeast of the genus Pichia ) containing:

i) a safe and effective amount of one or more extracts, or sources of extracts, of the yeast of the genus Pichia to achieve a concentration of the extract of the yeast of the genus Pichia in corneal fluid in corneal tissues of the eye of at least about 0.3 mg/ml;

ii) optionally a safe and effective amount of a penetration enhancer; And

iii) optionally an ophthalmologically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

On FIG. 1 is bar graphs showing increased HA production in corneal epithelial cells after topical application of Pichia anomala yeast extract at 24 and 48 hours.

On FIG. 2 is a bar graph showing a statistically significant increase in HA production in corneal epithelial cells 48 hours after corneal epithelial cells were placed in a culture medium containing Pichia anomala extract.

DETAILED DESCRIPTION OF THE INVENTION

It is believed that in accordance with the description provided in this document, a person skilled in the art will be able to use this invention to the fullest extent. The following specific embodiments can only be considered as illustrative examples and do not limit the rest of the present description in any way.

Compositions of the present invention may contain, consist of, or consist essentially of the elements, steps, and limitations of the invention described herein, as well as any of the additional or optional ingredients, components, or limitations described herein.

In this document, the term "comprising" (and its grammatical variations) is used in a broad sense in the sense of "having" or "including", and not in a narrow sense in the sense of "consisting only of". In this document, the singular forms are considered to cover both the plural and the singular.

Unless otherwise indicated, all technical and scientific terms used in this document have a generally accepted meaning, understandable to any person skilled in the field to which the invention pertains. In addition, all publications, patent applications, patents and other references mentioned in this document are incorporated herein in their entirety by reference to the extent that they do not contradict the present description. In this document, all percentages are given by weight of the total composition, unless otherwise indicated.

As used herein, the term "cornea" or "corneal" includes and/or refers to the transparent anterior portion of the eye that covers the iris, pupil, and anterior chamber, wherein layers of this transparent anterior portion include the corneal epithelium layer (comprising corneal epithelial cells), Bowman's membrane (also known as the anterior limiting membrane), corneal stroma (also self-substance), Descemet's membrane (also posterior limiting membrane), and corneal endothelium (a simple squamous or low-cubic monolayer of mitochondrial-rich cells that is about 5 µm).

Following are the functions of the different layers.

Epithelium:

provides a barrier to chemicals and water;

provides a barrier to microbes;

provides a smooth optical surface as the inner part of the tear film interface and helps to increase the refractive power of the eye; And

contains Langerhans cells that perform important immunological functions.

Bowman's membrane:

helps maintain the shape of the cornea.

Corneal stroma:

provides mechanical strength to the cornea;

provides transparency of the cornea; And

acts as a refractive lens.

Descemet's membrane:

acts as a support layer for endothelial cells.

Corneal endothelium:

maintains the transparency of the cornea by removing water from the corneal stroma.

The corneal epithelium layer consists of 5-7 fairly evenly spaced layers of cells. The thickness of the corneal epithelium is about 50 microns. The epithelium is homogeneous to provide a smooth surface and consists of non-keratinized stratified squamous epithelium. Without being limited by theory, it is believed that the corneal epithelium layer additionally contains tight junction structures (i.e., structures [e.g., membranes or film barriers] that generally interfere with the penetration of chemicals into tissues) that can interfere with or protrude into as a barrier to diffusion of one or more extracts or sources of extracts of the yeast of the genus Pichia , reducing the diffusion of one or more extracts or sources of extracts of the yeast of the genus Pichia through the corneal epithelium into the corneal tissue, thus reducing concentrations of the extract that may accumulate in the corneal tissues and contact with the cellular regions of such tissues responsible for the production of hyaluronic acid.

As used herein, the phrase "reduced or low level of production/release/delivery/release of hyaluronic acid from the cornea and/or to the cornea" means a concentration of hyaluronic acid that is less than the concentration of hyaluronic acid in the tear fluid of a normal (i.e. healthy) person or in in some embodiments, less than 25 (or about 25) nanograms per milligram of proteins, as determined by the method described in Dreyfuss JL, Regatieri CV, Coelho B, et al. Altered hyaluronic acid content in tear fluid of patients with adenoviral conjunctivitis. An Acad Bras Cienc. 2015;87(1):455-462. This method (Dreyfus method) is reproduced below.

Sample selection

To collect tear fluid, Schirmer strips were placed on the temporal side of each eye under the eyelid for 5 minutes without the use of local anesthetics. The strips were dried at room temperature and stored at -20°C until analysis.

Tear sample preparation

Lacrimal compounds were eluted from the Schirmer strips using 100 μl of distilled water and analyzed for hyaluronic acid and protein content.

Measurement of hyaluronic acid

The content of hyaluronic acid in the lacrimal fluid was determined using a non-isotopic fluorescence assay (see Martins Jr, Passerotti CC, Maciel RM, Sampaio Lo, Dietrich CP and Nader HB. 2003.) Practical determination of hyaluronan by a new noncompetitive fluorescence-based assay on serum of normal and cirrhotic patients. Anal Biochem 319: 65-72.) Eluted tears and standard concentrations of hyaluronic acid (Sigma, St. Louis, MO) were added to 96-well plates (FluoNUNC Maxisorp Microtiterplates, Roskilde, Denmark) pre-coated with binder. hyaluronic acid protein. The plates were then sequentially incubated with biotinylated hyaluronic acid binding protein and europium labeled streptavidin (Amershan, Piscataway, NJ). Thereafter, the europium remaining in the solid phase was released with an amplifying solution, and fluorescence was measured using a time-resolved fluorometer (Perkin-Elmer Life Sciences-Wallac Oy, Turku, Finland). Data (counts/s) were processed automatically using MultiCalc software (Perkin-Elmer Life Sciences-Wallac Oy) and values were expressed in ng/mg protein.

Protein analysis

Total tear protein concentration was determined using a colorimetric assay kit according to the manufacturer's instructions (Protein Assay Kit from Bio-Rad, Hercules, CA). The protein profile was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, as previously described (see Laemmli UK. 1970.) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.). Briefly, 10 μg of protein from tear fluid samples was applied to a 3-20% linear gradient polyacrylamide gel under reducing conditions. After electrophoresis, the gels were stained with Coomassie blue (Bio-Rad, Hercules, CA). Each protein band was quantified by densitometry using ImageJ software version 10.2 for Mac (U.S. National Institutes of Health, Bethesda, MD, USA). Results are expressed in arbitrary densitometric units (ADU).

As used herein, a composition "substantially free" of an ingredient means a composition containing about 2% or less of that ingredient by weight, based on the total weight of the composition. Preferably, the substantially ingredient-free composition contains about 1% or less, more preferably about 0.5% or less, more preferably about 0.1% or less, more preferably about 0.05% or less, more preferably about 0 01% or less of an ingredient by weight, based on the total weight of the composition. In certain more preferred embodiments, a substantially ingredient-free composition does not contain an ingredient, i.e., the ingredient is not present in the composition.

As used herein, the term "ophthalmologically acceptable" means that the ingredients to which the term refers are acceptable for use in contact with tissues (e.g. soft tissues of the eye or periorbital skin tissues) without the side effects of non-specific toxicity, incompatibility, instability, irritation, allergic reaction, etc. As will be understood by any person skilled in the art, cosmetically/dermatologically acceptable salts are acid/anionic or alkaline/cationic salts.

As used herein, the term "safe and effective amount" means an amount of the described extract, compound or composition sufficient to induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid from one or more layers of the cornea and/or into one or more more layer of the cornea, but low enough to prevent serious side effects. The safe and effective amount of a compound, extract or composition will vary depending on, for example, the age, health and environmental conditions of the end user, the duration and nature of the treatment, the particular extract, ingredient or composition used, the particular ophthalmologically acceptable carrier used, and to similar factors.

In certain embodiments, the present invention described herein may be practiced in the absence of any compound or element (or group of compounds or elements) not specifically referenced herein.

In general, this document applies the rules of the International Union of Pure and Applied Chemistry (IUPAC) nomenclature in accordance with the following definitions of terms.

The term "C1-8 alkyl", whether used alone or as part of a substituent group, refers to a saturated aliphatic branched or straight chain monovalent hydrocarbon radical having 1-8 carbon atoms. For example, "C1-8 alkyl" specifically includes methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, t-butyl, 1-butyl, 1-pentyl, 2-pentyl, 3 -pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 1-octyl, 2-octyl, 3-octyl, etc. This term may also refer to the corresponding alkyldiyl radical. The alkyl and alkyldiyl radicals can be attached to the parent molecule via a terminal carbon atom or via an intrachain carbon atom. Similarly, any number of variable substituents allowed by the available valences may be attached to the alkyl or alkyldiyl radical.

