Dry eye treatment

Abstract

Disclosed are ophthalmic compositions for removing biofilms from the eye and for treating inflammation, blepharitis and dry eye of the eye, the ophthalmic compositions comprising an alpha-amylase and optionally other ingredients. The blepharitis is inflammation of eyelids and areas around the eyelids. The blepharitis generally affects both eyes, including areas along the edges of the eyelids. The blepharitis is considered to occur when tiny oil glands near the roots of the eyelashes become clogged, causing stimulation and redness. Several diseases and conditions may be associated with blepharitis.

Description

[0001] Background

[0002] In the following discussion, for background and introductory purposes, certain articles and methods will be described. Nothing contained herein should be construed as an "admission" of prior art. Applicants expressly reserve the right to demonstrate, in an appropriate case, that the articles and methods cited herein do not constitute prior art under applicable statutory provisions.

[0003] Blepharitis is an inflammation of the eyelids and the surrounding area. Blepharitis typically affects both eyes and includes the area along the eyelid margins. Blepharitis is considered to occur when the tiny oil glands near the base of the eyelashes become blocked, causing irritation and redness. Several diseases and conditions may be associated with blepharitis. In addition to being uncomfortable and unsightly, blepharitis typically occurs as a chronic condition that is difficult to treat, mainly because current existing treatments, including artificial tears, immunosuppressive drugs (Restasis), short-term use of antibiotics, short-term use of steroids, lymphocyte function-associated antigen-1 (LFA-1) antagonists (Xiidra), warm compresses, intense pulsed light therapy, or mechanically expressing meibum from the meibomian glands, do not address the root cause of the disease.

[0004] There remains a significant gap in the arsenal of treatments for blepharitis, mainly due to the limitations of existing treatments in addressing the underlying causes. The present disclosure addresses this need first by identifying biofilm formation on the eyelids as an important factor driving the prognosis of blepharitis. Second, the present disclosure provides compositions and methods of using the compositions that provide effective therapeutic treatment for blepharitis and dry eye.

[0005] Overview

[0006] This overview is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This overview is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written detailed description, which includes aspects illustrated in the accompanying drawings and defined in the appended claims.

[0007] In some aspects, the present disclosure provides a method for treating one or more of blepharitis and dry eye, the method comprising administering to a subject a therapeutically effective amount of a composition comprising optionally with a certain concentration of CaCl 2A stabilized active α - amylase or a functional fragment thereof and one or more pharmaceutically acceptable excipients. In some cases, administration promotes the removal of biofilm in or around the eyes of a subject. In some cases, a therapeutically effective amount of the composition is administered daily for a period of at least 1 second, at least 5 seconds, at least 1 day, 1 week, or for a period of at least 1 month.

[0008] In some aspects, the present disclosure provides an ophthalmic composition comprising a therapeutically effective amount of a functionally active α - amylase for treating blepharitis or dry eye conditions, a certain concentration of CaCl for stabilizing the α - amylase 2 and one or more pharmaceutically acceptable excipients.

[0009] In some aspects, the present disclosure provides compositions and methods, the compositions comprising α - amylase. In some aspects, the present disclosure provides a method for treating one or more of blepharitis and dry eye disease, the method comprising administering to a subject a therapeutically effective amount of a composition comprising a stabilized active α - amylase or a functional fragment thereof and one or more pharmaceutically acceptable excipients stabilized with a certain concentration of CaCl 2 In some formulations, the composition is in a wipe, such as a pre - moistened wipe or a wipe containing dry ingredients. In many cases, the composition is applied to the eyelids or used as a facial wipe. In some cases, administration promotes the removal of biofilm in or around the eyes of a subject. The composition can be administered daily, for example, for a period long enough to provide contact with the biofilm, at least 1 second, at least 5 seconds, at least 10 seconds. The composition can be administered daily for a period of at least 1 week. In some aspects, the α - amylase shares at least 90% sequence homology, at least 95% sequence homology, or at least 99% sequence homology with an α - amylase peptide sequence from Aspergillus oryzae. In some aspects, the composition comprises from 1 mg / mL (w / v) to 20 mg / mL (w / v) of active α - amylase, from 1 mg / mL (w / v) to 10 mg / mL (w / v) of active α - amylase, or another suitable amount. In some aspects, the therapeutically effective amount comprises one or more drops of a formulation having an active α - amylase between 1 I.U. / mg and 3000 I.U. / mg. In some aspects, the concentration of CaCl 2 is in the range from 0.2% (w / v) to 0.8% (w / v). In some aspects, the composition further comprises a citrate buffer.

