RU2802625C2 - Compositions and methods of treating eye disorders - Google Patents

Abstract

FIELD: medicine, ophthalmology.

SUBSTANCE: invention is intended for the prevention or treatment of symptoms associated with dry eye. The microemulsion composition contains a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of eyes, selected from one or more plant extracts or sources of extracts from plants of the genus Acronychia acidula; source of extracts of bacteria of the genus Actinomyces; and a compound selected from (2E,4E,6E)-7-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)-3-methylocta-2,4,6-trienoic acid and 4-(1-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)vinyl)benzoic acid and mixtures thereof. The composition also contains an oily component selected from castor oil, corn oil, sunflower oil, mineral oil, silicone oil and mixtures thereof; an emollient or sedative, which is hyaluronic acid or its salt; and an ophthalmologically acceptable vehicle. Microemulsion drops or particles have a maximum size of less than .

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.

9 cl, 6 dwg, 5 tbl, 11 ex

Description

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of previously filed United States Provisional Patent Applications Nos. 62/937,450 and 62/937,467 dated November 19, 2019, which are incorporated herein by reference in their entirety.

FIELD OF APPLICATION OF THE INVENTION

The present invention relates to compositions containing one or more extracts and/or compounds having retinol-like activity and properties, and methods of using these compositions for the treatment of eyes.

PREREQUISITES FOR CREATION OF THE INVENTION

“Dry eye is a multifactorial ocular surface disease that is characterized by loss of tear film homeostasis and is accompanied by ocular symptoms in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, 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 may result from abnormal or insufficient tear production and deficiency of mucin secretion (ie, keratoconjunctivitis sicca). Symptoms of dry eye syndrome can result from a variety of 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 typically involves rapid breakdown of the preocular tear film, resulting in dehydration of the exposed outer surface. Normal tear production is necessary to maintain hydration of the cornea and conjunctiva, which in turn helps prevent ulceration and also maintain corneal clarity. In addition, tears facilitate the movement of the eyelid along the surface of the eye (for example, blinking) and the removal of foreign substances from the eye. Tear fluid also usually contains lysozyme, which helps prevent eye infections. Dry eye syndrome can cause mild discomfort to severe pain in the eye. When this happens for extended periods of time, blurred vision, graininess, and/or a burning and itching sensation may occur. If left untreated, the condition can also lead to corneal ulcers and/or scarring.

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

Eye drops are an effective way to treat dry eye syndrome. These eye drops usually contain active ingredients to treat dry eye syndrome—a common active ingredient in these 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 properties and high threading ability, and is used as a humectant in topical products for the treatment of various types of skin problems, etc. In the case of dry eye syndrome caused by Sjogren'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 effects of hyaluronic acid eye drops only last about 2 to 3 hours, which means the patient must use the drops more frequently (e.g., 5 to 10 times per day).

Hyaluronic acid (HA) is produced by the epithelial cells of the cornea of the eye. Notably, significantly higher concentrations of hyaluronic acid were found in the corneas of young adults. (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).

In addition, the tear film is the body's natural defense against dry eyes. The tear film contains ocular mucins and is essential for maintaining ocular moist surface homeostasis. Mucins are produced, among other things, by the epithelial cells of the cornea. Mucins are glycoproteins expressed by mucosal epithelial tissues. They protect tissue by functioning as antioxidants and providing lubrication. Mucin genes associated with the tear film include MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16.

Mucin is also used as an antimicrobial agent, in addition, mucin, like hyaluronic acid, promotes the healing of any wounds and is necessary 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 and/or mucin from and/or into the cornea.

The present inventors have discovered compounds and/or extracts having retinol-like properties and/or beneficial effects that can induce, stimulate and/or improve the production/release/delivery/release of hyaluronic acid and/or mucin from and/or into the cornea.

Accordingly, one aspect of the present invention relates to compositions containing a safe and effective amount of one or more compounds and/or extracts having retinol-like properties and/or beneficial effects for the treatment of dry eye syndrome.

Another aspect of the present invention relates to microemulsion compositions containing a safe and effective amount of one or more compounds and/or extracts having retinol-like properties and/or beneficial effects for inducing, stimulating and/or improving the production/release/delivery/release of hyaluronic acid and/ or mucin from and/or into the cornea.

Another aspect of the present invention relates to methods of preventing and/or treating (for example, reducing) symptoms of ocular disorders associated with dry eye syndrome and/or resulting from decreased or low levels of production/release/delivery/release of hyaluronic acid and/or mucin from cornea and/or into the cornea, by administering compositions containing a safe and effective amount of one or more compounds and/or extracts having retinol-like properties and/or beneficial effects.

In particular, one aspect of the present invention relates to compositions containing a safe and effective amount of one or more compounds and/or extracts having retinol-like properties and/or beneficial effects for inducing, stimulating and/or improving the production/release/delivery/release of hyaluronic acid and/or mucin from and/or into the cornea, wherein the compositions can be administered to patients with a concentration of hyaluronic acid in the tear fluid of less than 10 (or about 10) nanograms, in some cases less than 15 (or about 15) nanograms, in some cases less than 20 (or about 20) nanograms, or in some cases less than 25 (or about 25) nanograms, per milligram of proteins such that the concentration of hyaluronic acid in their tear fluid increases to (or becomes equal to) 10 (or about 10) or more, in in some cases 15 (or about 15), in some cases 20 (or about 20), in some cases 25 (or about 25) nanograms, in some cases 30 (or about 30), in some cases 35 (or about 35) or more, in some cases 40 (or about 40) or more, or in some cases 45 (or about 45) or more nanograms per milligram of proteins up to 100 (or about 100), in some cases 90 (or about 90), in some in some cases 80 (or about 80), in some cases 70 (or about 70), or in some cases 60 (or about 60) nanograms per milligram of proteins.

In some embodiments, the above-described concentration of hyaluronic acid in the patient's tear fluid, due to compounds and/or extracts having retinol-like properties and/or beneficial effects, is maintained for a period of up to at least about 2 hours, in some cases about 4 hours, in some cases about 6 hours, in some cases about 8 hours, in some cases about 10 hours, in some cases about 12 hours, or in some cases from about 12 to about 24 hours.

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

A similar aspect of the present invention relates to compositions containing a safe and effective amount of one or more compounds and/or extracts having retinol-like properties and/or beneficial effects for inducing, stimulating and/or improving the production/release/delivery/release of hyaluronic acid and/or mucin from and/or into the cornea, wherein the compositions can be administered to patients having a tear MUC5AC concentration of less than 6 (or about 6), in some cases 8 (or about 8) nanograms per milligram of proteins such that the tear MUC5AC concentration is increased to (or becoming equal to) 8 (or about 8) or more nanograms to 15 (or about 15) nanograms, in some cases from 9 (or about 9) nanograms to 12 (or about 12) nanograms per milligram of proteins.

In certain embodiments, the above-described concentration of MUC5AC in tear fluid (i.e., equal to or greater than 8 (or about 8) nanograms to 15 (or about 15) nanograms, in some cases from 9 (or about 9) nanograms to 12 (or about 12) nanograms per milligram of proteins), resulting from the effects of compounds and/or extracts having retinol-like properties and/or beneficial effects for inducing, stimulating and/or improving the production/release/delivery/release of hyaluronic acid and/or mucin from the cornea and into the cornea, maintained for a period of up to at least about 2 hours, in some cases about 4 hours, in some cases about 6 hours, in some cases about 8 hours, in some cases about 10 hours, in some cases about 12 hours or in some cases from about 12 to about 24 hours.

MUC5AC concentrations in tear fluid, detailed above, were determined by the Uchino method (described below in definitions).

Another aspect of the present invention relates to methods of promoting healing or increasing the rate of healing of wounds in and/or on the surface of the eye (for example, non-dry eye syndrome, ocular trauma, post-operative surgical or non-specific wounds) of a patient by administering compositions containing a safe and effective an amount of one or more compounds and/or extracts, or sources of extracts, having retinol-like properties and/or beneficial effects for inducing, stimulating and/or improving the production/release/delivery/release of hyaluronic acid and/or mucin from and into the cornea (and namely, to enhance the production/release/delivery/release of hyaluronic acid or mucin from and/or into the cornea) in certain embodiments, in addition to the corresponding concentrations of hyaluronic acid or mucin that would normally be produced in such patient without (or in the absence of) administration of the compositions containing a safe and effective amount of one or more compounds and/or extracts having retinol-like properties and/or beneficial effects).

Another further aspect of the present invention relates to methods of improving the antimicrobial properties of the tear fluid (or tear film of the eye) of a patient by administering compositions containing a safe and effective amount of one or more compounds and/or extracts having retinol-like properties and/or beneficial effects for inducing, stimulating and/or enhancing the production/release/delivery/release of hyaluronic acid and/or mucin from and into the cornea (namely, to enhance the production/release/delivery/release of mucin from and/or into the cornea in certain embodiments beyond the level concentration of mucin normally produced by such patient, without (or in the absence of) administration of compositions containing a safe and effective amount of such one or more compounds and/or extracts having retinol-like properties and/or beneficial effects).

SUMMARY OF THE INVENTION

The present invention relates to microemulsion compositions for eye treatment containing:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of eyes, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; optionally one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally a methyl (-CH3) or methylene (=CH2) functional group; a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally from 1 to 10 carbon atoms; optionally 6 carbon atoms; optionally an aromatic functional group, optionally a phenyl functional group; optionally 2-methyl-prop-1,3-diene; And

ii) an ophthalmologically acceptable carrier,

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

The present invention provides methods for preventing or treating symptoms associated with dry eye (optionally in a patient in need of such prevention and treatment), comprising the step of administering a microemulsion composition comprising:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella ; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; optionally one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally a methyl (-CH3) or methylene (=CH2) functional group; a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally from 1 to 10 carbon atoms; optionally 6 carbon atoms; optionally an aromatic functional group, optionally a phenyl functional group; optionally 2-methyl-prop-1,3-diene; And

ii) an ophthalmologically acceptable carrier,

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

The present invention relates to methods of treating patients with reduced or low production/release/delivery/release of hyaluronic acid from the cornea and/or into the cornea, comprising the step of locally administering to the patient a microemulsion composition containing:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella ; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; optionally one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally a methyl (-CH3) or methylene (=CH2) functional group; a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally from 1 to 10 carbon atoms; optionally 6 carbon atoms; optionally an aromatic functional group, optionally a phenyl functional group; optionally 2-methyl-prop-1,3-diene; And

ii) in some cases an ophthalmologically acceptable carrier,

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

The present invention provides methods for preventing or treating symptoms of dry eye syndrome, comprising the step of locally administering to a patient (in some cases, a patient in need of such prevention or reduction of dry eye symptoms) a microemulsion composition comprising:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella ; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; optionally one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally a methyl (-CH3) or methylene (=CH2) functional group; a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally from 1 to 10 carbon atoms; optionally 6 carbon atoms; optionally an aromatic functional group, optionally a phenyl functional group; optionally 2-methyl-prop-1,3-diene; And

ii) in some cases an ophthalmologically acceptable carrier,

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

The present invention relates to methods of promoting healing or increasing the rate of healing of wounds in and/or on the surface of a patient's eye (in some cases when the patient requires such treatment of ocular wounds) by administering compositions (i.e., that increase the production/release/delivery of release of hyaluronic acid from and/or within the cornea, in some embodiments, in addition to the concentration of hyaluronic acid produced by such patient without (or in the absence of) administration of emulsion compositions containing a safe and effective amount of one or more compounds and/or extracts having retinol-like properties and/or beneficial effects) containing:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella ; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; preferably one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably methyl

(-CH3) or methylene (=CH2) functional group;

A represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably from 1 to 10 carbon atoms; more preferably 6 carbon atoms; preferably an aromatic functional group, preferably a phenyl functional group; preferably 2-methylprop-1,3-diene.

ii) in some cases an ophthalmologically acceptable carrier,

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

BRIEF DESCRIPTION OF THE FIGURES

In fig. Figure 1 shows bar graphs showing the expression of the MUC1, MUC4 and MUC16 genes induced by lemon aspen extract in corneal epithelial cells.

In fig. Figure 2 shows bar graphs showing mucin-1 secretion induced by lemon aspen extract in corneal epithelial cells.

In fig. Figure 3 shows bar graphs showing HAS3 gene expression induced by lemon aspen extract in corneal epithelial cells.

In fig. Figure 4 shows bar graphs showing HA secretion induced by lemon aspen extract in corneal epithelial cells.

In fig. Figure 5 shows bar graphs showing HAS3 gene expression induced by lemon aspen extract in corneal epithelial cells.

In fig. Figure 6 shows bar graphs showing HA secretion induced by lemon aspen extract in corneal epithelial cells.

DETAILED DESCRIPTION OF THE INVENTION

It is believed that, based on the description provided herein, one skilled in the art will be able to use this invention to its fullest extent. The following specific embodiments are to be considered as illustrative examples only and are not intended to limit the remainder of the present disclosure in any way.