The term "C _1-4 alkyl", whether used alone or as part of a substituent group, refers to a saturated aliphatic branched or straight chain monovalent hydrocarbon radical or alkyldiyl linking group having a specified number of carbon atoms, the radical derived from the removal of one hydrogen atom from a carbon atom , and the alkyldiyl linking group is obtained by removing one hydrogen atom from each of the two carbon atoms in the chain. The term "C _1-4 alkyl" refers to a radical having 1-4 carbon atoms in a linear or branched configuration. For example, "C _1-4 alkyl" specifically includes methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, t-butyl, 1-butyl, and the like radicals. Alkyl and alkyldiyl radicals can be attached to the main molecule via a terminal carbon atom or via an intrachain carbon atom. Similarly, any number of variable substituents allowed by the available valences may be attached to the alkyl or alkyldiyl radical.

The term "C _2-4 alkenyl" refers to an alkenyl radical having 2-4 carbon atoms. For example, specifically includes ethenyl, propenyl, allyl (2-propenyl), butenyl, and the like radicals. As described above, an alkenyl radical can be similarly attached to the parent molecule and further substituted where indicated.

The term "halogen", alone or in combination with other terms, means a halogen atom such as fluorine, chlorine, bromine or iodine.

The term "substituted" refers to a base molecule in which one or more hydrogen atoms have been replaced by the number of substituents allowed by the available valences. Substitution is not limited to the parent molecule, but can also occur on a substituent radical, whereby the radical is converted to a linking group.

The term "independently selected" refers to two or more substituents which may be selected from a group of variable substituents, the selected substituents being the same or different.

The term "dependently selected" refers to one or more variable substituents that are given in the specified combination for substitution in the main molecule (for example, variables that refer to groups of substituents listed in the tabular list of compounds).

Acceptable salts prepared using inorganic bases include, for example, sodium or potassium salts, and the like. Acceptable salts prepared using organic bases include, for example, salts formed by reaction with primary, secondary, or tertiary amines, etc.

Compounds and/or extracts that induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid in the cornea.

The present invention includes one or more compounds and/or extracts that induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid from and/or to the cornea.

In certain embodiments, the compounds and/or extracts that induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid from and/or to the cornea are, or contain, extracts, or sources of extracts, of the yeast of the genus Pichia .

Pichia is a genus of yeast in the Saccharomycetaceae family. More than 100 species of this genus are known. Suitable species for use in the compositions of the present invention include (selected from the group consisting of) Pichia anomala , Pichia guilliermondii , Pichia norvegensis and Pichia ohmeri . Pichia anomala (formerly Hansenula anomala ) can be found in unboiled milk and cheese. Pichia yeast extracts are high in mannans, polysaccharides composed of mannose monomers. Extracts or sources of extracts of yeasts of the genus Pichia can be isolated from the fruit or other aerial parts of the plant. Any ophthalmologically acceptable yeast extract of the genus Pichia can be used. Mixtures of extracts, or extract sources, of the aforementioned species of the genus Pichia can also be used.

In certain embodiments, the extracts, or sources of extracts, of the yeast of the genus Pichia used in the present invention are extracts of Pichia anomala. In some embodiments, a suitable extract of Pichia anomala is obtained from a strain of Pichia anomala found on kiwi fruits or leaves. In another embodiment, the Pichia anomala extract is available from Silab-France under the trade names PRO-LIPISKIN, or UNFLAMAGYL®, where the extract is obtained from a strain of Pichia anomala found on sugar cane.

In one embodiment, the extract of Pichia anomala is obtained in accordance with the following method, as described in FR2897266 and FR2938768, which are incorporated herein by reference.

In certain embodiments, the extraction method described below removes most of the protein from the Pichia extract and increases the concentration of active mannan. The method includes at least one step of enzymatic hydrolysis of proteins to produce peptides and small proteins, and in certain embodiments, an additional step of removing these small peptides and proteins by filtering by selecting such molecules by size.

In certain embodiments, Pichia yeast extracts, including Pichia anomala extract, are produced by an extraction process involving one or more hydrolysis enzymes to hydrolyze proteins in the Pichia yeast, either sequentially or simultaneously.

In certain embodiments, enzymatic hydrolysis is used to cleave the proteins in the Pichia yeast extract into protein fractions with a weight average molecular weight of less than 5000 Da. Suitable hydrolysis enzymes include, without limitation, at least one peptidase, which in certain embodiments is selected from papain, trypsin, chymotrypsin, subtilisin, pepsin, thermolysin, pronase, flavastacin, enterokinase, factor Xa protease, turine, bromelain, proteinase K, genenase I, termitase, carboxypeptidase A, carboxypeptidase B, collagenase, or mixtures thereof.

In certain embodiments, the enzymes used to prepare the extract of the yeast of the genus Pichia are inactivated before separating the resulting soluble and insoluble phases.

In one embodiment, the Pichia anomala extract is characterized in that it has:

- solids content from 5 to 300 g/l,

- pH from 4 to 9,

- protein content from 2 to 170 g/l, and

- sugar content in the range from 1 to 100 g/l.

In certain embodiments, the Pichia yeast extract has a mannan content greater than or equal to 30% by weight of the total weight of the dried Pichia yeast extract, or optionally a mannan content of at least 50% by weight of the total weight of the dried Pichia yeast extract.

In certain embodiments, the Pichia yeast extract phase is dried (and the solids content is measured) by passing the phase containing the Pichia yeast extract through an oven at 105°C (or about 105°C) in the presence of sand until a constant masses.

In certain embodiments, the solids content of the dried Pichia yeast extract is 10 to 200 g/L, or optionally 26 to 40 g/L.

In certain embodiments, the pH, measured potentiometrically at room temperature (25° C.), is between 4.5 and 8.5, optionally between 6.0 and 7.0.

The DUBOIS method can be used to determine the total sugar content. In the presence of concentrated sulfuric acid and phenol, reducing sugars give an orange-yellow compound. The total sugar content of a sample can be determined from the standard range. In certain embodiments, the total sugar content of the dried Pichia yeast extract is 7 to 145 g/L, or optionally 18 to 29 g/L. In certain embodiments, the dried Pichia yeast extract contains at least 30% total sugars by weight of the dried Pichia yeast extract compared to the total solids content by weight of the dried Pichia yeast extract, optionally at least 50% by weight of the dried yeast extract genus Pichia .

In certain embodiments, the carbohydrate fraction of the dried Pichia yeast extract consists of mannose and glucose in the form (or substantially in the form) of oligosaccharides and polysaccharides having a weight average molecular weight of from about 180 to about 800,000 Da, optionally from about 5,000 to about 515,000 Yes, optionally from about 6,000 to about 270,000 Da. In certain embodiments, at least 70% (or about 70%), optionally at least 75% (or about 75%), at least 80% (or about 80%), optionally at least 85% (or about 85%), at least 90% (or about 90%), optionally at least 95% (or about 95%) or 100% (or about 100%) of the oligosaccharides and polysaccharides in the dried extract of the Pichia yeast fall within the above ranges weight average molecular weight.

Determination of protein content is carried out by the method of Kjeldahl (Kjeldahl). In certain embodiments, the dried Pichia yeast extract has a protein content of 4 to 90 g/L, or optionally 12 to 18 g/L. The dried Pichia yeast extract contains less than 45% proteins, or optionally less than 30% of the total solids content of the dried Pichia yeast extract.

In certain embodiments, the dried Pichia yeast extract contains mannans polymerized as oligosaccharides and polysaccharides having a weight average molecular weight of from about 180 to about 800,000 Da, optionally from about 5,000 to about 515,000 Da, optionally from about 6,000 up to about 270,000 Da in a dried extract of the yeast of the genus Pichia . In certain embodiments, at least 70% (or about 70%), optionally at least 75% (or about 75%), at least 80% (or about 80%), optionally at least 85% (or about 85%), at least 90% (or about 90%), optionally at least 95% (or about 95%) or 100% (or about 100%) of the oligosaccharides and polysaccharides in the dried extract of the Pichia yeast fall within the above ranges weight average molecular weight.

In certain embodiments, after the step of enzymatic hydrolysis of the proteins, the extraction method includes the step of removing (eg, by filtration) proteins having a weight average molecular weight of less than 5000 Da. Accordingly, the Pichia yeast extracts are free or substantially free of proteins and/or peptides having a weight average molecular weight of less than 5000 Da.

In certain embodiments, the extraction method described above may include the step of deodorizing, bleaching, and/or stabilizing the Pichia yeast extract prior to filtration. Types of filtration can be as follows: press filtration and sterilizing filtration.