[0010] In some aspects, the present disclosure provides an ophthalmic composition that comprises a therapeutically effective amount of a functional active α-amylase for treating blepharitis or a dry eye condition, a certain concentration of CaCl for stabilizing the α-amylase 2 and one or more pharmaceutically acceptable excipients. The α-amylase in the composition may share at least 90%, at least 95% or at least 99% sequence homology with the α-amylase peptide sequence from Aspergillus oryzae. The composition may comprise from 1 mg / ml (w / v) of functional active α-amylase to 20 mg / ml (w / v) of functional active α-amylase. The composition may comprise active α-amylase between 1 I.U. / mg and 3000 I.U. / mg. In some aspects, the concentration of CaCl 2 is in the range from 0.2% (w / v) to 0.8% (w / v). The composition may further comprise one or more of an ophthalmic astringent, an ophthalmic demulcent, an ophthalmic emollient, an ophthalmic hypertonicity agent or an ophthalmic vasoconstrictor. In some formulations, the demulcent may be sodium carboxymethylcellulose (CMC), such as from 0.2% to 3.5% CMC. In some formulations, the demulcent may be polyvinyl alcohol (PVA), such as 0.1% to 5% PVA. In some aspects, the composition further comprises a citrate buffer.

[0011] In some formulations, the composition further comprises hydroxyethyl cellulose between 0.2% (v / v) and 3.5% (v / v), hypromellose between 0.2% (v / v) and 3.5% (v / v), methylcellulose between 0.2% (v / v) and 3.5% (v / v), dextran between 0.01% (v / v) and 1.0% (v / v), gelatin between 0.001% (v / v) and 0.1% (v / v), glycerol between 0.01% (v / v) and 1.5% (v / v), polyethylene glycol 300 between 0.05% (v / v) and 1.5% (v / v), polyethylene glycol 400 between 0.05% (v / v) and 1.5% (v / v), polysorbate between 0.2% (v / v) and 1.0% (v / v), polypropylene glycol between 0.2% (v / v) and 2.0% (v / v), polyvinyl alcohol between 0.2% (v / v) and 6.0% (v / v), povidone between 0.1% (v / v) and 4.0% (v / v), benzalkonium chloride (BAK) between 0.001% (v / v) and 0.1% (v / v) and / or polyquad (polyquaternium-1) between 0.0001% (v / v) and 0.01% (v / v). In some formulations, the composition further comprises stabilized oxychlorocomplex, sodium perborate, disodium edetate and sorbic acid, borate, sorbitol, propylene glycol and zinc ion buffer, polyhexanide (polyhexamethylene biguanide). In a preferred case, the composition can be stabilized in a citrate buffer.

[0012] The composition can be an ophthalmic composition, i.e., a composition in which the administration is ophthalmic administration. In some aspects, the administration is topical administration to the external eye. In some aspects, the composition is formulated as a liquid. In other aspects, the composition is formulated as a gel. However, in certain preferred cases, the composition is formulated in a dry powder form or in a wet wipe.

[0013] In some aspects, the present disclosure provides a composition comprising functional active α-amylase at a concentration in the range from 1 mg / mL to 20 mg / mL, CaCl 2 at a concentration in the range from 0.4% (w / v) to 0.8% (w / v) for stabilizing α-amylase, polyvinyl alcohol (PVA) at a concentration in the range from 0.1% to 5%, and at least one pharmaceutically acceptable excipient. In some aspects, the concentration of functional active α-amylase is 10 mg / mL, and / or the concentration of polyvinyl alcohol (PVA) is 4%, and / or the concentration of CaCl 2 is 0.4%.