The 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.

As used herein, the term “comprising” (and its grammatical variations) is used broadly to mean “having” or “including”, rather than narrowly meaning “consisting only of”. As used herein, the singular forms are considered to include both the plural and the singular.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention relates. In addition, all publications, patent applications, patents and other references mentioned herein are incorporated herein in their entirety by reference to the extent consistent with the disclosure herein. All percentages herein are by weight of the total composition unless otherwise noted.

As used herein, the term “cornea” or “corneal” includes and/or refers to the clear anterior portion of the eye that covers the iris, pupil, and anterior chamber, the layers of this clear anterior portion including a layer of corneal epithelium (containing corneal epithelial cells), Bowman's membrane (also known as the anterior limiting membrane), corneal stroma (also substantia propria), Descemet's membrane (also posterior limiting membrane), and corneal endothelium (a simple squamous or low cubic monolayer of mitochondria-rich cells whose thickness is approximately 5 µm).

As used herein, the phrase “reduced or low production/release/delivery/release of hyaluronic acid from and/or into 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 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.

Preparing a tear sample

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

Hyaluronic acid measurement

The content of hyaluronic acid in tear fluid was determined using non-isotopic fluorescence analysis (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 (Practical determination of hyaluronan using a new non-competitive fluorescent assay in the blood serum of healthy people and patients with cirrhosis). Anal Biochem 319: 65-72.) Eluted tear fluids 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 with protein. The plates were then sequentially incubated with biotinylated hyaluronic acid binding protein and europium-labeled streptavidin (Amershan, Piscataway, NJ). The europium remaining in the solid phase was then released with an intensifying solution and fluorescence was measured using a time-resolved fluorimeter (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 as ng/mg protein.

Protein Analysis

Total tear protein concentration was determined using a colorimetric assay kit according to the manufacturer's instructions (Bio-Rad Protein Assay Kit, Hercules, CA, USA). The protein profile was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (see Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 bacteriophage T4 heads. Nature 227: 680-685.). Briefly, 10 μg of protein from tear fluid samples was loaded onto a 3–20% linear gradient polyacrylamide gel under reducing conditions. After electrophoresis, the gels were stained with Coomassie blue (Bio-Rad, Hercules, CA, USA). 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, the phrase “reduced or low production/release/delivery/release of mucin from and/or into the cornea” means a concentration of MUC5AC that is less than the concentration of MUC5AC in the tear fluid of a normal (i.e., healthy) person, or, in some embodiments implementation, less than 6, in some cases less than 8 nanograms per milligram of proteins, as determined by the method described in Uchino Y, Uchino M, Yokoi N, et al. Alteration of Tear Mucin 5AC in Office Workers Using Visual Display Terminals: The Osaka Study. JAMA Ophthalmol. n2014;132(8):985-992. This method (Uchino method) is reproduced below:

Concentration of MUC5AC in tear fluid

The concentration of secreted mucin MUC5AC in tear fluid samples was quantified using an enzyme-linked immunosorbent assay (E90756Hu; USCN Life Science). (See Maker AV, Katabi N, Gonen M, et al. Pancreatic cyst fluid and serum mucin levels predict dysplasia in intraductal papillary mucinous neoplasms of the pancreas glands. Ann Surg Oncol. 2011;18(1):199-206.) All samples were analyzed according to the manufacturer's instructions. Absorbance was measured at 450 nm, and the standard solutions in the kit were recombinant human MUC5AC. A protein assay reagent kit (BCA protein assay kit; Pierce) was used to determine protein concentrations in tear fluid samples. MUC5AC concentration was normalized to tear protein content and expressed as MUC5AC protein (nanograms) per total tear protein (milligrams).

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 the ingredient by weight, based on the total weight of the composition. In certain more preferred embodiments, the substantially ingredient-free composition is ingredient-free, i.e., the ingredient is not present in the composition.

As used herein, the term “microemulsion” means emulsions having one or more of the following characteristics: i) they form spontaneously or substantially spontaneously when their components are brought into contact, that is, without a significant source of energy, for example, without heat or the use of high-energy equipment. shear force or other form of significant mixing; ii) they demonstrate thermodynamic stability; iii) they are single-phase; iv) they practically transmit light, i.e. transparent or opalescent when observed with optical microscopic means; and/or v) in the undisturbed state they are optically isotropic, although an anisotropic structure can be observed, for example, using x-ray techniques. Microemulsion particles can be spherical, although other structures are possible, such as liquid crystals with lamellar, hexagonal or isotropic symmetry. Typically, microemulsions contain droplets or particles with a maximum size (eg diameter) of less than 1500 Å, preferably less than 1000 Å, preferably less than 500 Å but greater than 100 Å. Self-emulsifying drug delivery systems (SEDDS) fall under this definition of microemulsion. SEDDS are isotropic mixtures of oil, surfactant (with or without co-surfactant) and co-solvent that spontaneously emulsify when exposed to an aqueous environment with gentle stirring. SEDDS can be used to improve the oral bioavailability of poorly water-soluble drugs. The addition of an auxiliary solvent promotes the formation of a self-emulsifying system, since it significantly reduces the interfacial tension. Thus, it creates an interfacial fluid that is flexible enough to accept the varying curvatures required to form a microemulsion in a wide range of compositions. Additional information regarding SEDDS can be found in US Patent Publication No. US2018/0036233A1, Shabaik et al., which is incorporated herein by reference in its entirety.

As used herein, the term "ophthalmically acceptable" means that the ingredients to which the term refers are suitable for use in contact with tissues (e.g., the soft tissues of the eye or periorbital skin tissues) without adverse effects of nonspecific toxicity, incompatibility, instability, irritation, allergic reaction, etc. As will be appreciated by one skilled in the art, ophthalmologically acceptable salts are acidic/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 and/or mucin from one or more layers of the cornea and/or or into one or more layers 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 pharmaceutically acceptable carrier used and similar factors.

The term “retinol-like properties and/or beneficial effects” means the properties and/or beneficial effects caused by retinol.

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 nomenclature of the International Union of Pure and Applied Chemistry (IUPAC) in accordance with the following definitions of terms.

The term "C1-8 alkyl", 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 the radicals methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, tert-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. The term may also refer to the corresponding alkyldiyl radical. Alkyl and alkyldiyl radicals can attach to the main molecule through a terminal carbon atom or through an intrachain carbon atom. Similarly, any number of variable substituents allowed by the available valences can be attached to an alkyl or alkyldiyl radical.

The term "C1-4 alkyl", 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 being formed by the removal of one hydrogen atom from a carbon atom, and the alkyldiyl linking group is made by removing one hydrogen atom from each of the two carbon atoms in the chain. The term "C1-4 alkyl" refers to a radical having 1-4 carbon atoms in a linear or branched configuration. For example, "C1-4 alkyl" specifically includes the radicals methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, tert-butyl, 1-butyl, etc. Alkyl and alkyldiyl radicals can attach to the main molecule via a terminal carbon atom or via a carbon atom within the chain. Similarly, any number of variable substituents allowed by the available valences can be attached to an alkyl or alkyldiyl radical.

The term "C2-4 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 may 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 parent 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 main molecule, but can also occur in the substituting radical, with the radical being converted into a linking group.

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

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

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

Compounds/extracts with retinol-like properties and/or beneficial effects

The present invention includes compounds and/or extracts having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, including those selected from or selected from the group consisting of one or more plant extracts or sources of plant extracts of the genus Acronychia, Licaria , Calendula and/or Trigonella; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; optionally one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally a methyl (-CH3) or methylene (=CH2) functional group; a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally from 1 to 10 carbon atoms; optionally 6 carbon atoms; optionally an aromatic functional group, optionally a phenyl functional group; optionally 2-methyl-prop-1,3-diene.

Acronychia, Licaria, Calendula and/or Trigonella plant extract(s)

In certain embodiments, the compounds/extracts having retinol-like properties and/or beneficial effects are or contain extracts or sources of extracts from Acronychia, Licaria, Calendula and/or Trigonella plants. Acronychia, Licaria, Calendula or Trigonella extracts, or sources of such extracts, are obtained from plants of the genus Acronychia, Licaria, Calendula or Trigonella.

Plants of the genus Acronychia from which extracts used in the present invention are obtained include, for example, Acronychia aberrans, Acronychia acidula (also referred to herein as lemon aspen), Acronychia acronychioides, Acronychia acuminate, Acronychia baeuerlenii, Acronychia chooreechillum, Acronychia crassipetala, Acronychia eungellensis, Acronychia imperforate, Acronychia laevis, Acronychia laurifolia, Acronychia littoralis, Acronychia oblongifolia, Acronychia octandra, Acronychia parviflora, Acronychia pauciflora, Acronychia pedunculata, Acronychia pubescens, Acronychia species (Batavia Downs), Acronychia suberosa, Acronychia vestita, Acronychia wilcoxiana and combinations two or more of them. In one embodiment, the extract used in the present invention was obtained from Acronychia acidula.

Plants of the genus Licaria from which the extracts used in the present invention are obtained include, for example, Licaria vernicosa, Licaria brittoniana, Licaria canella, Licaria cubensis, Licaria velutina and Licaria triandra, as well as combinations of two or more of them. There are about 40 species of the genus Licaria that are endemic to Central and South America. In one embodiment, the extract used in the present invention is obtained from Licaria vernicosa.

There are about 15-20 species of the genus Calendula, which have been found in Southwest Asia, Western Europe, Macaronesia and the Mediterranean. Plants of the genus Calendula from which extracts useful in the present invention are obtained include, for example, Calendula arvensis (field marigold); Calendula maderensis (Madeira marigold); and Calendula officinalis (medicinal marigold) and combinations of two or more of them. In one embodiment, the extract used in the present invention is obtained from Calendula officinalis.

Plants of the genus Trigonella include 36 known species of the genus Trigonella, from which extracts useful in the present invention are obtained, include, for example, Trigonella foenum-graecum, Trigonella balansae, Trigonella corniculata, Trigonella maritima, Trigonella spicata, Trigonella caerulea, Trigonella occulta, Trigonella polycerata, Trigonella Callices, Trigonella Cretica and combinations of two or more of them. Trigonella foenum-graecum, or fenugreek, is the best known member of the Trigonella genus. In one embodiment, the extract used in the present invention is obtained from Trigonella foenum-graecum.

In some embodiments, the extract used in the present invention is a mixture of extracts obtained from plants of the genus Acronychia, Licaria, Calendula and/or Trigonella.

Licaria vernicosa extracts used in the present invention are available from the Baruch S. Blumerg Institute, Doylestown, Pennsylvania (formerly known as IVHR). In some embodiments, one extract is obtained from the woody part of the plant (E2) and the second extract is obtained from the roots of the plant (E3). In the IHVR collection nomenclature, the two extracts are labeled IHVR_40256_G10=X-005348-001E002 and IHVR_40256_E10=X-005346-001M002, respectively. Woody parts and roots of Licaria vernicosa (Mez) Kosterm. can be purchased in Guyana. 504.3 g of dried ground woody plant material can be extracted with a sufficient amount of methanol, which can be dried in a vacuum to obtain 10.54 g of raw methanol extract (E2) for X-005348-001E002. 403.8 g of dried, ground root material can be extracted with sufficient methanol, which can be vacuum dried to produce 18.11 g of raw methanol (E3) for X-005346-001M002.

In some embodiments, the Acronychia and/or Licaria extracts used in the present invention contain compounds having Formula II:

Where:

R1 is selected from the group consisting of C1 - C20 alkyl, C2 - C20 alkenyl, C2 - C20 alkynyl and C3 - C8 cycloalkyl or aryl;

R2 is selected from the group consisting of hydrogen, hydroxyl, C1 - C6 alkyl, C2 - C6 alkenyl, C2 - C6 alkynyl, C3 - C8 cycloalkyl or aryl, - OC1 - C6 alkyl, - OC2 - C6 alkenyl, - OC2 - C6 alkynyl , - OC3 - C8 cycloalkyl or aryl, thiol, - SC1 - C6 alkyl, - SC2 - C6 alkenyl, - SC2 - C6 alkynyl, - SC3 - C8 cycloalkyl or aryl, - NR4C1 - C6 alkyl, - NR4C2 - C6 alkenyl, - NR4C2 - C6 alkynyl and - NR4C3 - C8 cycloalkyl or aryl;

R3 is selected from -CO2H, -CO2R4 or the isosteric equivalent of a carboxyl group, where R4 is C1-C6 alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl or aryl; And

Y represents -(CH2 - CH2)-, -(CH = H - or -(C ≡ C)-;

or an ophthalmologically acceptable salt thereof.