In certain embodiments, the extracts used are of the Pichia anomala variety. A specific, non-limiting example of a manufacturing process is described below:

- extracts (or yeast) of Pichia anomala are cultivated in a culture medium adapted to their development, then centrifuged to extract the biomass,

- then the biomass is ground in a ball mill; then the ground material is resuspended in water at a concentration of 50 grams per liter before enzymatic hydrolysis in an alkaline medium at 30 °C for 6 hours,

- after hydrolysis, the product is centrifuged and filtered before sterilization,

- by successive filtrations on filters of different sizes, a hydrolyzate is obtained containing at least 30% of mannans relative to the total weight of solids, and/or proteins with a certain weight average molecular weight are obtained. (The resulting hydrolyzate is in the form of a light yellow clear liquid aqueous solution.)

In certain embodiments, the extractPichia anomala receive according to the following manufacturing examples.

1. Pichia anomala A extract

I. Extraction of Pichia anomala A extract

Obtaining an extract of Pichia anomala A includes the following steps:

- cultivation of the yeast Pichia anomala in an environment adapted for their development,

- centrifugation to extract biomass,

- biomass solubilization,

- enzymatic hydrolysis at alkaline pH,

- separation of soluble and insoluble phases,

- heat treatment,

- filtering, and

- sterile filtration.

II. Characteristics of Pichia anomala A extract

The extract of Pichia anomala A obtained above is characterized by the following parameters:

- solids content from 48 to 84 g/l,

- pH from 4 to 9,

- protein content from 19 to 48 g/l, and

- total sugar content from 10 to 42 g/l.

2. Pichia anomala B extract

I. Extraction of Pichia anomala B extract

Obtaining an extract of Pichia anomala B includes the following steps:

- cultivation of the yeast Pichia anomala in an environment adapted for their development,

- centrifugation to extract biomass,

- biomass solubilization,

- enzymatic hydrolysis at acidic pH,

- separation of soluble and insoluble phases,

- heat treatment,

- filtering, and

- sterile filtration.

II. Characteristics of Pichia anomala B extract

The extract of Pichia anomala B obtained above is characterized by the following parameters:

- solids content from 58 to 95 g/l,

- pH from 4 to 9,

- protein content from 23 to 54 g/l, and

- total sugar content from 12 to 32 g/l.

3. Pichia anomala C extract

I. Extraction of Pichia anomala C extract

Obtaining an extract of Pichia anomala C includes the following steps:

- cultivation of the yeast Pichia anomala in an environment adapted for their development,

- centrifugation to extract biomass,

- biomass solubilization,

- successive operations of enzymatic hydrolysis in an alkaline medium,

- separation of soluble and insoluble phases,

- heat treatment,

- filtering, and

- sterile filtration.

II. Characteristics of Pichia anomala C extract

The extract of Pichia anomala C obtained above is characterized by the following parameters:

- solids content from 91 to 195 g/l,

- pH from 4 to 9,

- protein content from 36 to 111 g/l, and

- total sugar content from 18 to 65 g/l.

4. Pichia anomala D extract

I. Extraction of Pichia anomala D extract

Obtaining an extract of Pichia anomala D includes the following steps:

- cultivation of the yeast Pichia anomala in an environment adapted for their development,

- centrifugation to extract biomass,

- biomass solubilization,

- hydrolyze simultaneously with at least two enzymes at acidic pH,

- separation of soluble and insoluble phases,

- heat treatment,

- filtering, and

- sterile filtration.

II. Characteristics of Pichia anomala D extract

The extract of Pichia anomala D obtained above is characterized by the following parameters:

- solids content from 5 to 53 g/l,

- pH from 4 to 9,

- protein content from 2 to 30 g/l, and

- total sugar content from 1 to 18 g/l.

5. Pichia anomala E extract

I. Extraction of Pichia anomala E extract

Obtaining the extract of Pichia anomala E includes the following steps:

- cultivation of the yeast Pichia anomala in an environment adapted for their development,

- centrifugation to extract biomass,

- solubilization of biomass in a water-glycol environment,

- hydrolyze simultaneously with at least two enzymes at acidic pH,

- separation of soluble and insoluble phases,

- heat treatment,

- filtering, and

- sterile filtration.

II. Characteristics of Pichia anomala E extract

The extract of Pichia anomala E obtained above is characterized by the following parameters:

- solids content from 172 to 300 g/l,

- pH from 4 to 9,

- protein content from 69 to 170 g/l, and

- total sugar content from 34 to 100 g/l.

In certain embodiments, the extract, or source of extracts, of yeast of the genus Pichia contains oligosaccharides and polysaccharides with an average degree of polymerization from DP 1 to DP 4444, optionally from DP 30 to DP 2860, optionally from DP 35 to DP 1500. In certain embodiments, at least at least 70% (or about 70%), optionally at least 75% (or about 75%), at least 80% (or about 80%), optionally at least 85% (or about 85%), at least at least 90% (or about 90%), optionally at least 95% (or about 95%), or 100% (or about 100%) of the oligosaccharides and polysaccharides in the dried Pichia yeast extract fall within the above average degree of polymerization ranges.

In certain embodiments, an extract, or source of extracts, of yeast of the genus Pichia is present in the compositions of the present invention to provide, after topical application to the user, in the cornea or tissue cells, or in contact with the cornea or tissue cells (i.e., in the context of internal corneal fluid levels ), a Pichia extract concentration of at least 0.3 mg/mL (or about 0.3 mg/mL), optionally at least 0.5 mg/mL (or about 0.5 mg/mL), optionally at least at least 1 mg/ml (or about 1 mg/ml), optionally at least 1.5 mg/ml (or about 1.5 mg/ml), optionally at least 2 mg/ml (or about 2 mg/ml ), optionally at least 2.5 mg/ml (or about 2.5 mg/ml), optionally at least 3 mg/ml (or about 3 mg/ml), optionally at least 3.5 mg/ml (or about 3.5 mg/ml), optionally at least 4 mg/ml (or about 4 mg/ml), optionally at least 4.5 mg/ml (or about 4.5 mg/ml), optionally at least 5 mg/ml (or about 5 mg/ml), or at least 5.5 mg/ml (or about 5.5 mg/ml), optionally at least 6 mg/ml (or about 6 mg/ml), or optionally at least 6.5 mg/ml (or about 6.5 mg/ml), optionally at least 7 mg/ml (or about 7 mg/ml), optionally at least 7.5 mg/ml ( or about 7.5 mg/ml), optionally at least 8 mg/ml (or about 8 mg/ml), optionally at least 8.5 mg/ml (or about 8.5 mg/ml), optionally at least 9 mg/ml (or about 9 mg/ml), optionally at least 8.5 mg/ml (or about 8.5 mg/ml), optionally at least 9 mg/ml (or about 9 mg/ml) ml), optionally at least 9.5 mg/ml (or about 9.5 mg/ml), or optionally at least 10 mg/ml (or about 10 mg/ml) to 100 mg/ml (or about 100 mg/ml), optionally up to 95 mg/ml (or about 95 mg/ml), optionally up to 90 mg/ml (or about 90 mg/ml), optionally up to 85 mg/ml (or about 85 mg/ml), optional up to 80 mg/ml (or about 80 mg/ml), optional up to 75 mg/ml (or about 75 mg/ml), optionally up to 70 mg/ml (or about 70 mg/ml), optionally up to 65 mg/ml (or about 65 mg/ml), optionally up to 60 mg/ml (or about 60 mg/ml), optionally up to 55 mg/ml (or about 55 mg/ml), optionally up to 50 mg/ml (or about 50 mg/ml), optionally up to 45 mg/ml (or about 45 mg /ml), optionally up to 40 mg/ml (or about 40 mg/ml), optionally up to 35 mg/ml (or about 35 mg/ml), optionally up to 30 mg/ml (or about 30 mg/ml), optionally up to 25 mg/ml (or about 25 mg/ml), optionally up to 20 mg/ml (or about 20 mg/ml) or optionally up to 15 mg/ml (or about 15 mg/ml).