[0014] In some aspects, the present disclosure provides a topical applicator, such as a wipe, that includes functional active α-amylase at a concentration in the range from 1 mg / mL to 20 mg / mL, CaCl at a concentration in the range from 0.4% (w / v) to 0.8% (w / v) for stabilizing the α-amylase, polyvinyl alcohol (PVA) at a concentration in the range from 0.1% to 5%, and at least one pharmaceutically acceptable excipient. In some formulations, the topical applicator, such as a wipe, includes functional active α-amylase at a concentration of about 10 mg / mL, CaCl at a concentration of about 0.4% (w / v), and polyvinyl alcohol (PVA) at a concentration of about 4% (w / v). In some aspects, the wipe is individually wrapped. 2 and polyvinyl alcohol (PVA) at a concentration in the range from 0.1% to 5%. In some formulations, the topical applicator, such as a wipe, includes functional active α-amylase at a concentration of about 10 mg / mL, CaCl 2 and polyvinyl alcohol (PVA) at a concentration of about 4% (w / v). In some aspects, the wipe is individually wrapped.

[0015] These aspects and other features and advantages of the invention are described in more detail below. Brief Description of the Drawings

[0017] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0018] The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings, in which:

[0019] Figure 1A and Figure 1B ( Figure 1A and Figure 1B ) are photographs depicting two different regions of the eye of a subject suffering from meibomian gland obstruction, a shiny biofilm at the base of the eyelashes, a shiny biofilm on the eyelashes, and misdirected eyelash growth. After the photographs were taken, the subject was treated with Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation.

[0020] Figure 2A and Figure 2B ( Figure 2A and Figure 2B ) are photographs depicting two different regions of the eye of the subject approximately 6 months after the photographs of Figure 1A and Figure 1B were taken. After the photographs were taken, the subject received 1 treatment with Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation. Subsequently, the subject was prescribed Avenova (0.01% hypochlorous acid (HOCl)) for daily use.

[0021] Figure 3A and Figure 3B ( Figure 3Aand Figure 3B ) are photographs of two different areas of the subject's eyes taken approximately 6 months after the photographs of Figure 2A and Figure 2B . After the photographs were taken, the subject received 1 treatment of Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation. Subsequently, the subject was prescribed daily use of Avenova (0.01% hypochlorous acid (HOCl)).

[0022] Figure 4A and Figure 4B ( Figure 4A and Figure 4B ) are photographs of two different areas of the subject's eyes taken approximately 4 months after the photographs of Figure 3A and Figure 3B . After the photographs were taken, the subject received 1 treatment of Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation. Subsequently, the subject continued daily treatment with Avenova (0.01% hypochlorous acid (HOCl)).

[0023] Figure 5A and Figure 5B ( Figure 5A and Figure 5B ) are photographs of two different areas of the subject's eyes taken approximately 1 month after the photographs of Figure 4A and Figure 4B and after the subject received several applications of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid.

[0024] Figure 6A and Figure 6B ( Figure 6A and Figure 6B ) are photographs of two different areas of the subject's eyes taken approximately 6 months after the photographs of Figure 5A and Figure 5B . After the photographs of Figure 5A and Figure 5B were taken, the subject received regular daily use of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. As Figure 6A and Figure 6B show, the biofilm was significantly eliminated, there was no biofilm on the eyelashes, the inflammation and redness in the eyelids were reduced, and the patient reported a significant improvement in dry eye symptoms.

[0025] Figure 7A and Figure 7B ( Figure 7A and Figure 7B ) are photographs of two different areas of the subject's eyes taken approximately 6 months after the photographs of Figure 5A and Figure 5BPhotographs of two different regions of the eyes of a subject approximately 6 months after the photograph and over a time course of approximately 6 months after daily routine use of a composition comprising 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid.