In some embodiments, the Acronychia and/or Licaria extracts used in the present invention contain compounds having Formula II:

Where:

R1 is selected from the group consisting of C5 - C16 alkyl, C5 - C16 alkenyl and C5 - C16 alkynyl, more preferably C5 - C16 alkenyl, including, for example, farnesyl;

R2 is selected from the group consisting of hydrogen, hydroxyl, - OC1 - C6 alkyl, - OC2 - C6 alkenyl, - OC2 - C6 alkynyl, - OC3 - C8 cycloalkyl, more preferably hydrogen, hydroxyl, - OC1 - C6 alkyl, even more preferably - hydrogen or - OC1 - C3 alkyl;

R3 is selected from - CO2H, - CO2R4, where R4 is a C1 - C6 alkyl or the isosteric equivalent of a carboxyl group; And

Y represents -(CH2 - CH2)- or -(CH = CH)-;

or an ophthalmologically acceptable salt thereof.

In some embodiments, at least one of the compounds of Formula II is present in the Acronychia and/or Licaria extract at a concentration of from at least 1% (or about 1%) to about 20%, or in some cases from about 7% (or about 7% ) to about 10% (or about 10%) by weight of the Acronychia and/or Licaria extract.

In some embodiments, the compounds of formula II used in the present invention are in the form of an acid or alkyl ester selected from (or selected from the group consisting of) 3-(4-farnesyloxyphenyl)propionic acid, 3-(4-farnesyloxy-3 -hydroxyphenyl)-propionic acid, 3-(4-farnesyloxy-3-methoxyphenyl)-propionic acid, alkyl esters thereof, in particular ethyl esters thereof, and combinations of two or more of them.

In some embodiments, the compound of Formula II used in the present invention is 3-(4-farnesyloxyphenyl)propionic acid and/or its ethyl ester.

In some embodiments, the compound of Formula II used in the present invention is 3-(4-farnesyloxy-3-hydroxyphenyl)-propionic acid and/or an ethyl ester thereof.

In some embodiments, the compound of Formula II used in the present invention is 3-(4-farnesyloxy-3-methoxyphenyl)-propionic acid and/or its ethyl ester.

Compounds and extracts obtained from Acronychia species are described in US Patent No. 9,220,928, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, 3-(4-farnesyloxyphenyl)-propionic acid and/or its ethyl ester is present in the Acronychia and/or Licaria extract at a concentration of at least 1% (or about 1%) to about 20%, or in some cases from about 7% (or about 7%) to about 10% (or about 10%) wt. Acronychia and/or Licaria extract.

Any of a variety of Acronychia and/or Licaria extracts can be used for embodiments in which the method includes applying such an extract. The extract can be obtained from any part of the plant, such as, for example, fruits, seeds, bark, leaves, flowers, roots and wood.

In some embodiments, the extract is obtained from the fruit of the plant. Suitable extracts of the fruits, seeds, bark, leaves, flowers, roots and wood of Acronychia or Licaria can be obtained using standard methods, including, without limitation, direct extraction of material from biomass by grinding, maceration, pressing, squeezing, crushing, centrifugation and/or processes such as cold percolation, stirring/distillation, microwave extraction, supercritical/subcritical CO2 gas extraction with or without polar modifiers, pressurized solvent extraction, accelerated solvent extraction, hot water extraction at elevated or normal pressure, hot water extraction under elevated pressure in the presence of a surfactant, oil extraction, membrane extraction, Soxhlet extraction, distillation/golden finger extraction, and/or methods disclosed, for example, in US Patent Nos. 7442391, 7473435 and 7537791 issued to Integrated Botanical Technologies, LLC, incorporated herein by reference, etc. or using other methods such as solvent extraction and the like. In methods involving solvent extraction, any of a variety of solvents may be used, including polar solvents, non-polar solvents, or combinations of two or more of them.

Suitable polar solvents include polar inorganic solvents such as water and the like, polar organic solvents such as alcohols and corresponding organic acids, for example C1-C8 alcohols including methanol, ethanol, propanol, butanol and the like, as well as organic acids, including acetic acid, formic acid, propanoic acid, etc., polyols and glycols, including C1-C8 polyols/glycols, etc., and combinations of two or more of them. Suitable non-polar solvents include non-polar organic solvents such as alkanes, including C1-C8 alkanes, cycloalkanes, including C1-C8 cycloalkanes, alkyl ethers, including C1-C8 alkyl ethers, petroleum ethers, ketones, including C1-C8 ketones, methylene chloride, ethyl acetate, xylene , toluene, chloroform, vegetable oil, mineral oil, etc. In another embodiment, the extraction can be carried out using non-polar solvents described above, or by supercritical fluid extraction in the presence or absence of a polar modifier such as C1-C8 alcohols, water, C1-C8 polyols/glycols or C1-C8 organic acids.

In one embodiment, the extract contains an extract of Acronychia acidula. In another embodiment, the extract of the present invention contains a combination of polar and non-polar extracts from the fruit of Acronychia acidula. In another embodiment, the extract of the invention contains alcoholic or glycolic extracts of Acronychia acidula fruit.

In one embodiment, the extract comprises an extract of Licaria vernicosa. In another embodiment, the extract of the present invention contains a combination of polar and non-polar extracts from Licaria vernicosa wood or Licaria vernicosa root. In another embodiment, the extract of the present invention contains alcoholic extracts of Licaria vernicosa wood or Licaria vernicosa root.

In another embodiment, the extract of the present invention contains a polar extract obtained by extracting from the fruit of Acronychia acidula, wood of Licaria vernicosa or root of Licaria vernicosa using a polar solvent containing water, C1 - C8 alcohols, C1 - C8 polyols, C1 - C8 glycols and combinations of two or more of them. In some embodiments, the extract is extracted using one or more C1-C4 alcohols, C1-C4 polyols, and/or C1-C4 glycols. In some embodiments, the extract is prepared using a solvent containing methanol, ethanol, or a combination thereof, in the presence or absence of water. In another embodiment, the extract is a polar extract obtained from the fruit of Acronychia acidula using a combination of alcohol and water. In yet another embodiment, the extract is a polar extract obtained from ground Licaria vernicosa wood or ground Licaria vernicosa root using methanol.

In yet another embodiment, the extract comprises a non-polar extract obtained by extracting from the fruit of Acronychia acidula, the wood of Licaria vernicosa or the root of Licaria vernicosa using a non-polar solvent containing one or more C1 - C8 alkanes, C1 - C8 cycloalkanes, C1 - C8 alkyl esters, C1 - C8 ethers and/or chloroform, more preferably one or more C1 - C8 alkanes, C1 - C8 alkyl esters and/or chloroform. In yet another embodiment, a non-polar extract is prepared from the fruit of Acronychia acidula, wood of Licaria vernicosa, or root of Licaria vernicosa using hexanes, ethyl acetate, chloroform, or mixtures of two or more of these. In yet another embodiment, a non-polar extract is prepared from the fruit of Acronychia acidula using ethyl acetate.

In one embodiment, the extract comprises Calendula officinalis extract. In another embodiment, the extract of the present invention contains a combination of polar and non-polar extracts of Calendula officinalis petals. In another embodiment, the extract of the present invention comprises a non-polar extract of Calendula officinalis petals.

In one embodiment, the extract comprises Trigonella foenum-graecum extract. In another embodiment, the extract of the present invention contains a combination of polar and non-polar extracts of Trigonella folium leaves. In another embodiment, the extract of the invention contains a non-polar extract of Trigonella foenum-graecum leaves.

In yet another embodiment, the Calendula and/or Trigonella extract is a non-polar extract prepared using a non-polar solvent containing one or more C1 - C8 alkanes, C1 - C8 cycloalkanes, C1 - C8 alkyl ethers, C1 - C8 alkyl esters and /or chloroform, more preferably one or more C1 - C8 alkanes, C1 - C8 alkyl esters and/or chloroform.

In yet another embodiment, the Calendula and/or Trigonella extract is a non-polar extract prepared using hexanes, ethyl acetate, chloroform, or mixtures of two or more of them. In yet another embodiment, the extract is a non-polar extract prepared using ethyl acetate.

In one embodiment, plant extracts can be obtained by extraction of cell cultures of various plants, including cell cultures of the genera Acronychia, Licaria, Calendula or Trigonella. The cell cultures that are extracted to obtain plant extracts for use in the present invention may be in any form, including suspension cell cultures and the like.

Extracts of Calendula officinalis and Trigonella foenum-graecum can be purchased from Caithness Biotechnologies Ltd, UK (http://www.caithnessbiotechnologies.com/). These extracts are part of the Phytotitre Natural Product Library, which is publicly available for purchase. Alternatively, extracts can be prepared using a preparation method described by Caithness Biotechnologies Ltd. as non-polar and prepared from a mixture of methanol and methylene chloride. For a detailed description, please refer to http://caithnessbiotechnologies.com/contact.html. Typically, in extraction, pre-weighed dried powdered biomass is suspended and stirred in methanol/methylene chloride (1:1) overnight at ambient temperature. The suspension is then filtered and the filtrate is dried under reduced pressure to a solvent-free residue.

In some embodiments, the extract of Acronychia, Licaria, Calendula and/or Trigonella is present in the compositions of the present invention in an amount of from about 0.001% to about 10%, in some cases from about 0.001% to about 5%, or in some cases from about 0.01 % to about 1% wt. compositions.

Extract(s) of bacteria of the genus Actinomyces with retinol-like properties and/or beneficial effects

In some embodiments, the compounds/extracts demonstrating retinol-like properties and/or beneficial effects are or contain extract(s) of bacteria of the genus Actinomyces. Bacteria of the genus Actinomyces include many fully characterized species as well as poorly characterized ones, such as one species collected from the United States and labeled species A5640. A sample of these bacteria was collected, grown in culture and converted into an extract. The extract is part of a collection of natural products now operated by the Baruch S. Blumerg Institute, Doylestown, Pennsylvania, USA (formerly known as the Institute for Hepatitis Virus Research (IHVR)). In the nomenclature of the IHVR collection, the extract is labeled as IHVR_39565_F7.

In one embodiment, the extract used in the present invention is obtained from an Actinomyces species capable of producing a chemical composition similar to that obtained with the A5640 extract. In another embodiment, the bacteria are collected in the United States and the strain is identical to previously isolated species A5640.

In some embodiments, the Actinomyces extract is contained in the composition of the present invention in an amount of from about 0.001% to about 10%, optionally from about 0.001% to about 5%, or optionally from about 0.01% to about 1% by weight. compositions.

Compound(s) of formula (I) having retinol-like properties and/or beneficial effects.

In some embodiments, the compounds/extracts having retinol-like properties and/or beneficial effects are or contain compounds of formula (I).

(I),

Where -

dotted lines indicate a single or double bond; optionally one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally a methyl (-CH3) or methylene (=CH2) functional group; a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally from 1 to 10 carbon atoms; optionally 6 carbon atoms; optionally an aromatic functional group, optionally a phenyl functional group; preferably 2-methylprop-1,3-diene.

In some embodiments, compounds of formula (I) include corresponding salts of metal ions such as, but not limited to, Li+, Na+, K+, Ca2+, or Mg2+.

In some embodiments, the compounds of formula I are:

(1)

(2E,4E,6E)-7-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)-3-methylocta-2,4,6- trienoic acid

or

(2)

4-(1-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)vinyl)benzoic acid and, in each case, their derivatives exhibiting retinoid-like activity . These compounds are further referred to in Examples 8 and 9 as Compound 1 and Compound 2, respectively.

The present invention relates to compounds such as (2E,4E,6E)-7-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)-3- methylocta-2,4,6-trienoic acid and 4-(1-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)vinyl)benzoic acid and their derivatives, which demonstrate retinoid-like activity, and mixtures thereof.

Mixtures of any of the above compounds/extracts/extract sources having retinol-like properties and/or beneficial effects may also be used.

The compounds and extracts of Formula I are described in US Patent Publication 2019/0091122, which is incorporated herein by reference in its entirety.

In some embodiments, the compound(s) of Formula I is present in the composition of the present invention in an amount of from about 0.0001% to about 20%, in some cases from about 0.001% to about 10%, in some cases from about 0.01% to about 5%, or in some cases from about 0.2 to about 2% by weight. compositions. In some embodiments, the compound(s) of Formula I is contained in the composition of the present invention in an amount of from about 0.0001% to about 1%, in some cases from about 0.001% to about 1%, or in some cases from about 0.01% up to about 1% wt. compositions.

Compositions The present inventors have discovered that compounds and/or extracts having retinol-like properties and/or beneficial effects can stimulate and/or improve the delivery/release of hyaluronic acid from corneal epithelial cells.

Permeability enhancer

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

Suitable permeability enhancers include (selected from or 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, decame bromide tonium 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'-disuccinic 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 permeability enhancer administered should be the minimum amount necessary to sufficiently increase absorption of the compound and/or extract across the mucous membrane or other protective membranes of the eye. Typically, concentrations ranging from 0.01% (or about 0.01%), optionally from 0.05% (or about 0.05%), optionally from 0.1% (or about 0.05%), can be used in the compositions of the present invention. 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%), optional up to 16% (or about 16%), optional up to 16.5% (or about 16.5%), optional up to 17% (or about 17%), optional up to 17.5% (or about 17.5%), optional up to 18% (or about 18%), optional up to 18.5% (or about 18.5%), optional 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 amount of the composition (w/v).