In certain embodiments, an extract, or source of extracts, of yeast of the genus Pichia is present in the compositions of the present invention at a concentration of from 0.01% (or about 0.01%), optionally from 0.05% (or about 0.05%), optionally from 0.1% (or about 0.1%), optionally from 0.5% (or about 0.5%), optionally from 1% (or about 1%), optionally from 1.5% (or about 1 .5%), optionally from 2% (or about 2%), optionally from 2.5% (or about 2.5%), optionally from 3% (or about 3%), optionally from 3.5%, ( or about 3.5%), optionally from 4% (or about 4%), optionally from 4.5% (or about 4.5%), optionally from 5% (or about 5%), optionally from 5.5 % (or about 5.5%), optionally from 6% (or about 6%), optionally from 6.5% (or about 6.5%), optionally from 7% (or about 7%), optionally from 7 .5% (or about 7.5%), optionally from 8% (or about 8%), optionally from 8.5% (or about 8.5%), optionally from 9% (or about 9%), optionally from 9.5% (or about 9.5%), optional preferably from 10% (or about 10%), optionally from 10.5% (or about 10.5%), optionally from 11% (or about 11%), optionally from 11.5% (or about 11.5% ), optionally from 12% (or about 12%), optionally from 12.5% (or about 12.5%), optionally from 13% (or about 13%), optionally from 13.5% (or about 13. 5%), optionally from 14% (or about 14%), optionally from 14.5% (or about 14.5%), optionally from 15% (or about 15%), optionally from 15.5% (or about 15.5%), optionally from 16% (or about 16%), optionally from 16.5% (or about 16.5%), optionally from 17% (or about 17%), optionally from 17.5% ( or about 17.5%), optionally from 18% (or about 18%), optionally from 18.5% (or about 18.5%), optionally from 19% (or about 19%), optionally from 19.5 % (or about 19.5%), optionally from 20% (or about 20%), optionally from 20.5% (or about 20.5%) to 30% (or about 30%), optionally up to 35% ( or about 35%), optionally up to 40% (or about 40%), optionally up to 45% (or about about 45%), optionally up to 50% (or about 50%), optionally up to 55% (or about 55%), optionally up to 60% (or about 60%), optionally up to 65% (or about 65%), optionally up to 70% (or about 70%), optionally up to 75% (or about 75%), optionally up to 80% (or about 80%), optionally up to 85% (or about 85%), optionally up to 90% (or about 90%), optionally up to 95% (or about 95%), or optionally up to 100% (or about 100%) by weight of the total weight of the composition.

Penetration enhancer

In certain embodiments, the compositions of the present invention optionally contain a penetration enhancer.

Suitable penetration enhancers include (selected from the group consisting of), alone or in combination, surfactants such as saponins, polyoxyethylene, polyoxyethylene fatty acid ethers such as polyoxyethylene 4-, 9-, 10 - and 23-lauryl ether, polyoxyethylene 10- and 20-cetyl ether, polyoxyethylene 10- and 20-stearyl ether, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene monolaurate, polyoxyethylene sorbitans such as polyoxyethylene sorbitan monolaurate, decamethonium, decamethonium bromide and dodecyltrimethylammonium bromide; chelators such as natural polyacids (eg citric acid), phosphate salts (eg disodium pyrophosphate), phosphonates, bisphosphonates (eg etidronic acid), aminocarboxylic acids (eg ethylenediaminetetraacetic acid (EDTA) and disodium EDTA) and ethylenediamine-N , N'-succinic acid (EDDS)); bile acids and salts such as cholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium cholate, sodium glycocholate, glycocholate, sodium deoxycholate, sodium taurocholate, sodium glycodeoxycholate, sodium taurodeoxycholate, chenodeoxycholic acid, and ursodeoxycholic acid ; fusidic acid derivatives, glycyrrhizic acid and ammonium glycyrrhizinate with saponin-EDTA, fusidic acid, polyoxyethylene 9-lauryl ether, polyoxyethylene 20-stearyl ether, glycocholate, or mixtures of any of the above.

The concentration of penetration enhancer administered should be the minimum amount necessary to sufficiently increase absorption of the compound and/or extract through the mucosa or other protective membranes of the eye. As such, concentrations ranging from 0.01% (or about 0.01%), optionally from 0.05% (or about 0.05%), optionally from 0.1% (or about 0 .1%), optionally from 0.15% (or about 0.15%), optionally from 0.2% (or about 0.2%), optionally from 0.25% (or about 0.25%) to 2% (or about 2%), optionally up to 2.5% (or about 2.5%), optionally up to 3% (or about 3%), optionally up to 3.5%, (or about 3.5%) , optionally up to 4% (or about 4%), optionally up to 4.5% (or about 4.5%), optionally up to 5% (or about 5%), optionally up to 5.5% (or about 5.5 %), optionally up to 6% (or about 6%), optionally up to 6.5% (or about 6.5%), optionally up to 7% (or about 7%), optionally up to 7.5% (or about 7 .5%), optionally up to 8% (or about 8%), optionally up to 8.5% (or about 8.5%), optionally up to 9% (or about 9%), optionally up to 9.5% (or about 9.5%), optionally up to 10% (or about 10%), optionally up to 10.5% (or about 10.5%), optionally up to 11% (or about 11%), optionally up to 11.5% (or about 11.5%), optionally up to 12% (or about 12%), optionally up to 12.5% (or about 12.5% ), optionally up to 13% (or about 13%), optionally up to 13.5% (or about 13.5%), optionally up to 14% (or about 14%), optionally up to 14.5% (or about 14. 5%), optionally up to 15% (or about 15%), optionally up to 15.5% (or about 15.5%), optionally up to 16% (or about 16%), optionally up to 16.5% (or about 16.5%), optionally up to 17% (or about 17%), optionally up to 17.5% (or about 17.5%), optionally up to 18% (or about 18%), optionally up to 18.5% ( or about 18.5%), optionally up to 19% (or about 19%), optionally up to 19.5% (or about 19.5%), optionally up to 20% (or about 20%) of the total weight of the composition (wt. ./about.).

Ophthalmologically acceptable carrier

The compositions of the present invention also contain an aqueous carrier, an oil-in-water emulsion carrier or a water-in-oil emulsion carrier. The carrier is ophthalmologically acceptable. Useful oil-in-water and water-in-oil carriers can be found in US Patent Publication No. 20030165545A1 and US Pat.

The ophthalmologically acceptable carrier (or compositions of the present invention) may optionally contain one or more additional excipients and/or one or more additional active ingredients. Examples of such optional components are described below.

Excipients commonly used in ophthalmic compositions include, without limitation, emollients, soothing, tonic, preservative, chelating, buffering agents (other than and in addition to the organic acids of the present invention), and surfactants. Other excipients contain solubilizing, stabilizing, comforting agents, polymers, emollients, pH adjusters (other than and in addition to the organic acids of the present invention), and/or lubricants. Any of a variety of excipients may be used in the compositions of the present invention, including water, mixtures of water and water-miscible solvents such as vegetable oils or mineral oils containing 0.5% to 5% non-toxic water-soluble polymers, natural products such as agar and gum arabic, starch derivatives such as starch acetate and hydroxypropyl starch, as well as other synthetic products such as polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methyl ether, polyethylene oxide, and preferably cross-linked polyacrylic acid, and mixtures thereof.

Emollients or sedatives useful in embodiments of the present invention include, without limitation, cellulose derivatives (such as hydroxyethylcellulose, methylcellulose, hypromellose, or mixtures thereof), hyaluronic acid, tamarind seed extract, glycerin, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, propylene glycol and polyacrylic acid and mixtures thereof. In some embodiments, one or more of hyaluronic acid, propylene glycol, tamarind seed extract, glycerin, and/or polyethylene glycol 400 are emollients or sedatives. In some embodiments, the emollient and sedative is selected from hyaluronic acid, tamarind seed extract, or mixtures thereof.

Compositions of the present invention are ophthalmologically acceptable for instillation into the eye of a patient. The term "aqueous" typically means an aqueous formulation in which the excipient is water, by weight > about 50%, more preferably > about 75%, and especially > about 90%. In some embodiments, the implementation of the compositions of the present invention essentially do not contain compounds that irritate the eyes. In some embodiments, the implementation of the compositions of the present invention essentially do not contain free fatty acids and C ₁ -C ₄ alcohols. In some embodiments, the compositions of the present invention contain less than 40% (or about 40%), optionally less than 35% (or about 35%), optionally less than 30% (or about 30%), optionally less than 25% (or about 25%) ; optionally less than 20% (or about 20%), optionally less than 15% (or about 15%), optionally less than 10% (or about 10%), or optionally less than 5% (or about 5%) of non-alcoholic, organic excipient or solvent from the total weight of the composition. These drops may be dispensed from a single dose ampoule, which may preferably be sterile, in which case the bacteriostatic components in the formulation are redundant. Alternatively, drops may be dispensed from a multi-dose vial, which may preferably contain a device that removes any preservative from the composition as it is dispensed, and such devices are known in the art.

In certain embodiments, compositions of the present invention are of the isotonic or mildly hypotonic type to overcome any tear hypertonicity caused by dehydration and/or disease. This may require a tonicity regulator to bring the osmolality level of the formulation to or near 210-320 milliosmoles per kilogram (mOsm/kg). Compositions of the present invention essentially have an osmolality essentially in the range of 220-320 mOsm/kg, or optionally have an osmolality in the range of 235-300 mOsm/kg. The composition of the ophthalmic compositions will essentially correspond to the composition of sterile aqueous solutions.