[0026] Figure 8A and Figure 8B ( Figure 8A and Figure 8B ) are photographs depicting two different regions of the eyes of a subject approximately 4 months after the photograph of Figure 6A and Figure 6B and after daily routine use of a composition comprising 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid.

[0027] Figure 9A and Figure 9B ( Figure 9A and Figure 9B ) are photographs depicting regions of the eyes of a subject after receiving a 10 mg / ml dose of α-amylase in 0.01% hypochlorous acid.

[0028] Figure 10A and Figure 10B ( Figure 10A and Figure 10B ) are photographs depicting a direct comparison of regions of the eyes before and after treatment with a composition comprising 10 mg / ml of α-amylase in 0.01% hypochlorous acid and Ca 2+ .

[0029] Figure 11A ( Figure 11A ) is a graph illustrating the results of testing various α-amylase compositions containing a calcium cofactor (CaCl 2 ).

[0030] Figure 11B ( Figure 11B ) is a graph quantifying the percentage of biofilm removed within 10 min by various compositions containing a range of CaCl 2 .

[0031] Figure 12A ( Figure 12A ) is a graph of the analytical results quantifying the effect of various preservatives, namely BAK (benzalkonium chloride), 0.1% oxy-chloride, 0.001% polyquad (polyquaternium-1), and bleach control, on enzyme stability. The α-amylase solution was 10 mg / mL of ∼30 U / mg α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0032] Figure 12B ( Figure 12B ) is a graph showing the analysis results of the effects of multiple preservatives, namely BAK (benzalkonium chloride) and GenAqua / Dequest (sodium perborate), on enzyme stability. The α-amylase solution is 10 mg / mL of ~30 U / mg α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0033] Figure 13 ( Figure 13 ) is a graph showing the analysis results of the effect of 0.01% hypochlorous acid preservative on enzyme activity.

[0034] Figure 14A ( Figure 14A ) is a graph showing the analysis results of multiple moderators, including 1% polyethylene glycol 400 (PEG 400), 2.5% sodium carboxymethylcellulose (CMC), 1% glycerol, 2% polyvinylpyrrolidone (PVP), 1% propylene glycol, 4% polyvinyl alcohol (PVA), 0.1% dextran 70 + 0.3% hypromellose. The α-amylase solution is 10 mg / mL of ~30 U / mg α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0035] Figure 14B ( Figure 14B ) is a graph showing the analysis results of multiple moderators, including polyvinyl alcohol (PVA), PVA + α-amylase, sodium carboxymethylcellulose (CMC), and sodium carboxymethylcellulose (CMC) + α-amylase. The α-amylase solution is 10 mg / mL of ~30 U / mg α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0036] Figure 14C ( Figure 14C ) is a graph showing the results of testing α-amylase stabilized in 4% PVA after 36 days. The α-amylase solution is 10 mg / mL of ~30 U / mg α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0037] Figure 15 (Figure 15 ) is a chart showing the results of a comparison of ophthalmic antibiotics for Staphylococcus aureus biofilm reduction. The α - amylase solution is 10 mg / mL of ~30 U / mg α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0038] Figure 16 ( Figure 16 ) is a chart showing the results of various heat - stable sugars and moderators (including combinations of trehalose and sucrose with moderators) on biofilm reduction. The α - amylase solution is 10 mg / mL of ~30 U / mg α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0039] Figure 17 ( Figure 17 ) is a chart showing the results tested with various concentrations of trehalose (including 3%, 6.8% and 8%). The α - amylase solution is 10 mg / mL of ~30 U / mg α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0040] Figure 18A ( Figure 18A ) is a chart showing the results of testing 50 mM citrate buffer in the presence of sugar stabilizers and moderators. The α - amylase solution is 10 mg / mL of ~30 U / mg α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0041] Figure 18B ( Figure 18B ) is a chart showing the results of testing 100 mM citrate buffer in the presence of sugar stabilizers and moderators. The α - amylase solution is 10 mg / mL of ~30 U / mg α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0042] Figure 19 ( Figure 19) is a chart showing the results of testing the effects of hyaluronic acid and tea tree oil on a composition containing α-amylase. The α-amylase solution is 10 mg / mL to 30 U / mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number #10065) supplemented with 0.4% CaCl 2 and 0.56% NaCl.