Ophthalmologically acceptable carrier

The compositions of the present invention also contain an aqueous oil-in-water emulsion, a water-in-oil emulsion vehicle, an oil-in-water microemulsion, or a water-in-oil microemulsion vehicle. The carrier is ophthalmologically acceptable. Useful oil-in-water and water-in-oil media can be found in US Patent Publication No. 20030165545A1 and US Patent Nos. 9480645, 8828412 and 8496976, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the compositions of the present invention include emulsions, in some cases self-emulsifying emulsions, including an oily component, such as, for example, one or more oils, without limitation, mineral oil and/or one or more other conventional well known and/or commercially available oils suitable for use in the present invention; a surfactant component that includes three or more surfactants; and an aqueous component, which includes an aqueous phase. In addition, several additional components may be used in the present compositions. The compositions of the present invention are substantially non-toxic and/or non-irritating and/or non-damaging to the eye, and may provide a protective function for ocular cells and tissues.

One or more oils or oily substances are used to form the present compositions. Any suitable oil, oily substance, or combinations of oils or oily substances may be used, provided that such oils and/or oily substances are effective in the present compositions and do not cause significant or significant harmful effects on the person or animal to which the composition is administered, or on the contact being treated. lens or the wearer of the contact lens being treated or the wearer of the contact lens being treated. The oily component may, for example, without limitation, be a glyceride of a higher fatty acid, such as castor oil, corn oil, sunflower oil, etc., and mixtures thereof. The oily component may include one or more non-polar oils, such as mineral oil, silicone oil, etc., as well as mixtures thereof.

The compositions of the present invention may in some cases contain one or more additional excipients and/or one or more additional active ingredients. Examples of such optional components are described below.

Excipients that are commonly used in ophthalmic compositions include, but are not limited to, emollients, tonicity regulators, preservatives, chelating agents, buffering agents (other than and complementary to the organic acids of the present invention), and surfactants. Other excipients contain solubilizing, stabilizing, comfort enhancing agents, polymers, emollients, pH adjusters (different from and complementary to the organic acids of the present invention) and/or lubricants. The compositions of the present invention can use any of a variety of excipients, including water, mixtures of water and water-miscible solvents such as vegetable oils or mineral oils containing from 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, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, and preferably cross-linked polyacrylic acid, and mixtures thereof.

Emollients and soothing agents used in embodiments of the present invention include, but are not limited to, cellulose derivatives (such as hydroxyethylcellulose, methylcellulose, hypromellose, or mixtures thereof), hyaluronic acid or a salt thereof (for example, sodium hyaluronate), 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 and soothing agents. In some embodiments, the emollient and soothing agent is selected from hyaluronic acid, tamarind seed extract, or mixtures thereof.

The compositions of the present invention are ophthalmologically acceptable for instillation into the eyes of a patient. The term “aqueous” generally means an aqueous composition in which the excipient constitutes more than about 50%, preferably more than about 75%, or in particular more than about 90% by weight of water. In some embodiments, the compositions of the present invention are substantially free of eye irritant compounds. In some embodiments, the compositions of the present invention are substantially free of free fatty acids and C1-C4 alcohols. In some embodiments, the compositions of the present invention contain less than 40% (or about 40%), in some cases less than 35% (or about 35%), in some cases less than 30% (or about 30%), in some cases less than 25% (or about 25%); in some cases less than 20% (or about 20%), in some cases less than 15% (or about 15%), in some cases less than 10% (or about 10%) or in some cases less than 5% (or about 5% ) non-alcoholic, organic filler or solvent from the total weight of the composition. These drops can be dispensed from a single dose ampoule, which may preferably be sterile, eliminating the need for bacteriostatic components in the formulation. Alternatively, the 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, the 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 formulation's osmolality level to or around 210-320 milliosmoles per kilogram (mOsm/kg). The compositions of the present invention have an osmolality substantially in the range of 220-320 mOsm/kg, or optionally have an osmolality in the range of 235-300 mOsm/kg. The composition of 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 normal tear fluid, which is equivalent to approximately 0.9% w/v sodium chloride in water. Examples of suitable tonicity regulators include, but are not limited to, 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 administer pharmaceutically active compounds. Such compounds include, but are not limited to, glaucoma therapeutics, analgesics, anti-inflammatory and antiallergic drugs, and antimicrobial agents. 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 therapeutics such as PDE4 inhibitors; and antiallergic drugs such as H1/H4 inhibitors, H4 inhibitors, olopatadine, or mixtures thereof.

It is also contemplated 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 subtraction of ingredients in a given formulation.

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

In some embodiments, various conventional buffer solutions may be used, such as phosphate, borate, citrate, acetate, histidine, tris, bis-tris, etc., 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 salt of boric acid in solution. Hydrated salts such as sodium borate decahydrate can also be used. Phosphate buffers include phosphoric acid and its salts; for example, M2HPO4 and MH2PO4, where M represents an alkali metal such as sodium and potassium. Salt hydrates can also be used. In one embodiment of the present invention, Na2HPO4.7H2O and NaH2PO2.H2O 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 concentration of the buffer solution generally ranges from about 0.01 to 2.5% w/v, and 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 cP, in some cases from about 10 to about 200 cP, or in some cases from about 10 to about 100 cP when measured on a TA Instruments AR 2000 rheometer. The TA Instruments AR 2000 Rheometer must be used with the TA Rheological Advantage Software AR2000 Flow Test Method with 40mm steel plate geometry; Viscosity ranges should be obtained by measuring steady state flow at a controlled shear rate from 0 s-¹ to 200 s-¹.

In some embodiments, the compositions of the present invention are used as an eye drop solution, an eye wash solution, a contact lens lubricant and/or moisturizer solution, a spray solution, an irrigation solution, or any other method of administering the composition to the eye.

The compositions of the present invention can also be used as packaging solutions for contact lenses. In some embodiments, as packaging solutions, the compositions of the present invention can be sealed in blister packs 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; US Patents No. D435966; 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 the solution before sealing the package and then sterilizing the package. This method is the preferred method for treating ophthalmic products with antiallergic substances.

Sterilization can be carried out at different temperatures and for different periods of time. Preferred sterilization conditions range from about 100°C for about 8 hours to about 150°C 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 approximately 124°C for approximately 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 solutions, other buffer solutions, and deionized water. In some embodiments, the packaging solution is an aqueous solution in deionized water or saline containing salts, including, without limitation, sodium chloride, sodium borate, sodium phosphate, sodium hydrogenphosphate, sodium dihydrogenphosphate, or their corresponding potassium salts. These ingredients typically combine to form buffer solutions containing an acid and a conjugate base, so 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 will contain about 500-18,500 ppm sodium borate, most preferably about 1,000 ppm sodium borate.

If any ingredients contained in packaging solutions undergo 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 units and catenanes. See David A. Leigh et al Angew. Chem Int. Ed., 2001, 40, No. 8, pgs. 1538-1542 and Jean-Claude Chambron et al. Pure & Appl. Chem., 1990, Vol. 62, No. 6, pgs. 1027-1034. Stabilizers that prevent oxidation can also be other substances that inhibit oxidation, for example, 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(octadecycloxy)-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 thereof.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 Pharmaceutical Compositions That Are Unstable to Oxidation,” and the corresponding unconditional application, which is incorporated herein by reference in its entirety. In some embodiments, the concentration of oxidation stabilizers in the solution is from about 2.5 µmol/L to about 5000 µmol/L, in some cases from about 20 µmol/L to about 1000 µmol/L, in some cases from about 100 µmol/L up to about 1000 µmol/L or in some cases 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 once a day, five times a day, six times a day, eight times a day, every hour or more often. This dosing frequency is also maintained for periods of varying duration depending on the therapeutic needs of the patient. The duration of a particular therapeutic regimen can vary from a single-dose 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 this invention can be prepared using techniques well known to one 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 may be made by one skilled in the art without departing from the spirit and scope of the present invention.

The following test methods were used in the examples.

Example 1

Lemon aspen extract compositions showed increased expression of MUC1, MUC4 and MUC16 genes in human epicorneal tissues when treated in the medium.

Human 3D epicorneal tissues were purchased from MatTek Company (Ashland, MA, USA). Once 3D human epicorneal tissues were obtained, they were incubated in MatTek assay medium overnight according to the manufacturer's instructions. Three-dimensional human epicorneal tissues were divided into three experimental groups with a minimum of three tissues in each group. Lemon aspen extract was respectively added to the culture medium containing human epicorneal tissues from the two experimental groups to obtain medium concentrations of 0.001% or 0.01% (w/v), respectively. Epicorneal tissues from all four experimental groups were incubated for two days. The lemon aspen extract used was supplied by Southern Cross Botanicals (Knockrow Nsw, Australia). After incubation for two days, the expression of mucin 1 (MUC1), mucin 4 (MUC4) and mucin 16 (MUC16) genes was analyzed. After two days of incubation, human 3D epicorneal tissues were cut in half and half the tissue was lysed in 350 μl of lysis buffer consisting of 100 parts RLT buffer (RNeasy Mini' Kit, Qiagen, Valencia, CA, USA) per part 2-mercaptoethanol. RNA was extracted from the solutions using the RNeasy mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions, and RNA was eluted in 25 μl of RNase-free water.

Reverse transcription (RT) was performed using a Biosystems High Throughput Reverse Transcription Kit (ThermoFisher Scientific, Bridgewater, NJ, USA). Gene expression assay kits sold under the trade name TAQMAN for mucin-1 (MUC1), mucin-4 (MUC4) and mucin-16 (MUC16), polypeptide A (RNA) polymerase (RNA) II (POLR2A) and master mix purchased from ThermoFisher Scientific (Bridgewater, NJ). The qPCR assay was performed using TaqMan® Master Mix (ThermoFisher Scientific, Bridgewater, NJ, USA) and performed on a real-time PCR system commercially available under the trade name QUANTSTUDIO 7 Flex System (ThermoFisher Scientific, NJ, USA). Bridgewater, New Jersey, USA). The expression of the MUC1, MUC4 and MUC16 genes was normalized to the expression of the constitutive human RNA gene POLR2A. Fold changes were calculated compared to untreated control (UT) and a two-tailed two-sample Student's t test (Microsoft Office Excel 2007; Microsoft, Redmond, WA, USA). The results are shown in Fig. 1.

Example 2

Lemon aspen extract compositions showed an increase in mucin-1 secretion in human epicorneal tissues when treated in the medium.

Human 3D epicorneal tissues were purchased from MatTek Company (Ashland, MA, USA). Once 3D human epicorneal tissues were obtained, they were incubated in MatTek assay medium overnight according to the manufacturer's instructions. Three-dimensional human epicorneal tissues were divided into three experimental groups with a minimum of three tissues in each group. Lemon aspen extract was respectively added to the culture medium containing human epicorneal tissues from the two experimental groups to obtain medium concentrations of 0.001% or 0.01% (w/v), respectively. Epicorneal tissues from all four experimental groups were incubated for two days. Two days later, the culture medium was collected to measure mucin 1 secretion using a human mucin-1 (CA15-3) enzyme-linked immunosorbent assay (ELISA) kit (EHMUC1, ThermoFisher Scientific, Bridgewater, NJ) 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 standard enzyme-linked immunosorbent assay method and therefore there is a linear correlation between the concentration of mucin-1 in the sample and the colorimetric change. A standard curve was generated with mucin-1 concentration on the x-axis and absorbance on the y-axis indicating the corresponding mucin-1 concentration. The results are shown below in Fig. 2.

Example 3

Lemon aspen (Acronychia acidula) extract, when applied topically, demonstrated increased induction of HAS3 gene expression in human epicorneal tissues.

Human 3D epicorneal tissues were purchased from MatTek Company (Ashland, MA, USA). Once 3D human epicorneal tissues were obtained, they were incubated in MatTek assay medium overnight according to the manufacturer's instructions. Human epicorneal tissues with three-dimensional structure were divided into two experimental groups with a minimum of three tissues in each group. A solution of lemon aspen extract (0.1% w/v extract in a vehicle containing 30/70 (v/v) polypropylene glycol/ethanol) was topically applied to human epicorneal tissue in one of the experimental groups. Epicorneal tissues in both experimental groups were incubated for two days. After two days, the expression of hyaluronic acid synthase 3 (HAS3) genes was analyzed. HAS3 is an enzyme involved in the synthesis of hyaluronic acid. The lemon aspen extract used was supplied by Southern Cross Botanicals (Knockrow Nsw, Australia). After two days of incubation, human 3D epicorneal tissues were cut in half and half the tissue was lysed in 350 μl of lysis buffer consisting of 100 parts RLT buffer (RNeasy Mini' Kit, Qiagen, Valencia, CA, USA) per part 2-mercaptoethanol. RNA was extracted from the solutions using the RNeasy mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions, and RNA was eluted in 25 μl of RNase-free water.