The osmolality of the compositions of the present invention can be adjusted with tonicity regulators to a value compatible with the intended use of the compositions. For example, the osmolality of the composition can be adjusted to approximate the osmotic pressure of a normal tear fluid, which is equivalent to about 0.9% w/v sodium chloride in water. Examples of acceptable tonicity regulators include, without limitation, sodium, potassium, calcium and magnesium chloride; dextrose; glycerol; propylene glycol; mannitol; sorbitol, etc., as well as mixtures thereof. In one embodiment, a combination of sodium chloride and potassium chloride is used to control the tonicity of the composition.

The compositions of the present invention can also be used to apply pharmaceutically active compounds. Such compounds include, without limitation, glaucoma drugs, analgesics, anti-inflammatory and antiallergic drugs, and antimicrobials. More specific examples of pharmaceutically active compounds include betaxolol, timolol, pilocarpine, carbonic anhydrase inhibitors and prostaglandins; dopaminergic antagonists; postoperative antihypertensive agents such as para-aminoclonidine (apraclonidine); anti-infectives such as ciprofloxacin, moxifloxacin and tobramycin; non-steroidal and steroidal anti-inflammatory drugs such as naproxen, diclofenac, nepafenac, suprofen, ketorolac, tetrahydrocortisol and dexamethasone; dry eye medications such as PDE4 inhibitors; and antiallergic drugs such as H1/H4 inhibitors, H4 inhibitors, olopatadine, or mixtures thereof.

It is also expected that the concentrations of the ingredients included in the compositions of the present invention may vary. One of ordinary skill in the art will appreciate that concentrations may vary depending on the addition, substitution and/or withdrawal of ingredients in a given formulation.

In some embodiments, the compositions of the present invention may have a pH that is compatible with the intended use, and often in the range of 4 (or about 4) to 10 (or about 10), optionally from 6 (or about 6) to 8 (or about 8) , optionally from 6.5 (or about 6.5) to 7.5 (or about 7.5) or optionally from 6.8 (or about 6.8) to 7.2 (or about 7.2).

In some embodiments, various conventional buffer solutions can be used, such as phosphate, borate, citrate, acetate, histidine, tris, bis-tris, and the like, as well as mixtures thereof. Borate buffer solutions include boric acid and its salts such as sodium or potassium borate. Potassium tetraborate or potassium metaborate can also be used, which produce boric acid or a boric acid salt in solution. In addition, hydrated salts such as sodium borate decahydrate can be used. Phosphate buffers include phosphoric acid and its salts; for example, M ₂ HPO ₄ and MH ₂ PO4, where M is an alkali metal such as sodium and potassium. Salt hydrates may also be used. In one embodiment of the present invention, Na ₂ HPO ₄ ·7H ₂ O and NaH ₂ PO ₂ ·H ₂ O are used as buffer solutions. The term "phosphate" also includes compounds that form phosphoric acid or a salt of phosphoric acid in solution. In addition, organic counterions can also be used in the above buffer solutions. The buffer solution concentration essentially ranges from about 0.01 to 2.5% w/v, and more preferably ranges from about 0.05% to about 0.5% w/v.

In some embodiments, the viscosity of the compositions of the present invention ranges from about 1 to about 500 centipoise, optionally from about 10 to about 200 centipoise, or optionally from about 10 to about 100 centipoise, as measured by a TA Instruments AR 2000 rheometer. TA Instruments AR 2000 rheometer must be used with TA Rheological Advantage software AR2000 flow test method with 40 mm steel plate geometry; Viscosity ranges should be obtained by measuring steady state flow with a controlled shear rate from 0 s¯¹ to 200 s¯¹.

In some embodiments, the compositions of the present invention are used as and in the form of an eye drop solution, an eye wash solution, a solution for lubricating and/or moistening contact lenses, a solution for spraying, spraying, or any other method of administering the composition to the eye.

The compositions of the present invention may also be used as and in the form of contact lens packaging solutions. In some embodiments, as packaging solutions, the compositions of the present invention can be sealed in a blister pack and are also suitable for sterilization.

Examples of blister packs and sterilization methods are described in the following references, which are incorporated herein by reference in their entirety; U.S. Patent Nos. D435,966; 4,691,820; 5,467,868; 5,704,468; 5,823,327; 6,050,398, 5,696,686; 6,018,931; 5,577,367; and 5,488,815. This part of the manufacturing process is another way of treating ophthalmic products with an antiallergic agent, namely adding antiallergic agents to a solution before sealing the package and then sterilizing the package. This method is the preferred method for treating ophthalmic products with antiallergic agents.

Sterilization can be carried out at different temperatures and for different periods of time. Preferred sterilization conditions range from about 100 ºC for about 8 hours to about 150 ºC for about 0.5 minutes. Preferred sterilization conditions range from about 115°C for about 2.5 hours to about 130°C for about 5.0 minutes. Preferred sterilization conditions are around 124 ºC for about 18 minutes.

When used as packaging solutions, the compositions of the present invention may be aqueous. Typical packaging solutions include, but are not limited to, saline, other buffer solutions, and deionized water. In some embodiments, the packaging solution is an aqueous solution in deioinized water or saline containing salts, including but not limited to sodium chloride, sodium borate, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, or their respective potassium salts. These ingredients are typically combined to form buffer solutions comprising an acid and a conjugate base such that the addition of acids and bases results in only a relatively small change in pH. In some embodiments, the pH of the packaging solution is as described above. Buffer solutions may further include 2-(N-morpholino)ethanesulfonic acid (MES), sodium hydroxide, 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol, n-tris(hydroxymethyl)methyl- 2-aminoethanesulfonic acid, citric acid, sodium citrate, sodium carbonate, sodium bicarbonate, acetic acid, sodium acetate, ethylenediaminetetraacetic acid, etc., and combinations thereof Preferably, the solution is borate or phosphate buffered saline or deionized water A particularly preferred solution would contain about 500-18,500 ppm sodium borate, most preferably about 1000 ppm sodium borate.

If any of the ingredients in the packaging solutions are subjected to oxidative degradation, agents may be added to stabilize the packaging solutions containing such ingredients. Such "oxidation stabilizers" include, but are not limited to, chelating agents such as EDTA, Dequest, desferal, silica, chitin derivatives such as chitosan, cellulose and its derivatives, and N, N, N', N', N" , N"-hexa(2-pyridyl)-1,3,5-tris(aminomethyl)benzene, as well as some macrocyclic ligands such as crown ethers, ligand-containing nodes and catenanes. See David A. Leigh et al Angew. Chem Int. Ed., 2001, 40, no. 8, pgs. 1538-1542, as well as Jean-Claude Chambron et al. Pure & Appl. Chem., 1990, Vol. 62, no. 6, pgs. 1027-1034. Anti-oxidation stabilizers may include other substances which inhibit oxidation, such as substances from the group consisting of 2,2',2'',6,6',6''-hexa-(1,1-dimethyl) 4,4',4''-[(2,4,6-trimethyl-1,3,5-benztriyl)-trismethylene]-triphenol (Irganox 1330), 1,3,5tris[3,5-di(1 ,1-dimethylethyl)4-hydroxybenzyl]-1H,3H,5H-1,3,5-triazine-2,4,6-trione, pentaerythrityl tetrakis-[3-[3,5-di(1,1-dimethylethyl )-4-hydroxyphenyl]-propionate], octadecyl-3-[3,5-di(1,1-dimethylethyl)-4-hydroxyphenyl]-propionate, tris[2,4-di(1,1-dimethylethyl)- phenyl]-phosphite, 2,2'-di(octadecyclooxy)-5,5'-spirobi(1,3,2-dioxaphosphorine), dioctadecyl disulfide, didodecyl-3,3'-thiodipropionate, dioctadecyl-3,3'- thiodipropionate, butylhydroxytoluene, ethylene bis[3,3-di[3-(1,1-dimethylethyl)-4-hydroxyphenyl]butyrate] and mixtures of these substances. Preferred oxidation stabilizers are diethylenetriaminepentaacetic acid (DTPA) or DTPA salts such as CaNa3DTPA, ZnNa3DTPA and Ca2DTPA. See US Patent Application No. 60/783,557, filed March 17, 2006, entitled Methods for Stabilizing Oxidatively Unstable Pharmaceutical Compositions and its corresponding unqualified application, which are hereby incorporated by reference in their entirety. In some embodiments, the concentration of oxidation stabilizers in solution is from about 2.5 µmol/L to about 5000 µmol/L, optionally from about 20 µmol/L to about 1000 µmol/L, optionally from about 100 µmol/L to about 1000 µmol/L /l or optionally from about 100 µmol/l to about 500 µmol/l.