[0043] It should be understood that the figures and pictures are not necessarily to scale.

[0044] Incorporated by reference

[0045] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference.

[0046] Definitions

[0047] The terms “protein,” “polypeptide,” and “peptide,” which are used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The term also includes polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any portion of a protein or polypeptide that has a specific function or structure, e.g., the α-amylase or lysozyme catalytic domain refers to the domain that is capable of breaking peptide bonds.

[0048] A protein is said to have an “N-terminus” and a “C-terminus.” The term “N-terminus” refers to the start of a protein or polypeptide terminated by an amino acid having a free amine group (-NH 2 ). The term “C-terminus” refers to the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH).

[0049] As used herein, the term “biofilm” refers to an assemblage of microbial cells associated with the surface of the eyelid and encapsulated in a matrix typically made of a polysaccharide material. The lower layer of the biofilm contains microorganisms bound together with other organic components such as DNA, protein, and inorganic materials in the polysaccharide matrix. The upper layer is a more loosely amorphous layer that extends into the surrounding medium. The fluid layer adjacent to the biofilm typically has a stationary sublayer and a dynamic sublayer.

[0050] The term "conservative amino acid substitution" refers to the replacement of an amino acid that is normally present in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitutions include the replacement of a nonpolar (hydrophobic) residue such as isoleucine, valine, or leucine with another nonpolar residue. Similarly, examples of conservative substitutions include the replacement of one polar (hydrophilic) residue with another polar residue, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the replacement of a basic residue such as lysine, arginine, or histidine with another basic residue, or the replacement of an acidic residue such as aspartic acid or glutamic acid with another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the replacement of a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine and / or the replacement of a polar residue with a nonpolar residue. Typical amino acid classifications are summarized below.

[0051]

[0052] In the context of two polynucleotide or polypeptide sequences, "sequence identity" or "identity" refers to the residues that are the same in both sequences when aligned for maximum correspondence over a specified comparison window. When using the percent sequence identity with respect to a protein, the positions of non-identical residues are often different due to conservative amino acid substitutions, where an amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and thus the functional properties of the molecule are not changed. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making such an adjustment are well known. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percent sequence identity. Thus, for example, in a situation where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1. The scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0053] "Percent sequence identity" includes the value determined by comparing two optimally aligned sequences (the maximum number of residue matches) over a comparison window, where the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences. The percent is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Unless otherwise stated (e.g., the shorter sequence includes linked heterologous sequences), the comparison window is the full length of the shorter of the two sequences being compared.

[0054] Unless otherwise stated, sequence identity / similarity values include those obtained using GAP Version 10 with the following parameters: % identity and % similarity for nucleotide sequences using a gap weight of 50 and a length weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a gap weight of 8 and a length weight of 2 and the BLOSUM62 scoring matrix; or any equivalent program. "Equivalent program" includes any sequence comparison program that, for any two sequences being discussed, generates an alignment having the same nucleotide or amino acid residue matches and the same percent sequence identity as the corresponding alignment generated by GAP Version 10.

[0055] The term "in vitro" includes an artificial environment and processes or reactions that occur within an artificial environment (e.g., a test tube). The term "in vivo" includes a natural environment (e.g., a cell or an organism or a body) and processes or reactions that occur within a natural environment. The term "ex vivo" includes cells that have been removed from an individual's body and processes or reactions that occur within such cells.