Reverse transcription (RT) was performed using a Biosystems High Throughput Reverse Transcription Kit (ThermoFisher Scientific, Bridgewater, NJ, USA). Gene expression assay kits sold under the trade name TAQMAN for hyaluronic acid synthase 3 (HAS3), polymerase (RNA) polypeptide A (RNA) II (POLR2A) and master mix were purchased from ThermoFisher Scientific (Bridgewater, NJ ). The qPCR assay was performed using TaqMan® Master Mix (ThermoFisher Scientific, Bridgewater, NJ, USA) and performed on a real-time PCR system commercially available under the trade name QUANTSTUDIO 7 Flex System (ThermoFisher Scientific, NJ, USA). Bridgewater, New Jersey, USA). HAS3 gene expression was normalized to the expression of the constitutive human RNA gene POLR2A. Fold changes were calculated compared to untreated control (UT) and a two-tailed two-sample Student's t test (Microsoft Office Excel 2007; Microsoft, Redmond, WA, USA). The results are shown in Fig. 3.

Example 4

Lemon aspen extract, when applied topically, showed an increase in the biological activity of hyaluronic acid in the tissues of the human epicorneal membrane.

Human 3D epicorneal tissues were purchased from MatTek Company (Ashland, MA, USA). Once 3D human epicorneal tissues were obtained, they were incubated in MatTek assay medium overnight according to the manufacturer's instructions. Human epicorneal tissues with three-dimensional structure were divided into two experimental groups with a minimum of three tissues in each group. A solution of lemon aspen extract (0.1% w/v extract in a vehicle containing 30/70 (v/v) polypropylene glycol/ethanol) was topically applied to human epicorneal tissue in one of the experimental groups. Epicorneal tissues in both experimental groups were incubated for two days. Two days later, the culture medium was collected to measure hyaluronic acid (HA) secretion using an enzyme-linked immunosorbent assay (ELISA) reagent kit (K-1200, Echelon, Salt Lake City, Utah, USA) according to the manufacturer's protocol. . The lemon aspen extract used in this Example 4 is similar to the lemon aspen extract used in Example 3. To assess activity, the colorimetric change was measured at 405 nm using a microplate photometer (SpectraMax M2E, Molecular Devices, Sunnyvale, CA, USA). This assay uses a competitive enzyme-linked immunosorbent assay method and therefore there is an inverse correlation between the HA concentration in the sample and the colorimetric change. A standard curve was generated with HA concentration on the x-axis and absorbance on the y-axis indicating the corresponding HA concentration. The results are shown in Fig. 4.

Example 5

Lemon aspen extract showed an increase in HAS3 gene expression in human epicorneal tissues when treated in the medium.

Human 3D epicorneal tissues were purchased from MatTek Company (Ashland, MA, USA). Once 3D human epicorneal tissues were obtained, they were incubated in MatTek assay medium overnight according to the manufacturer's instructions. Three-dimensional human epicorneal tissues were divided into four experimental groups with a minimum of three tissues in each group. Lemon aspen extract was respectively added to the culture medium containing human epicorneal tissues from the three experimental groups to obtain medium concentrations of 0.0001%, 0.001% or 0.01% (w/v), respectively. Epicorneal tissues from all four experimental groups were incubated for two days. The lemon aspen extract used in Example 5, available from Southern Cross Botanicals (Knockrow Nsw, Australia), was extracted using a different extraction process from the extraction process used in Example 3. After two days of incubation, the expression of hyaluronic acid synthase 3 genes ( HAS3) as described below. Human epicorneal tissues were cut in half, and half the tissue was lysed in 350 μl of lysis buffer consisting of 100 parts RLT buffer (RNeasy Mini Qiagen kit, Valencia, CA, USA) to one part 2-mercaptoethanol. RNA was extracted from the solutions using the RNeasy mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions, and RNA was eluted in 25 μl of RNase-free water.

Reverse transcription (RT) was performed using a Biosystems High Throughput Reverse Transcription Kit (ThermoFisher Scientific, Bridgewater, NJ, USA). Gene expression assay kits sold under the trade name TAQMAN for hyaluronic acid synthase 3 (HAS3), polymerase (RNA) polypeptide A (RNA) II (POLR2A) and master mix were purchased from ThermoFisher Scientific (Bridgewater, NJ ). The qPCR assay was performed using TaqMan® Master Mix (ThermoFisher Scientific, Bridgewater, NJ, USA) and performed on a real-time PCR system commercially available under the trade name QUANTSTUDIO 7 Flex System (ThermoFisher Scientific, NJ, USA). Bridgewater, New Jersey, USA). HAS3 gene expression was normalized to the expression of the constitutive human RNA gene POLR2A. Fold changes were calculated compared to untreated control (UT) and a two-tailed two-sample Student's t test (Microsoft Office Excel 2007; Microsoft, Redmond, WA, USA). The results are shown in Fig. 5.

Example 6

Lemon aspen extract showed an increase in the biological activity of hyaluronic acid in human epicorneal tissues when processed in the medium.

Human 3D epicorneal tissues were purchased from MatTek Company (Ashland, MA, USA). Once 3D human epicorneal tissues were obtained, they were incubated in MatTek assay medium overnight according to the manufacturer's instructions. Three-dimensional human epicorneal tissues were divided into four experimental groups with a minimum of three tissues in each group. Lemon aspen extract was respectively added to the culture medium containing human epicorneal tissues from the three experimental groups to obtain medium concentrations of 0.0001%, 0.001% or 0.01% (w/v), respectively. Epicorneal tissues from all four experimental groups were incubated for two days. The lemon aspen extract used in this Example 6 is similar to the lemon aspen extract used in Example 5. Two days later, the culture medium was collected to measure hyaluronic acid (HA) secretion using an enzyme-linked immunosorbent assay (ELISA) reagent kit (K-1200, Echelon, Salt Lake City, Utah, 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 and therefore there is an inverse correlation between the HA concentration in the sample and the colorimetric change. A standard curve was generated with HA concentration on the x-axis and absorbance on the y-axis indicating the corresponding HA concentration. The results are shown in Fig. 6.

Example 7

Table 1 sets forth the components of such formulations (as shown in Formulations 7A-7D) that can be formulated as described below using conventional mixing technology.

Table 1

7A
Used to relieve irritation caused by dry eyes 7B
Used to relieve irritation caused by dry eyes 7C
Used to relieve irritation caused by dry eyes (with or without contact lenses) 7D
Used to relieve irritation caused by dry eyes (with or without contact lenses) INGREDIENT % w/w Quantity per batch (grams) % w/w Quantity per batch (grams) % w/w Quantity per batch (grams) % w/w Quantity per batch (grams) Sodium hyaluronate 0.20 2.0 0.30 3.0 0.15 1.5 0.15 1.5 Lemon Aspen Extract 0.10 1.0 0.01 0.10 0.10 1.0 0.01 0.10 Polysorbate 80 1.0 10.0 0.2 2.0 1.0 10.0 0.2 2.0 Polysorbate 20 5.0 50.0 1.0 10.0 2.0 20.0 1.0 10.0 Polyethylene glycol 400 0.25 2.5 0.25 2.5 0 0 0 0 Boric acid 0.60 6.0 0.60 6.0 0.60 6.0 0.60 6.0 Sodium borate 0.05 0.50 0.05 0.50 0.05 0.50 0.05 0.50 Sodium chloride* Potassium chloride 0.10 1.0 0.10 1.0 0.10 1.0 0.10 1.0 Calcium chloride dihydrate 0.006 0.06 0.006 0.06 0.006 0.06 0.006 0.06 Magnesium chloride 0.006 0.06 0.006 0.06 0.006 0.06 0.006 0.06 Sodium chlorite dihydrate 0.014 0.14 0.014 0.14 0.014 0.14 0.014 0.14 Polyquaternium-42 (33% aqueous solution) 0.0015 0.015 0.0015 0.015 0.0015 0.015 0.0015 0.015 Sodium chlorite dihydrate 0.014 0.14 0.014 0.14 0.014 0.14 0.014 0.14 1N sodium hydroxide solution** 1N hydrochloric acid solution** Purified water*** General 100.00% 1000.0 g 100.00% 1000.0 g 100.00% 1000.00 g 100.00% 1000.00 g

* can be adjusted to tonicity 280-290 mOsm/kg

**can be adjusted to pH 7.2

*** optionally in sufficient quantities up to 100% w/w.

For examples 7A-7D: Sodium hyaluronate can be supplied by CONTIPRO A.S. (DOLNI DOBRUCZ, CZECH REPUBLIC).

For Examples 7A-7D: Lemon Aspen Extract (Abacross™ Acronychia Fruit Extract) can be supplied by SOUTHERN CROBOTANICALS (KNOCKROW NSW, AUSTRALIA).

For Examples 7A-7D: Polysorbate 20 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 7A-7D: Polysorbate 80 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 7A-7B: Polyethylene glycol 400 can be supplied by Clariant Produkte (BURGKIRCHEN, GERMANY).

For Examples 7A-7D: Boric acid can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 7A-7D: Sodium borate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 7A-7D: Sodium chloride can be supplied by Caldic (DUSSELDORF, GERMANY).

For Examples 7A-7D: Potassium chloride can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 7A-7D: Calcium chloride dihydrate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 7A-7D: Magnesium chloride can be supplied by KGaA (DARMSTADT, GERMANY).

For Examples 7A-7D: Polyquaternium-42 (33% aqueous solution) can be supplied by DSM BIOMEDICAL (BERKELEY, CA, USA).

For Examples 7A-7D: Sodium chlorite dihydrate can be supplied by Oxychem (WICHITA, KANSAS, USA)

For Examples 7A-7D: 1N sodium hydroxide can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

For Examples 7A-7D: 1N hydrochloric acid can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

Solution 7A 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 both ingredients are completely mixed and dissolved.

3. Add 1.0 g of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen extract dissolves.

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

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

6. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.5 grams polyethylene glycol-400, 6.0 grams boric acid, 0.05 grams sodium borate, 1.0 grams sodium chloride, 0.06 grams chloride calcium, 0.06 grams of magnesium chloride and 0.0015 grams of polyquaternium-42 (aqueous).

7. While continuing to stir, 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 composition is adjusted to 7.2 using 1N sodium hydroxide and/or 1N hydrochloric acid.

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

11. Filter the solution through a 0.22 micron filter.

Solution 7B can be prepared as follows:

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

2. Add 2 g of polysorbate 80 and 10 g of polysorbate 20 to the above solution. Stir the solution until both ingredients are completely mixed and dissolved.

3. Add 0.1 g of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen extract dissolves.

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

5. Add 3.0 grams of sodium hyaluronate to the solution from step 4. The solution is stirred until the sodium hyaluronate is completely dissolved.

6. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.5 grams polyethylene glycol-400, 6.0 grams boric acid, 0.05 grams sodium borate, 1.0 grams sodium chloride, 0.06 grams chloride calcium, 0.06 grams of magnesium chloride and 0.0015 grams of polyquaternium-42 (aqueous).

7. While continuing to stir, 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 composition is adjusted to 7.2 using 1N sodium hydroxide and/or 1N hydrochloric acid.

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

11. Filter the solution through a 0.22 micron filter.

Solution 7C can be prepared as follows:

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

2. Add 10 g of polysorbate 80 and 20 g of polysorbate 20 to the above solution. Stir the solution until both ingredients are completely mixed and dissolved.

3. Add 1.0 g of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen extract dissolves.

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

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

6. Then add the following ingredients in succession, allowing each to dissolve completely before adding the next: 6.0 grams boric acid, 0.05 grams sodium borate, 1.0 grams potassium chloride, 0.06 grams calcium chloride dihydrate, 0.06 grams chloride magnesium and 0.0015 grams of polyquaternium-42 (aqueous).

7. While continuing to stir, 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 composition is adjusted to 7.2 using 1N sodium hydroxide and/or 1N hydrochloric acid.

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

11. Filter the solution through a 0.22 micron filter.

Solution 7D can be prepared as follows:

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

2. To the above solution add 2 g of polysorbate 10 and 50 g of polysorbate 20. The solution is stirred until both ingredients are completely mixed and dissolved.

3. Add 0.1 g of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen extract dissolves.

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

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

6. Then add the following ingredients in succession, allowing each to dissolve completely before adding the next: 6.0 grams boric acid, 0.05 grams sodium borate, 1.0 grams potassium chloride, 0.06 grams calcium chloride dihydrate, 0.06 grams chloride magnesium and 0.0015 grams of polyquaternium-42 (aqueous).

7. While continuing to stir, 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 composition is adjusted to 7.2 using 1N sodium hydroxide and/or 1N hydrochloric acid.