In specific embodiments, the compositions of the present invention are formulated for administration at any frequency of administration, including once a week, once every five days, once every three days, once every two days, twice a day, three times a day, four times a day, five times a day, six times a day, eight times a day, every hour or more. This dosing frequency is also maintained for a period of time of varying duration depending on the therapeutic needs of the patient. The duration of a particular therapeutic regimen may vary from a single dosing regimen to a regimen lasting several months or years. One of ordinary skill in the art will be familiar with the definition of a therapeutic regimen for specific indications.

The composition and products containing such compositions of the present invention can be prepared using techniques well known to those of ordinary skill in the art.

EXAMPLES

Any of the compositions of the present invention described in the examples below illustrate, but are not limited to, specific embodiments of the compositions of the present invention. Other modifications can be made by a person skilled in the art without departing from the essence and scope of this invention.

In the examples, the following test methods were used.

Example 1

Enzymatic extracts of Pichia anomala (hyalurodin) induced hyaluronic acid secretion in human epicorneal tissues with a three-dimensional structure when applied topically to corneal tissue cells. Without being limited by theory, it is believed that such topical application requires diffusion of enzymatic extracts of Pichia anomala through dense junction structures of human epicorneal tissue (i.e., structures [e.g., membranes or film barriers] that typically interfere with the penetration of chemicals into the tissue) to reach the inner surface of the epicorneal tissue.

Three-dimensional human epicorneal tissues were purchased from MatTek Company (Ashland, Massachusetts, USA). After obtaining human epicorneal tissues with a three-dimensional structure, they were incubated in the MatTek assay medium overnight according to the manufacturer's instructions. Human epicorneal tissues were divided into five experimental groups with three tissues in each group. Four concentrations of enzymatic extracts of Pichia anomala (hyalurodin), 1.95 mg/mL, 7.8 mg/mL, 19.5 mg/mL and 39 mg/mL aqueous phosphate buffer solution (PBS) were topically applied, respectively, to the surface of the corneal epithelium of four experimental groups (so that Pichia anomala enzyme extracts are delivered through the aforementioned tight junction structures). Epicorneal tissues in all five experimental groups were incubated for two days. The enzymatic extract of Pichia anomala supplied by Silab, Saint Viance, France was used. Two days later, the culture medium was harvested to measure hyaluronic acid (HA) secretion using an enzyme-linked immunosorbent assay (ELISA) kit (K-1200, Echelon, Salt Lake City, UT, USA) according to the manufacturer's protocol. . To assess activity, colorimetric change was measured using a microplate photometer (SpectraMax M2E, Molecular Devices, Sunnyvale, CA, USA). This assay uses a competitive enzyme-linked immunosorbent assay method, so there is an inverse correlation between the HA concentration in the sample and the colorimetric change. A standard curve was plotted with HA concentration on the x-axis and absorbance on the y-axis, indicating the corresponding HA concentration. The results are shown in FIG. 1.

Although the results indicate that topical application at the concentration studied is directed towards the induction of HA production in corneal cells, factors such as tight junction structures (or other surface or film layer barriers) may reduce the concentration of Pichia anomala reaching the inner corneal tissues. In such situations, embodiments of the present invention including penetration enhancers can be used.

Example 2

To reduce the obstacles potentially caused by the above tight junction structures (either membrane or film barriers), the induction of hyaluronic acid secretion in human epicorneal tissues with a three-dimensional structure was observed by bringing the corneal tissues into contact with a growth medium containing enzymatic extracts of Pichia anomala (hyalurodin ) (i.e. "treated" media) in such a way that the lower layer of corneal tissue cells (i.e. cell layers that do not have tight junctions that prevent the penetration of chemicals into the tissue) was immersed in a nutrient medium. By contacting the lower cell layer of corneal tissue with a growth medium containing enzymatic extracts of Pichia anomala , the extract can directly contact and move upward into the inner tissues of the cornea without having to first pass through any tight junction structures or other surface barrier, providing increased bioavailability of Pichia anomala for corneal tissue cells.

Three-dimensional human epicorneal tissues were purchased from MatTek Company (Ashland, Massachusetts, USA). After obtaining human epicorneal tissues, these tissues were incubated in MatTek assay medium overnight according to the manufacturer's instructions. Human epicorneal tissues were divided into five experimental groups with three tissues in each group. Enzymatic extracts of Pichia anomala (hyalurodin) were added to the culture medium of the four experimental groups to obtain media with concentrations of 0.39 mg/ml, 0.78 mg/ml, 1.56 mg/ml and 1.95 mg/ml, respectively, for contact with the lower cell layer of human epicorneal tissues. Epicorneal tissues from all five experimental groups were incubated for two days. The enzymatic extract of Pichia anomala supplied by Silab, Saint Viance, France was used. Two days later, the culture medium was harvested to measure hyaluronic acid (HA) secretion using an enzyme-linked immunosorbent assay (ELISA) kit (K-1200, Echelon, Salt Lake City, UT, USA) according to the manufacturer's protocol. . To assess activity, colorimetric change was measured using a microplate photometer (SpectraMax M2E, Molecular Devices, Sunnyvale, CA, USA). This assay uses a competitive enzyme-linked immunosorbent assay method, so there is an inverse correlation between the HA concentration in the sample and the colorimetric change. A standard curve was plotted with HA concentration on the x-axis and absorbance on the y-axis, indicating the corresponding HA concentration. The results are shown in FIG. 2.

The results show that a statistically significant increase in HA production in corneal tissue cells was observed at surrounding tissue concentrations of Pichia anomala of at least 0.3 mg/mL (i.e., at concentrations of Pichia anomala extract in contact with such cells of the inner corneal tissue [e.g. , corneal fluid concentration]).

Can be prepared solutions containing one or more compounds and/or extracts of the present invention, as shown in examples 3-4.

Example 3

Table 1 lists the components of such formulations (as shown in formulations 3A and 3B) that can be formulated as described below using conventional blending techniques.

Table 1

3A
Used to relieve irritation caused by dry eyes 3B
Used to relieve irritation caused by dry eyes (in the case of contact lenses) INGREDIENT wt %/
wt. quantity per batch (gram) wt %/
wt. quantity per batch (gram) sodium hyaluronate 0.20 2.0 0.15 1.5 Pichia anomala extract 2.0 20.0 5.0 50.0 Polysorbate 80 1.0 10.0 2.0 20.0 Polysorbate 20 5.0 50.0 10.0 100.0 Polyethylene glycol 400 0.25 2.5 0 0 Boric acid 0.60 6.0 0.60 6.0 sodium borate 0.05 0.50 0.05 0.50 Sodium chloride* potassium chloride 0.10 1.0 0.10 1.0 Calcium chloride dihydrate 0.006 0.06 0.006 0.06 magnesium chloride 0.006 0.06 0.006 0.06 Sodium chlorite dihydrate 0.014 0.14 0.014 0.14 Polyquaternium-42 (33% aqueous solution) 0.0015 0.015 0.0015 0.015 Sodium chlorite dihydrate 0.014 0.14 0.014 0.14 1N sodium hydroxide solution** 1N hydrochloric acid solution** Purified water*** total 100.00% 100.00%

* bringing tonicity 280-290 mOsm/kg

** adjustment to pH 7.2

*** in sufficient quantity up to 100% wt./mass.

For examples 3A-3B: sodium hyaluronate can be supplied by CONTIPRO A.S. (DOLNI DOBRUCH, CZECH REPUBLIC).

For examples 3A and 3B, the Pichia anomala extract can be supplied by SILAB (SAINT-VIANSE, FRANCE).

For examples 3A-3B: Polysorbate 20 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 3A-3B: Polysorbate 80 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 3A: polyethylene glycol 400 can be supplied by Clariant Produkte (G. BURGKIRCHEN, GERMANY).

For examples 3A-3B: boric acid can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 3A-3B: sodium borate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 3A-3B: sodium chloride can be supplied by Caldic (Düsseldorf, GERMANY).

For examples 3A-3B: potassium chloride can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 3A-3B: calcium chloride dihydrate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 3A-3B: magnesium chloride can be supplied by KGaA (G. DARMSTADT, GERMANY).

For examples 3A-3B: polyquaternium-42 (33% aqueous solution) can be supplied by DSM BIOMEDICAL (BERKELEY, CA, USA)

For examples 3A-3B: sodium chlorite dihydrate can be supplied by Oxychem (WICHITA, KS, USA)

For examples 3A-3B: 1N sodium hydroxide can be supplied by VWR (G. RADNER, PA, USA).

For examples 3A-3B: 1N hydrochloric acid can be supplied by VWR (G. RADNER, PA, USA).

Solution 3A can be prepared as follows .

1. Add 800 grams of USP purified water to a 1500 ml beaker.

2. To the above solution, add 10 g of polysorbate 80 and 50 g of polysorbate 20. The solution is stirred until complete mixing and dissolution of both ingredients.