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

11. Filter the solution through a 0.22 micron filter.

Example 8

Table 2 sets forth the components of the compositions of the present invention (as shown in Formulations 8A-8D) that can be formulated as described below using conventional mixing technology.

table 2

8A
Used to relieve irritation caused by dry eyes 8B
Used to relieve irritation caused by dry eyes (with or without contact lenses) 8C
Used to relieve irritation caused by dry eyes (with or without contact lenses) 8D
Used to relieve irritation caused by dry eyes INGREDIENT % wt./wt. Quantity per batch (grams) % wt./wt. Quantity per batch (grams) % wt./wt. Quantity per batch (grams) % wt./wt. Quantity per batch (grams) Sodium hyaluronate 0 0 0 0 0.120 1.20 0.120 1.20 Tamarind seed polysaccharide 0 0 0 0 0.200 2.00 0.200 2.00 Lemon Aspen Extract 0.10 1.0 0.10 1.0 0.01 0.10 0.01 0.10 Polysorbate 80 1.0 10.0 1.0 10.0 0.2 2.0 0.2 2.0 Polysorbate 20 5.0 50.0 5.0 50.0 1.0 10.0 1.0 10.0 Polyethylene glycol 400 0 0 0 0 0.25 2.5 0.25 2.5 Glycerol 0.25 2.5 0.25 2.5 0.25 2.5 0.25 2.5 Hypromellose E3 2910 0.198 1.98 0.198 1.98 0.198 1.98 0.198 1.98 Boric acid 0.40 4.0 0.40 4.0 0.40 4.0 0.40 4.0 Sodium borate 0.022 0.22 0.022 0.22 0.022 0.22 0.022 0.22 Sodium hydrogen phosphate 0.027 0.27 0.027 0.27 0.027 0.27 0.027 0.27 Sodium citrate dihydrate 0.40 4.0 0.40 4.0 0.40 4.0 0.40 4.0 Sodium chloride* Potassium chloride 0.10 1.0 0.10 1.0 0.10 1.0 0.10 1.0 50% aqueous sodium lactate solution 0.057 0.57 0.057 0.57 0.057 0.57 0.057 0.57 Magnesium chloride 0.013 0.13 0.013 0.13 0.013 0.13 0.013 0.13 Glucose 0.0036 0.036 0.0036 0.036 0.0036 0.036 0.0036 0.036 Glycine 0.00002 0.0002 0.00002 0.0002 0.00002 0.0002 0.00002 0.0002 Ascorbic acid 0.00001 0.0001 0.00001 0.0001 0.00001 0.0001 0.00001 0.0001 Edetate disodium 0.01 0.1 0.05 0.5 0.01 0.1 0.05 0.5 Polyquaternium-42 (33% aqueous solution) 0.0030 0.030 0.0015 0.015 0.0030 0.030 0.0015 0.015 Sodium chlorite dihydrate 0.014 0.14 0.014 0.14 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% 1000.00 g 100.00% 1000.00 g 100.00% 1000.00 g

* can be adjusted to tonicity 280-290 mOsm/kg

**can be adjusted to pH 7.2

*** optionally in sufficient quantities up to 100% w/w.

For examples 8C-8D: Sodium hyaluronate can be supplied by CONTIPRO A.S. (DOLNI DOBRUCZ, CZECH REPUBLIC).

For Examples 8C-8D: Tamarind seed polysaccharide can be supplied by INDENA (MILAN, ITALY).

For Examples 8A-8D: Lemon Aspen Extract (Abacross™ Acronychia Fruit Extract) can be supplied by SOUTHERN CROBOTANICALS (KNOCKROW NSW, AUSTRALIA).

For Examples 8A-8D: Polysorbate 20 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 8A-8D: Polysorbate 80 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 8C-8D: Polyethylene glycol 400 can be supplied by Clariant Produkte (BURGKIRCHEN, GERMANY).

For Examples 8A-8D: Boric acid can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 8A-8D: Sodium borate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 8A-8D: Sodium chloride can be supplied by Caldic (DUSSELDORF, GERMANY).

For Examples 8A-8D: Potassium chloride can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 8A-8D: Hypromellose E3 2910 can be supplied by DOW CHEMICAL (PLAQUEMINE, LOUISIANA, USA).

For Examples 8A-8D: Glycerin can be supplied by Emery Oleochemicals GmbH (DUSSELDORF, GERMANY).

For Examples 8A-8D: Disodium phosphate can be supplied by KGaA (DARMSTADT, GERMANY).

For Examples 8A-8D: Sodium citrate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 8A-8D: Sodium lactate can be supplied as sodium lactate (50% aqueous solution) from Merck KGaA (DARMSTADT, GERMANY).

For Examples 8A-8D: Glucose can be supplied by Roquette Freres (LASTREME, FRANCE).

For Examples 8A-8D: Glycine can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 8A-8D: Ascorbic acid can be supplied by DSM NUTRITIONAL Products (DRAKEMERE, SCOTLAND, UK).

For Examples 8A-8D: Polyquaternium-42 can be supplied as Polyquaternium-42 (33% aqueous solution) from DSM BIOMEDICAL (BERKELEY, CA, USA).

For Examples 8A-8D: Edetate disodium can be supplied by Merck NV/SA (OVEREYSE, BELGIUM).

For Examples 8A-8D: 1N sodium hydroxide can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

For Examples 8A-8D: 1N hydrochloric acid can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

For Examples 8A-8D: Sodium chlorite dihydrate can be supplied by Oxychem (WICHITA, KANSAS, USA)

Solution 8A 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 both ingredients are completely mixed and dissolved.

3. Add 1.0 g of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen 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 the hypromellose E3 Premium dissolves.

6. Then add the following ingredients in succession, allowing each to dissolve completely before adding the next: 2.50 grams glycerin, 4.0 grams boric acid, 0.22 grams sodium borate, 0.27 grams sodium hydrogen phosphate, 4.00 grams sodium citrate dihydrate , 1 gram potassium chloride, 0.57 grams sodium lactate (50% aqueous solution), 0.13 grams magnesium chloride, 0.036 grams glucose, 0.0002 grams glycine, 0.0001 grams ascorbic acid, 0.10 grams 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. Filter the solution through a 0.22 micron filter.

Solution 8B 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 both ingredients are completely mixed and dissolved.

3. Add 1.0 g of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen 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 the hypromellose E3 Premium dissolves.

6. Then add the following ingredients in succession, allowing each to dissolve completely before adding the next: 2.50 grams glycerin, 4.0 grams boric acid, 0.22 grams sodium borate, 0.27 grams sodium hydrogen phosphate, 4.00 grams sodium citrate dihydrate , 1 gram potassium chloride, 0.57 grams sodium lactate (50% aqueous solution), 0.13 grams magnesium chloride, 0.036 grams glucose, 0.0002 grams glycine, 0.0001 grams ascorbic acid, 0.05 grams 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. Filter the solution through a 0.22 micron filter.

The 8C solution can be prepared as follows:

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

2. Add 2 g of polysorbate 80 and 10 g of polysorbate 20 to the above solution. Stir the solution until both ingredients are completely mixed and dissolved.

3. Add 0.1 g of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen extract dissolves.

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

5. Add 1.2 grams of sodium hyaluronate to the solution from step 4. The solution is stirred until the sodium hyaluronate is completely dissolved.

6. Add 2.0 grams of tamarind seed polysaccharide to the above mixture. The solution is stirred until the tamarind seed polysaccharide is completely dissolved.

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

8. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.50 grams polyethylene glycol 400, 2.50 grams glycerin, 4.0 grams boric acid, 0.22 grams sodium borate, 0.27 grams sodium hydrogen phosphate, 4.00 grams sodium citrate dihydrate, 1 gram potassium chloride, 0.57 grams sodium lactate (50% aqueous solution), 0.13 grams magnesium chloride, 0.036 grams glucose, 0.0002 grams glycine, 0.0001 grams ascorbic acid acid, 0.10 grams of disodium edetate, 0.030 grams of polyquaternium-42 (33% aqueous solution) and 0.14 grams of sodium chlorite.

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

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

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

12. Filter the solution through a 0.22 micron filter.

Solution 8D can be prepared as follows:

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

2. Add 2 g of polysorbate 80 and 10 g of polysorbate 20 to the above solution. Stir the solution until both ingredients are completely mixed and dissolved.

3. Add 0.1 g of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen extract dissolves.

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

5. Add 1.2 grams of sodium hyaluronate to the solution from step 4. The solution is stirred until the sodium hyaluronate is completely dissolved.

6. Add 2.0 grams of tamarind seed polysaccharide to the above mixture. The solution is stirred until the tamarind seed polysaccharide is completely dissolved.

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

8. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.50 grams polyethylene glycol 400, 2.50 grams glycerin, 4.0 grams boric acid, 0.22 grams sodium borate, 0.27 grams sodium hydrogen phosphate, 4.00 grams sodium citrate dihydrate, 1 gram potassium chloride, 0.57 grams sodium lactate (50% aqueous solution), 0.13 grams magnesium chloride, 0.036 grams glucose, 0.0002 grams glycine, 0.0001 grams ascorbic acid acid, 0.10 grams of disodium edetate, 0.015 grams of polyquaternium-42 (33% aqueous solution) and 0.14 grams of sodium chlorite.

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

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

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

12. The solution is filtered through a 0.22 micron hydrophilic filter.

Example 9

Table 3 sets forth the components of the formulations of the present invention (as shown in Formulations 9A and 9B) that can be formulated as described below using conventional mixing technology.

Table 3

9A
Used to relieve irritation caused by dry eyes 9B
Used to relieve irritation caused by dry eyes INGREDIENT % wt./wt. Quantity per batch (grams) % wt./wt. Quantity per batch (grams) Sodium hyaluronate 0.20 2.0 0.10 1.0 Tamarind seed polysaccharide 0 0 0.20 2.0 Lemon Aspen Extract 0.01 0.1 0.10 1.0 Polysorbate 80 0.2 2.0 1.0 10.0 Polysorbate 20 1.0 10.0 5.0 50.0 Polyethylene glycol 400 0.25 2.5 0.25 2.5 Boric acid 0.60 6.0 0.60 6.0 Sodium borate 0.06 0.60 0.06 0.60 Sodium chloride* Specially refined castor oil 0.625 6.25 0.625 6.25 Lumuluse GRH-40 0.50 5.0 0.50 5.0 Ethyl linolenate 0.10 1.0 0 0 Retinyl palmitate 0.05 0.5 0 0 Polyquaternium 42
(33% aqueous solution) 0.0045 0.045 0.0045 0.045 1N sodium hydroxide solution** 1N hydrochloric acid solution** Purified water*** Total 100.00% 1000.0 g 100.00% 1000.0 g

* can be adjusted to tonicity 280-290 mOsm/kg

**can be adjusted to pH 7.2

*** optionally in sufficient quantities up to 100% w/w.

For examples 9A-9B: Sodium hyaluronate can be supplied by CONTIPRO A.S. (DOLNI DOBRUCZ, CZECH REPUBLIC).

For Examples 9A-9B: Lemon Aspen Extract (Abacross™ Acronychia Fruit Extract) can be supplied by SOUTHERN CROBOTANICALS (KNOCKROW NSW, AUSTRALIA).

For Examples 9A-9B: Polysorbate 20 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 9A-9B: Polysorbate 80 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 9A-9B: Polyethylene glycol 400 can be supplied by Clariant Produkte (BURGKIRCHEN, GERMANY).

For Examples 9A-9B: Boric acid can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 9A-9B: Sodium borate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 9A-9B: Sodium chloride can be supplied by Caldic (DUSSELDORF, GERMANY).

For Examples 9A-9B: 1N sodium hydroxide can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

For Examples 9A-9B: 1N hydrochloric acid can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

For Examples 9A-9B: Lumuluse GRH-40 can be supplied by VANTAGE (GURNEY, ILLINOIS, USA).

For Examples 9A-9B: Extra refined castor oil can be supplied by CRODA, EDISON, NJ, USA.

For Examples 9A-9B: Ethyl linolenate can be supplied by SIGMA-ALDRICH (ST. LOUIS, MISSOURI, USA).

For Examples 9A-9B: Retinyl palmitate can be supplied by SIGMA-ALDRICH (ST. LOUIS, MISSOURI, USA).

For Examples 9A-9B: Polyquaternium-42 (33% aqueous solution) can be supplied by DSM BIOMEDICAL (BERKELEY, CA, USA).

For Example 9B: Tamarind seed polysaccharide can be supplied by INDENA (MILAN, ITALY).

Solution 9A can be prepared as follows:

1. Add 5.0 grams of Lumuluse GRH-40 to a 50 ml beaker.

2. While stirring, add 6.25 grams of specially purified castor oil.

3. Next add 1 gram of ethyl inolenate and 0.5 grams

retinyl palmitate and stir until smooth.

4. Add 500 grams of purified water to a separate 1500 ml beaker.

5. To the above solution add 2 g of polysorbate 80 and 10 g of polysorbate 20. The solution is stirred until both ingredients are completely dissolved.

6. Add 0.1 grams of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen extract dissolves.

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

8. Add 2.0 grams of sodium hyaluronate to the solution in step 7. The solution is stirred until the sodium hyaluronate is completely dissolved.

9. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.5 grams polyethylene glycol 400, 6.0 grams boric acid, 0.06 grams sodium borate, and 0.045 grams polyquaternium-42 (33% aqueous solution) .

10. Add the mixture obtained in step 3 and mix until smooth using a homogenizer.

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

12. The pH of the composition is adjusted to 7.2 using 1N sodium hydroxide and/or 1N hydrochloric acid.

13. The solution is brought to a mass of 1000.0 grams by adding purified water according to USP and stirring for 10 minutes until completely homogeneous.

14. Filter the solution through a 0.22 micron filter.

Solution 9B can be prepared as follows:

1. Add 5.0 grams of Lumuluse GRH-40 to a 50 ml beaker.

2. While stirring, add 6.25 grams of specially purified castor oil.

3. The resulting homogeneous solution is stored for later use.

4. Add 500 grams of purified water to a separate 1500 ml beaker.

5. To the above solution add 10 g of polysorbate 80 and 50 g of polysorbate 20. The solution is stirred until both ingredients are completely dissolved.

6. Add 1.0 grams of lemon aspen extract to the above solution. The solution is stirred until the lemon aspen extract dissolves.

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

8. Add 1.0 grams of sodium hyaluronate to the solution in step 7. The solution is stirred until the sodium hyaluronate is completely dissolved.

9. Then add 2.0 grams of tamarind seed polysaccharide. The solution is stirred until the tamarind seed polysaccharide is completely dissolved.

10. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.5 grams polyethylene glycol 400, 6.0 grams boric acid, 0.06 grams sodium borate, and 0.045 grams polyquaternium-42 (33% aqueous solution) .

11. Add the mixture obtained in step 3 and mix until smooth using a homogenizer.

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

13. The pH of the composition is adjusted to 7.2 using 1N sodium hydroxide and/or 1N hydrochloric acid.

14. The solution is brought to a mass of 1000.0 grams by adding purified water according to USP and stirring for 10 minutes until completely homogeneous.

15. Filter the solution through a 0.22 micron filter.

Example 10

Table 4 sets forth the components of the compositions of the present invention (as shown in Formulations 10A and 10B) that can be formulated as described below using conventional mixing technology.

Table 4

10A
Used to relieve irritation caused by dry eyes 10B
Used to relieve irritation caused by dry eyes (with or without contact lenses) INGREDIENT % w/w Quantity per batch (grams) % w/w Quantity per batch (grams) Sodium hyaluronate 0.30 3.0 0.15 1.5 Actinomyces extract 5.0 50.0 0 0 Connection 1**** 0 0 5.0 50.0 Polysorbate 80 1.0 10.0 1.0 10.0 Polysorbate 20 10.0 100.0 7.5 75.0 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% 1000.0 g 100.00% 1000.00 g

* bringing to tonicity 280-290 mOsm/kg

** adjustment to pH 7.2

*** in sufficient quantity up to 100% w/w.

**** (2E,4E,6E)-7-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)-3-methylocta-2, 4,6-trienoic acid

For examples 10A-10B: Sodium hyaluronate can be supplied by CONTIPRO A.S. (DOLNI DOBRUCZ, CZECH REPUBLIC).

For Example 10A: An extract of Actinomyces species A5640 (a bacterial extract in the IHVR collection designated collection nomenclature IHVR_39565_F7) can be supplied by the Baruch S. Blumberg Institute, Doylestown, Pennsylvania.

For Example 10B: Compound 1 is available from Sigma-Aldrich.

For Examples 10A-10B: Polysorbate 20 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 10A-10B: Polysorbate 80 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 10A: Polyethylene glycol 400 can be supplied by Clariant Produkte (BURGKIRCHEN, GERMANY).

For Examples 10A-10B: Boric acid can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 10A-10B: Sodium borate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 10A-10B: Sodium chloride can be supplied by Caldic (DUSSELDORF, GERMANY).

For Examples 10A-10B: Potassium chloride can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 10A-10B: Calcium chloride dihydrate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 10A-10B: Magnesium chloride can be supplied by KGaA (DARMSTADT, GERMANY).

For Examples 10A-10B: Polyquaternium-42 (33% aqueous solution) can be supplied by DSM BIOMEDICAL (BERKELEY, CA, USA)

For Examples 10A-10B: Sodium chlorite dihydrate can be supplied by Oxychem (WICHITA, KANSAS, USA)

For Examples 10A-10B: 1N sodium hydroxide can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

For Examples 10A-10B: 1N hydrochloric acid can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

Solution 10A can be prepared as follows:

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

2. Add 10 g of polysorbate 80 and 100 g of polysorbate 20 to the above solution. Stir the solution until both ingredients are completely mixed and dissolved.

3. Add 50.0 g of Actinomyces extract to the above solution. The solution is stirred until the Actinomyces extract dissolves.

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

5. Add 3.0 grams of sodium hyaluronate to the solution from step 4. The solution is stirred until the sodium hyaluronate is completely dissolved.

6. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.5 grams polyethylene glycol-400, 6.0 grams boric acid, 0.05 grams sodium borate, 1.0 grams sodium chloride, 0.06 grams chloride calcium, 0.06 grams of magnesium chloride and 0.0015 grams of polyquaternium-42 (aqueous).

7. While continuing to stir, 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 composition is adjusted to 7.2 using 1N sodium hydroxide and/or 1N hydrochloric acid.

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

11. Filter the solution through a 0.22 micron filter.

Solution 10B 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 75 g of polysorbate 20. The solution is stirred until both ingredients are completely mixed and dissolved.

3. To the above, add 50.0 g of Compound I. The solution is stirred until Compound I dissolves.

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

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

6. Then add the following ingredients in succession, allowing each to dissolve completely before adding the next: 6.0 grams boric acid, 0.05 grams sodium borate, 1.0 grams potassium chloride, 0.06 grams calcium chloride dihydrate, 0.06 grams chloride magnesium and 0.0015 grams of polyquaternium-42 (aqueous).

7. While continuing to stir, 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 composition is adjusted to 7.2 using 1N sodium hydroxide and/or 1N hydrochloric acid.

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

11. Filter the solution through a 0.22 micron filter.

Example 11

Table 5 sets forth the components of the compositions of the present invention (as shown in Formulations 11A and 11B) that can be formulated as described below using conventional mixing technology.

Table 5

11A
Used to relieve irritation caused by dry eyes (with or without contact lenses) 11B
Used to relieve irritation caused by dry eyes INGREDIENT % wt./wt. Quantity per batch (grams) % wt./wt. Quantity per batch (grams) Sodium hyaluronate 0.120 1.20 0.120 1.20 Tamarind seed polysaccharide 0.200 2.00 0.200 2.00 Actinomyces extract 5.0 50.0 0 0 Connection 2**** 0 0 0.5 5.0 Polysorbate 80 1.0 10.0 1.0 10.0 Polysorbate 20 10.0 100.0 3.0 30.0 Polyethylene glycol 400 0.25 2.5 0.25 2.5 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*** General 100.00% 1000.00 g 100.00% 1000.00 g

* bringing to tonicity 280-290 mOsm/kg

** adjustment to pH 7.2

*** in sufficient quantity up to 100.00% volume

**** 4-(1-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)vinyl)benzoic acid.

For examples 11A-11B: Sodium hyaluronate can be supplied by CONTIPRO A.S. (DOLNI DOBRUCZ, CZECH REPUBLIC).

For Examples 11A-11B: Tamarind seed polysaccharide can be supplied by INDENA (MILAN, ITALY).

For Example 11A: An extract of Actinomyces species A5640 (a bacterial extract in the IHVR collection designated collection nomenclature IHVR_39565_F7) can be supplied by the Baruch S. Blumberg Institute, Doylestown, Pennsylvania.

For Example 11B: Compound 2 can be supplied by Sigma-Aldrich.

For Examples 11A-11B: Polysorbate 20 can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Polysorbate 80 is available from Merck KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Polyethylene glycol 400 can be supplied by Clariant Produkte (BURGKIRCHEN, GERMANY).

For Examples 11A-11B: Boric acid can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Sodium borate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Sodium chloride can be supplied by Caldic (DUSSELDORF, GERMANY).

For Examples 11A-11B: Potassium chloride can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Hypromellose E3 2910 can be supplied by DOW CHEMICAL (PLAQUEMINE, LOUISIANA, USA).

For Examples 11A-11B: Glycerin can be supplied by Emery Oleochemicals GmbH (DUSSELDORF, GERMANY).

For Examples 11A-11B: Disodium phosphate can be supplied by KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Sodium citrate can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Sodium lactate can be supplied as sodium lactate (50% aqueous solution) from Merck KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Glucose can be supplied by Roquette Freres (LASTREME, FRANCE).

For Examples 11A-11B: Glycine can be supplied by Merck KGaA (DARMSTADT, GERMANY).

For Examples 11A-11B: Ascorbic acid can be supplied by DSM NUTRITIONAL Products (DRAKEMERE, SCOTLAND, UK).

For Examples 11A-11B: Polyquaternium-42 can be supplied as Polyquaternium-42 (33% aqueous solution) from DSM BIOMEDICAL (BERKELEY, CA, USA).

For Examples 11A-11B: Edetate disodium can be supplied by Merck NV/SA (OVERYSE, BELGIUM).

For Examples 11A-11B: 1N sodium hydroxide can be supplied by VWR (RADNER, PENNSYLVANIA, USA).

For Examples 11A-11B: 1N hydrochloric acid can be supplied by VWR (RADNER, PENNSYLVANIA, USA). For Examples 11A-11B: Sodium chlorite dihydrate can be supplied by Oxychem (WICHITA, KANSAS, USA)

Solution 11A can be prepared as follows:

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

2. Add 10 g of polysorbate 80 and 100 g of polysorbate 20 to the above solution. Stir the solution until both ingredients are completely mixed and dissolved.

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

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

5. Add 1.2 grams of sodium hyaluronate to the solution from step 4. The solution is stirred until the sodium hyaluronate is completely dissolved.

6. Add 2.0 grams of tamarind seed polysaccharide to the above mixture. The solution is stirred until the tamarind seed polysaccharide is completely dissolved.

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

8. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.50 grams polyethylene glycol 400, 2.50 grams glycerin, 4.0 grams boric acid, 0.22 grams sodium borate, 0.27 grams sodium hydrogen phosphate, 4.00 grams sodium citrate dihydrate, 1 gram potassium chloride, 0.57 grams sodium lactate (50% aqueous solution), 0.13 grams magnesium chloride, 0.036 grams glucose, 0.0002 grams glycine, 0.0001 grams ascorbic acid acid, 0.10 grams of disodium edetate, 0.030 grams of polyquaternium-42 (33% aqueous solution) and 0.14 grams of sodium chlorite.

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

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

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

12. Filter the solution through a 0.22 micron filter.

Solution 11B 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 30 g of polysorbate 20. The solution is stirred until both ingredients are completely mixed and dissolved.

3. To the above, add 5.0 g of Compound I. The solution is stirred until Compound I dissolves.

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

5. Add 1.2 grams of sodium hyaluronate to the solution from step 4. The solution is stirred until the sodium hyaluronate is completely dissolved.

6. Add 2.0 grams of tamarind seed polysaccharide to the above mixture. The solution is stirred until the tamarind seed polysaccharide is completely dissolved.

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

8. Then add the following ingredients sequentially, allowing each to dissolve completely before adding the next: 2.50 grams polyethylene glycol 400, 2.50 grams glycerin, 4.0 grams boric acid, 0.22 grams sodium borate, 0.27 grams sodium hydrogen phosphate, 4.00 grams sodium citrate dihydrate, 1 gram potassium chloride, 0.57 grams sodium lactate (50% aqueous solution), 0.13 grams magnesium chloride, 0.036 grams glucose, 0.0002 grams glycine, 0.0001 grams ascorbic acid acid, 0.10 grams of disodium edetate, 0.015 grams of polyquaternium-42 (33% aqueous solution) and 0.14 grams of sodium chlorite.

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

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

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

12. The solution is filtered through a 0.22 micron hydrophilic filter.

Embodiments of the Present Invention

1. Microemulsion composition for eye treatment, containing:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of eyes, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; optionally one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally a methyl (-CH3) or methylene (=CH2) functional group; a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; optionally from 1 to 10 carbon atoms; optionally 6 carbon atoms; optionally an aromatic functional group, optionally a phenyl functional group; optionally 2-methyl-prop-1,3-diene.

ii) an ophthalmologically acceptable carrier,

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

2. The composition of embodiment 1 (or any of the following embodiments), wherein the compound and/or extract having retinol-like properties and/or beneficial effects is a plant extract or a source of extracts of plants of the genus Acronychia and/or Licaria.