3. To the above, add 20.0 g of Pichia anomala extract. The solution is stirred until the Pichia anomala extract dissolves.

4. The solution is filtered through a 0.45 micron filter and returned to a 1500 ml beaker.

5. To the solution from step 4 add 2.0 grams of sodium hyaluronate. The solution is stirred until the sodium hyaluronate is completely dissolved.

6. The following ingredients are then added in succession, allowing each to dissolve completely until the following is added: 2.5 grams of polyethylene glycol-400, 6.0 grams of boric acid, 0.05 grams of sodium borate, 1.0 grams of potassium chloride, 0.06 grams of dihydrate calcium chloride, 0.06 grams of magnesium chloride and 0.0015 grams of polyquaternium-42 (aqueous).

7. While stirring, add 0.14 grams of sodium chlorite dihydrate and stir until dissolved.

8. The tonicity of the composition is determined and adjusted to 280 mOsm/kg with sodium chloride.

9. The pH of the formulation is adjusted to 7.2 with 1N sodium hydroxide and/or 1N hydrochloric acid.

10. The solution is brought to a weight of 1000.0 grams by adding USP purified water and mixed for 10 minutes until completely homogeneous.

11. The solution is filtered through a 0.22 micron filter.

Solution 3B can be prepared as follows .

1. Add 800 grams of USP purified water to a 1500 ml beaker.

2. To the above solution, add 20 g of polysorbate 10 and 100 g of polysorbate 20. The solution is stirred until complete mixing and dissolution of both ingredients.

3. Add 50 g of Pichia anomala extract to the above. The solution is stirred until the Pichia anomala extract dissolves.

4. The solution is filtered through a 0.45 micron filter and returned to a 1500 ml beaker.

5. To the solution from step 4 add 1.5 grams of sodium hyaluronate. The solution is stirred until the sodium hyaluronate is completely dissolved.

6. The following ingredients are then added in succession, allowing each to dissolve completely until the following is added: 6.0 grams of boric acid, 0.05 grams of sodium borate, 1.0 grams of potassium chloride, 0.06 grams of calcium chloride dihydrate, 0.06 grams of chloride magnesium and 0.0015 grams of polyquaternium-42 (aqueous).

7. While stirring, add 0.14 grams of sodium chlorite dihydrate and stir until dissolved.

8. The tonicity of the composition is determined and adjusted to 280 mOsm/kg with sodium chloride.

9. The pH of the formulation is adjusted to 7.2 with 1N sodium hydroxide and/or 1N hydrochloric acid.

10. The solution is brought to a weight of 1000.0 grams by adding USP purified water and mixed for 10 minutes until completely homogeneous.

11. The solution is filtered through a 0.22 micron filter.

Example 4

Table 2 lists the components of the formulations of the present invention (as shown in formulations 4A and 4B) that can be formulated as described below using conventional mixing techniques.

table 2

4A
Used to relieve irritation caused by dry eyes 4B
Used to relieve irritation caused by dry eyes (in the case of contact lenses) INGREDIENT wt %/
wt. quantity per batch (gram) wt %/
wt. quantity per batch (gram) Pichia anomala extract 1.0 10.0 2.0 20.0 Polysorbate 80 1.0 10.0 1.0 10.0 Polysorbate 20 3.0 30.0 5.0 50.0 Glycerol 0.25 2.5 0.25 2.5 Hypromellose E3 2910 0.198 1.98 0.198 1.98 Boric acid 0.40 4.0 0.40 4.0 sodium borate 0.022 0.22 0.022 0.22 Sodium hydrogen phosphate 0.027 0.27 0.027 0.27 Sodium citrate dihydrate 0.40 4.0 0.40 4.0 Sodium chloride* potassium chloride 0.10 1.0 0.10 1.0 50% aqueous sodium lactate solution 0.057 0.57 0.057 0.57 magnesium chloride 0.013 0.13 0.013 0.13 Glucose 0.0036 0.036 0.0036 0.036 Glycine 0.00002 0.0002 0.00002 0.0002 Ascorbic acid 0.00001 0.0001 0.00001 0.0001 Edetate disodium 0.01 0.1 0.05 0.5 Polyquaternium-42 (33% aqueous solution) 0.0030 0.030 0.0015 0.015 Sodium chlorite dihydrate 0.014 0.14 0.014 0.14 1N sodium hydroxide solution** 1N hydrochloric acid solution** Purified water*** total 100.00% 1000.00
G 100.00%

* bringing tonicity 280-290 mOsm/kg

** adjustment to pH 7.2

*** in sufficient quantity up to a volume of 100.00%

For examples 4A and 4B: Pichia anomala extract can be supplied by SILAB (SAINT-VIANSE, FRANCE).

For examples 4A-4B: Polysorbate 20 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: Polysorbate 80 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: boric acid can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: sodium borate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: sodium chloride can be supplied by Caldic (Düsseldorf, GERMANY).

For examples 4A-4B: potassium chloride can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: hypromellose E3 2910 can be supplied by DOW CHEMICAL (G. PLAKEMIN, LA, USA).

For examples 4A-4B: glycerin can be supplied by Emery Oleochemicals GmbH (Düsseldorf, GERMANY).

For examples 4A-4B: sodium hydrogen phosphate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: sodium citrate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: sodium lactate can be supplied as sodium lactate (50% aqueous solution) from Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: glucose can be supplied by Roquette Freres (LASTREM, FRANCE).

For examples 4A-4B: glycine can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For examples 4A-4B: ascorbic acid can be supplied by DSM NUTRITIONAL Products (DRAKEMIRE, SCOTLAND, UK).

For Examples 4A-4B: Polyquaternium-42 can be supplied as Polyquaternium-42 (33% aqueous solution) from DSM BIOMEDICAL (BERKLEY, CA).

For examples 4A-4B: disodium edetate can be supplied by Merck NV/SA (OVEREISE, BELGIUM).

For examples 4A-4B: 1N sodium hydroxide can be supplied by VWR (G. RADNER, PA, USA).

For examples 4A-4B: 1N hydrochloric acid can be supplied by VWR (G. RADNER, PA, USA). For examples 4A-4B: sodium chlorite dihydrate can be supplied by Oxychem (WICHITA, KS, USA)

Solution 4A can be prepared as follows .

1. Add 800 grams of USP purified water to a 1500 ml beaker.

2. 10 g of polysorbate 80 and 30 g of polysorbate 20 are added to the above solution. The solution is stirred until complete mixing and dissolution of both ingredients.

3. To the above, add 10.0 g of Pichia anomala extract. The solution is stirred until the Pichia anomala extract dissolves.

4. The solution is filtered through a 0.45 micron filter and returned to a 1500 ml beaker.

5. Add 1.98 grams of Hypromellose E3 Premium to the above solution. The solution is stirred until hypromellose E3 Premium dissolves.

6. The following ingredients are then added in succession, allowing each to dissolve completely until the following is added: 2.50 grams of glycerol, 4.0 grams of boric acid, 0.22 grams of sodium borate, 0.27 grams of sodium hydrogen phosphate, 4.00 grams of sodium citrate dihydrate , 1 gram potassium chloride, 0.57 gram sodium lactate (50% aqueous solution), 0.13 gram magnesium chloride, 0.036 gram glucose, 0.0002 gram glycine, 0.0001 gram ascorbic acid, 0.10 gram edetate disodium, 0.030 grams of polyquaternium-42 (33% aqueous solution) and 0.14 grams of sodium chlorite.

7. The tonicity of the solution is determined and adjusted to 280 mOsm with sodium chloride.

8. Measure the pH of the solution and adjust it to 7.2 with 1N sodium hydroxide and/or 1N hydrochloric acid.

9. The solution is brought to a volume of 1000.00 grams with purified water and stirred for 10 minutes.

10. The solution is filtered through a 0.22 micron filter.

Solution 4B can be prepared as follows .

1. Add 800 grams of USP purified water to a 1500 ml beaker.

2. To the above solution, add 10 g of polysorbate 80 and 50 g of polysorbate 20. The solution is stirred until complete mixing and dissolution of both ingredients.

3. To the above, add 20.0 g of Pichia anomala extract. The solution is stirred until the Pichia anomala extract dissolves.

4. The solution is filtered through a 0.45 micron filter and returned to a 1500 ml beaker.

5. Add 1.98 grams of Hypromellose E3 Premium to the above solution. The solution is stirred until hypromellose E3 Premium dissolves.

6. The following ingredients are then added in succession, allowing each to dissolve completely until the following is added: 2.50 grams of glycerol, 4.0 grams of boric acid, 0.22 grams of sodium borate, 0.27 grams of sodium hydrogen phosphate, 4.00 grams of sodium citrate dihydrate , 1 gram potassium chloride, 0.57 gram sodium lactate (50% aqueous solution), 0.13 gram magnesium chloride, 0.036 gram glucose, 0.0002 gram glycine, 0.0001 gram ascorbic acid, 0.05 gram edetate disodium, 0.015 grams of polyquaternium-42 (33% aqueous solution) and 0.14 grams of sodium chlorite.

7. The tonicity of the solution is determined and adjusted to 280 mOsm with sodium chloride.

8. Measure the pH of the solution and adjust it to 7.2 with 1N sodium hydroxide and/or 1N hydrochloric acid.

9. The solution is brought to a volume of 1000.00 grams with purified water and stirred for 10 minutes.

10. The solution is filtered through a 0.22 micron filter.

Embodiments of the present invention

1. A method of treating a patient with a reduced or low level of production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea, comprising the step of locally administering to the patient's eye a composition containing:

i) a safe and effective amount of one or more extracts, or sources of extracts, of the yeast of the genus Pichia to achieve a concentration of the extract of the yeast of the genus Pichia in corneal fluid in corneal tissues of the eye of at least about 0.3 mg/ml;

ii) optionally a safe and effective amount of a penetration enhancer; And

iii) optionally an ophthalmologically acceptable carrier.

2. The method of Embodiment 1 (or any of the following embodiments), wherein the Pichia yeast extract in the composition contains oligosaccharides and polysaccharides with a weight average molecular weight of from about 180 to about 800,000 Da.

3. The method according to embodiment 1 and/or 2 (or any of the following embodiments), wherein the Pichia yeast extract in the composition contains oligosaccharides and polysaccharides with an average degree of polymerization from DP 1 to DP 4444.

4. The method according to any of embodiments 1-3 or combinations thereof (or any of the following embodiments), wherein the composition contains a penetration enhancer.

5. The method of any one of Embodiments 1-4, or combinations thereof (or any of the following embodiments), wherein the penetration enhancer is present at a concentration of from about 0.01% to about 20% (w/v) of total weight of the composition.

6. The method according to any of embodiments 1-5 or combinations thereof (or any of the following embodiments), wherein the penetration enhancer is present at a concentration of from about 0.1% to 10% (w/v) of the total weight compositions.

7. The method of any of Embodiments 1-6, or combinations thereof (or any of the following embodiments), wherein the penetration enhancer is present at a concentration of about 0.25% to 5% (w/v) of total weight compositions.

8. The method according to any one of Embodiments 1-7 or combinations thereof (or any of the following embodiments), wherein the penetration enhancer is selected from polyoxyethylene, polyoxyethylene fatty acid ethers, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene monolaurate, polyoxyethylene sorbitan monolaurate, fusidic acid and its derivatives, EDTA, disodium EDTA, cholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium cholate, sodium glycocholate, glycocholate, sodium deoxycholate, sodium taurocholate, sodium glycodeoxycholate, sodium taurodeoxycholate, chenodeoxycholic acid, ursodeoxycholic acid , saponins, glycyrrhizic acid, ammonium glycyrrhizinate, decamethonium, decamethonium bromide and dodecyltrimethylammonium bromide, or mixtures of any of the above.

9. The method of any one of Embodiments 1-8 or combinations thereof (or any of the following embodiments), wherein the composition is administered when the concentration of hyaluronic acid in the patient's lacrimal fluid is less than about 10 nanograms per milligram of proteins.

10. A method according to any one of Embodiments 1-9 or a combination thereof (or any of the following embodiments), wherein the composition is administered when the concentration of hyaluronic acid in the patient's lacrimal fluid is less than about 15 nanograms per milligram of proteins.

11. The method of any one of Embodiments 1-10 or a combination thereof (or any of the following embodiments), wherein the composition is administered when the concentration of hyaluronic acid in the patient's lacrimal fluid is less than about 20 nanograms per milligram of proteins.

12. The method of any or a combination of Embodiments 1-11 (or any of the following embodiments), wherein the composition is administered when the concentration of hyaluronic acid in the patient's tear fluid is less than about 25 nanograms per milligram of proteins.

13. A method according to any one of embodiments 1-12 or a combination thereof (or any of the following embodiments), wherein the composition is administered to the patient's eye to increase the concentration of hyaluronic acid in the patient's lacrimal fluid to a level equal to or greater than about 10 nanograms per milligram of proteins.

14. A method according to any one of embodiments 1-13 or a combination thereof (or any of the following embodiments), wherein the composition is administered to the eye of a patient to increase the concentration of hyaluronic acid in the patient's lacrimal fluid to a level equal to or greater than about 15 nanograms per milligram of proteins.

15. The method of any one of embodiments 1-14 or a combination thereof (or any of the following embodiments), wherein the composition is administered to the eye of a patient to increase the concentration of hyaluronic acid in the patient's lacrimal fluid to a level equal to or greater than about 20 nanograms per milligram of proteins.

16. The method of any one of embodiments 1-15 or a combination thereof (or any of the following embodiments), wherein the composition is administered to the eye of a patient to increase the concentration of hyaluronic acid in the patient's lacrimal fluid to a level equal to or greater than about 25 nanograms per milligram of proteins.

Claims (18)

1. A method of treating a patient with a reduced or low level of production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea, comprising the step of locally administering to the patient's eye a composition containing: i) a safe and effective amount of one or more extracts, or sources of extracts, of the yeast of the genus Pichia to achieve a concentration of the extract of the yeast of the genus Pichia in the corneal fluid in the tissues of the cornea of the eye of at least 0.3 mg/ml, in which the extract of the yeast of the genus Pichia is Pichia anomala ; ii) and an ophthalmologically acceptable carrier. 2. The method according to claim 1, wherein the Pichia yeast extract in the composition contains oligosaccharides and polysaccharides with a weight average molecular weight of 180 to 800,000 Da. 3. The method according to p. 1, in which the extract of yeast of the genus Pichia in the composition contains oligosaccharides and polysaccharides with an average degree of polymerization from DP 1 to DP 4444. 4. The method of claim 1 wherein the composition comprises a penetration enhancer. 5. The method according to claim 1 and/or 2, wherein the penetration enhancer is present at a concentration of from 0.01% to 20% (w/v) of the total weight of the composition. 6. The method of claim 3, wherein the penetration enhancer is present at a concentration of 0.1% to 10% (w/v) of the total weight of the composition. 7. The method of claim 4 wherein the penetration enhancer is present at a concentration of 0.25% to 5% (w/v) of the total weight of the composition. 8. The method of claim 4 wherein the penetration enhancer is selected from polyoxyethylene, polyoxyethylene fatty acid ethers, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene monolaurate, polyoxyethylene sorbitan monolaurate, fusidic acid and its derivatives, EDTA, disodium EDTA, cholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, taurodeoxycholic acid, sodium cholate, sodium glycocholate, glycocholate, sodium deoxycholate, sodium taurocholate, sodium glycodeoxycholate, sodium taurodeoxycholate, chenodeoxycholic acid, ursodeoxycholic acid, saponins, glycyrrhizic acid, ammonium glycyrrhizinate, decamethonium, decamethonium bromotrimidecyl dodecyl bromide and docamethonium bromide or mixtures of any of the above substances. 9. The method of claim 1, wherein the composition is administered when the concentration of hyaluronic acid in the patient's lacrimal fluid is less than 10 nanograms per milligram of proteins. 10. The method of claim 1 wherein the composition is administered when the concentration of hyaluronic acid in the patient's lacrimal fluid is less than 15 nanograms per milligram of proteins. 11. The method of claim 1 wherein the composition is administered when the concentration of hyaluronic acid in the patient's lacrimal fluid is less than 20 nanograms per milligram of proteins. 12. The method of claim 1, wherein the composition is administered when the concentration of hyaluronic acid in the patient's lacrimal fluid is less than 25 nanograms per milligram of proteins. 13. The method of claim 1, wherein the composition is administered to the patient's eye to increase the concentration of hyaluronic acid in the patient's lacrimal fluid to a level equal to or greater than 10 nanograms per milligram of proteins. 14. The method of claim 1, wherein the composition is administered to the patient's eye to increase the concentration of hyaluronic acid in the patient's lacrimal fluid to a level equal to or greater than 15 nanograms per milligram of proteins. 15. The method of claim 1, wherein the composition is administered to the patient's eye to increase the concentration of hyaluronic acid in the patient's lacrimal fluid to a level equal to or greater than 20 nanograms per milligram of proteins. 16. The method of claim 1, wherein the composition is administered to the patient's eye to increase the concentration of hyaluronic acid in the patient's lacrimal fluid to a level equal to or greater than 25 nanograms per milligram of proteins.

Images