3. The composition of embodiments 1 and/or 2 (or any of the following embodiments), wherein the compound and/or extract having retinol-like properties and/or beneficial effects is a plant extract or a source of plant extracts of the genus Acronychia.

4. The composition according to any of embodiments 1-3 (or any of the following embodiments) or a combination thereof, wherein the plant extract or source of plant extracts of the genus Acronychia is selected from the group consisting of Acronychia aberrans, Acronychia acidula, Acronychia acronychioides, Acronychia acuminate , Acronychia baeuerlenii, Acronychia chooreechillum, Acronychia crassipetala, Acronychia eungellensis, Acronychia imperforate, Acronychia laevis, Acronychia laurifolia, Acronychia littoralis, Acronychia oblongifolia, Acronychia octandra, Acronychia parviflora, Acronychia pauciflora, Acronychia pedunculata, Acronychia pubescens, Acronychia spp. (Batavia Downs), Acronychia suberosa, Acronychia vestita, Acronychia wilcoxiana and combinations of two or more of them.

5. The composition of any one of embodiments 1-4 (or any of the following embodiments) or a combination thereof, wherein the plant extract or source of plant extracts of the genus Acronychia is Acronychia acidula.

6. The composition of any one of embodiments 1-5 (or any of the following embodiments) or a combination thereof, wherein the plant extract of Acronychia and/or Licaria contains from about 1% to about 20% of a compound of formula II by weight of the extract

Where:

R1 is selected from the group consisting of C1 - C20 alkyl, C2 - C20 alkenyl, C2 - C20 alkynyl and C3 - C8 cycloalkyl or aryl;

R2 is selected from the group consisting of hydrogen, hydroxyl, C1 - C6 alkyl, C2 - C6 alkenyl, C2 - C6 alkynyl, C3 - C8 cycloalkyl or aryl, - OC1 - C6 alkyl, - OC2 - C6 alkenyl, - OC2 - C6 alkynyl , - OC3 - C8 cycloalkyl or aryl, thiol, - SC1 - C6 alkyl, - SC2 - C6 alkenyl, - SC2 - C6 alkynyl, - SC3 - C8 cycloalkyl or aryl, - NR4C1 - C6 alkyl, - NR4C2 - C6 alkenyl, - NR4C2 - C6 alkynyl and - NR4C3 - C8 cycloalkyl or aryl;

R3 is selected from -CO2H, -CO2R4 or the isosteric equivalent of a carboxyl group, where R4 is C1-C6 alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl or aryl; And

Y is -(CH2 - CH2)-, -(CH = H - or -(C ≡ C)-.

7. The composition of any one of embodiments 1-6 (or any of the following embodiments) or a combination thereof, wherein the plant extract of Acronychia and/or Licaria contains from about 1% to about 20% of a compound of formula II by weight of the extract.

Where:

R1 is selected from the group consisting of C5 - C16 alkyl, C5 - C16 alkenyl and C5 - C16 alkynyl, more preferably C5 - C16 alkenyl, including, for example, farnesyl;

R2 is selected from the group consisting of hydrogen, hydroxyl, - OC1 - C6 alkyl, - OC2 - C6 alkenyl, - OC2 - C6 alkynyl, - OC3 - C8 cycloalkyl, more preferably hydrogen, hydroxyl, - OC1 - C6 alkyl, even more preferably - hydrogen or - OC1 - C3 alkyl;

R3 is selected from - CO2H, - CO2R4, where R4 is a C1 - C6 alkyl or the isosteric equivalent of a carboxyl group; And

Y is -(CH2 - CH2)- or -(CH = CH)-.

8. The composition according to any of embodiments 1-7 (or any of the following embodiments) or a combination thereof, wherein the compound of formula (II) is in the form of an acid or an alkyl ester selected from 3-(4-farnesyloxyphenyl)propionic acid, 3-(4-farnesyloxy-3-hydroxyphenyl)-propionic acid, 3-(4-farnesyloxy-3-methoxyphenyl)-propionic acid, alkyl esters thereof and combinations of two or more of them.

9. The composition according to any one of embodiments 1-8 (or any of the following embodiments) or a combination thereof, wherein the compound of formula II used in the present invention is 3-(4-farnesyloxyphenyl)-propionic acid and/or its ethyl ether.

10. The composition according to any of embodiments 1-9 (or any of the following embodiments) or a combination thereof, wherein the compound and/or extract having retinol-like properties and/or beneficial effects is a plant extract or a source of extracts of plants of the genus Licaria .

11. The composition of any one of embodiments 1-10 (or any of the following embodiments) or a combination thereof, wherein the plant extract or source of plant extracts of the genus Licaria is selected from the group consisting of Licaria vernicosa, Licaria brittoniana, Licaria canella, Licaria cubensis , Licaria velutina and Licaria triandra and combinations of two or more of them.

12. The composition of any one of embodiments 1-11 (or any of the following embodiments) or a combination thereof, wherein the plant extract or source of plant extracts of the genus Licaria is Licaria vernicosa.

13. The composition according to any one of embodiments 1-12 (or any of the following embodiments) or a combination thereof, wherein the compound and/or extract having retinol-like properties and/or beneficial effects is an extract of bacteria or a source of extracts of the genus Actinomyces.

14. The composition of any one of embodiments 1-14 (or any of the following embodiments) or a combination thereof, wherein the compound and/or extract having retinol-like properties and/or beneficial effects is a bacterial extract or a source of extracts of Actinomyces species A5640 .

15. The composition according to any of embodiments 1-14 (or any of the following embodiments) or a combination thereof, wherein the compound and/or extract having retinol-like properties and/or beneficial effects contains a compound of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; optionally one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

16. The composition of any one of embodiments 1-15 (or any of the following embodiments) or a combination thereof, wherein the compound of formula I is selected from (2E,4E,6E)-7-(1,1,2,2,3 ,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)-3-methylocta-2,4,6-trienoic acid and 4-(1-(1,1,2,2,3,3 -hexamethyl-2,3-dihydro-1H-inden-5-yl)vinyl)benzoic acid and their derivatives exhibiting retinoid-like activity, and mixtures thereof.

17. A method of preventing or treating symptoms associated with dry eye (optionally in a patient in need of such prevention and treatment), comprising the step of administering a microemulsion composition containing:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella ; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably a methyl (-CH3) or methylene (=CH2) functional group;

A represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; And

ii) in some cases an ophthalmologically acceptable carrier.

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

18. A method of treating a patient with reduced or low production/release/delivery/release of hyaluronic acid and/or mucin from the cornea and/or into the cornea, comprising the step of local administration to the patient of a microemulsion composition containing:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella ; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; preferably one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably a methyl (-CH3) or methylene (=CH2) functional group;

A represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably from 1 to 10 carbon atoms; more preferably 6 carbon atoms; preferably an aromatic functional group, preferably a phenyl functional group; preferably 2-methylprop-1,3-diene.

ii) in some cases an ophthalmologically acceptable carrier.

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

19. A method of treating dry eye, including the stage of local administration to a patient of a microemulsion composition containing:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella ; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; preferably one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably a methyl (-CH3) or methylene (=CH2) functional group;

A represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably from 1 to 10 carbon atoms; more preferably 6 carbon atoms; preferably an aromatic functional group, preferably a phenyl functional group; preferably 2-methylprop-1,3-diene;

ii) one or more emollients or sedatives; And

iii) in some cases an ophthalmologically acceptable carrier.

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

20. A method of stimulating healing or increasing the rate of healing of wounds in and/or on the surface of the eye (optionally in a patient requiring such stimulated healing or increased rate of healing) by administering a microemulsion composition containing:

i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of dry eye syndrome, selected from one or more of plant extracts or sources of plant extracts of the genus Acronychia, Licaria, Calendula and/or Trigonella ; Bacterial extracts or sources of extracts of bacteria of the genus Actinomyces; and compounds of formula (I):

(I),

Where -

dotted lines indicate a single or double bond; preferably one of the dotted lines represents a double bond;

R1 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms;

R2 represents H, a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably a methyl (-CH3) or methylene (=CH2) functional group;

A represents a carbon chain, linear, cyclic or branched, saturated or unsaturated, containing from 1 to 20 carbon atoms; preferably from 1 to 10 carbon atoms; more preferably 6 carbon atoms; preferably an aromatic functional group, preferably a phenyl functional group; preferably 2-methylprop-1,3-diene.

ii) in some cases an ophthalmologically acceptable carrier.

in this case, microemulsion droplets or particles have a maximum size of less than 1500 Å.

Claims (24)

1. A microemulsion composition for the prevention or treatment of symptoms associated with dry eye, containing: i) a safe and effective amount of a compound and/or extract having retinol-like properties and/or beneficial effects for use in the treatment of eyes, selected from one or more plant extracts or sources of plant extracts of the genus Acronychia acidula; source of extracts of bacteria of the genus Actinomyces; and a compound selected from (2E,4E,6E)-7-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)-3-methylocta-2 ,4,6-trienoic acid and 4-(1-(1,1,2,2,3,3-hexamethyl-2,3-dihydro-1H-inden-5-yl)vinyl)benzoic acid and mixtures thereof; ii) an oily component selected from castor oil, corn oil, sunflower oil, mineral oil, silicone oil and mixtures thereof; iii) an emollient or soothing agent, which is hyaluronic acid or a salt thereof; And iv) an ophthalmologically acceptable carrier, in this case, microemulsion drops or particles have a maximum size of less than . 2. The composition of claim 1, wherein the plant extract of Acronychia acidula contains from about 1 to about 20% of a compound of formula II by weight of the extract where R ₁ is selected from the group consisting of C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl and C ₃ -C ₈ cycloalkyl or aryl; R ₂ is selected from the group consisting of hydrogen, hydroxyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl or aryl, -OC ₁ -C ₆ alkyl, -OC ₂ -C ₆ alkenyl, -OC ₂ -C ₆ alkynyl, -OC ₃ -C ₈ cycloalkyl or aryl, thiol, -SC ₁ -C ₆ alkyl, -SC ₂ -C ₆ alkenyl, -SC ₂ -C ₆ alkynyl, -SC ₃ -C ₈ cycloalkyl or aryl, -NR ₄ C ₁ -C ₆ alkyl, -NR ₄ C ₂ -C ₆ alkenyl, -NR ₄ C ₂ -C ₆ alkynyl and -NR ₄ C ₃ -C ₈ cycloalkyl or aryl; R ₃ is selected from -CO ₂ H, -CO ₂ R ₄ or the isosteric equivalent of a carboxyl group, where R ₄ represents C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₈ cycloalkyl or aryl; And Y represents -(CH ₂ -CH ₂ )-, -(CH ═ CH)- or -(C ≡ C)-. 3. The composition of claim 2, wherein the plant extract of Acronychia acidula contains from about 1 to about 20% of a compound of formula II by weight of the extract wherein R ₁ is selected from the group consisting of C ₅ -C ₁₆ alkyl, C ₅ -C ₁₆ alkenyl and C ₅ -C ₁₆ alkynyl, more preferably C ₅ -C ₁₆ alkenyl, including, for example, farnesyl; R ₂ is selected from the group consisting of hydrogen, hydroxyl, -OC ₁ -C ₆ alkyl, -OC ₂ -C ₆ alkenyl, -OC ₂ -C ₆ alkynyl and -OC ₃ -C ₈ cycloalkyl, more preferably hydrogen, hydroxyl, -OC ₁ -C ₆ alkyl, even more preferably hydrogen or -C ₁ -C ₃ alkyl; R ₃ is selected from -CO ₂ H and -CO ₂ R ₄ , where R ₄ represents a C ₁ -C ₆ alkyl or the isosteric equivalent of a carboxyl group; And Y represents -(CH ₂ - CH ₂ )- or -(CH ═ CH)-. 4. The composition according to claim 3, wherein the compound of formula (II) is in the form of an acid or an alkyl ester selected from the group consisting of 3-(4-farnesyloxyphenyl)-propionic acid, 3-(4-farnesyloxy-3-hydroxyphenyl) -propionic acid, 3-(4-farnesyloxy-3-methoxyphenyl)-propionic acid, alkyl esters thereof and combinations of two or more of them. 5. The composition according to claim 4, in which the compound of formula II is 3-(4-farnesyloxyphenyl)-propionic acid and/or its ethyl ester. 6. The composition according to claim 1, wherein the compound and/or extract having retinol-like properties and/or beneficial effects is a source of extracts of the genus Actinomyces. 7. The composition according to claim 6, wherein the compound and/or extract having retinol-like properties and/or beneficial effects is a source of extracts of Actinomyces species A5640. 8. The composition according to claim 1, wherein the oily component is castor oil. 9. The composition according to claim 1, in which the emollient or sedative is sodium hyaluronate.

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