[0056] A composition or method "comprising" or "including" one or more of the recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" a protein may contain the protein alone or in combination with other ingredients. The transitional phrase "consisting essentially of" means that the scope of a claim is to be interpreted to cover the specified elements recited in the claim and those elements that do not materially affect the basic and novel characteristics of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising".

[0057] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and means that the description includes both the case where the described event or circumstance occurs and the case where the described event or circumstance does not occur.

[0058] The specification of a value range includes all integers within or defining the range, as well as all sub-ranges defined by the integers within the range.

[0059] Unless otherwise apparent from the context, the term "about" encompasses values within the standard measurement error range (e.g., SEM) of the stated value.

[0060] The term "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

[0061] The term "or" means any one member of a particular list and also includes any combination of the members of that list.

[0062] Unless the context clearly indicates otherwise, the singular forms of the articles "a", "an", and "the" include plural referents. For example, the term "protein" or "at least one protein" may include more than one protein, including mixtures thereof.

[0063] Statistical significance means p ≤ 0.05.

[0064] Detailed Description

[0065] Overview

[0066] All functional intents described in connection with one embodiment of the methods, compositions or formulations described herein apply to additional embodiments of the methods, compositions or formulations described herein, unless in explicitly stated cases or where the feature or function is incompatible with the additional embodiment. For example, where a given feature or function of a component is explicitly described in connection with one embodiment but not explicitly mentioned in connection with an alternative embodiment, it should be understood that the feature or component can be deployed, utilized or implemented in connection with the alternative embodiment, unless the feature or component is incompatible with the alternative embodiment.

[0067] One of the main causes of dry eye disease is thought to be inflammation in the tissues that produce and support tear production. Blepharitis describes inflammation of the eyelid margins. This is usually an early sign of dry eye disease and is commonly observed in people with dry eye disease. When patients seek medical care for blepharitis, it is usually because they are starting to experience dry eye symptoms. Current treatments for dry eye disease include artificial tears, immunosuppressive drugs (Restasis), temporary use of antibiotics, temporary use of steroids, lymphocyte function-associated antigen-1 (LFA-1) antagonists (Xiidra), warm compresses, intense pulsed light treatment, or mechanically expressing meibum from the meibomian glands.

[0068] An important driver of blepharitis is thought to be bacterial colonization of the meibomian glands of the eyelids. People with dry eye disease have increased levels of bacteria and it is common to have biofilms present especially on the eyelids, eyes, and surrounding tissues. The eyelid margins are ideal for biofilm formation as they provide moisture, oil, and nutrients for bacterial survival. Analysis of biofilms from the eyelids of blepharitis patients indicates that the Staphylococcus genus is the most prevalent bacteria. Further analysis shows that 46% to 51% of patients with staphylococcal blepharitis test positive for Staphylococcus aureus. Staphylococcus aureus is considered to be a more pathogenic species than other Staphylococcus genus bacteria found on the eyelids. When these bacteria grow and multiply on the eyelids, they form biofilms.

[0069] Bacterial biofilms encapsulate bacteria in a polysaccharide matrix, which can make the biofilm more resistant to antibiotics and white blood cells. Bacteria in biofilms can also exchange genetic information to better survive in their environment. As the biofilm progresses, the bacteria start to release toxins to break down eyelid tissue for nutrients. This leads to an inflammatory response from the body. Since the bacteria are encapsulated in the biofilm, the body's immune response can hardly stop the bacteria's attack on the eyelid tissue. This leads to inflamed tissues that interfere with the production of, for example, tear components. Another complication occurs when Staphylococcus aureus and Staphylococcus epidermidis from the biofilm release lipases into the meibomian glands. This causes the clear oil in the meibomian glands to turn into a cloudy, sludge-like oil that clogs the meibomian glands, leading to meibomian gland dysfunction (MGD) and poor-quality tears. Current treatments for biofilms on the eyelid margins include mechanical scrubbing by an ophthalmologist with specialized tools and home eyelid cleansers and scrubs. The only FDA-approved method for removing biofilms from the eyelid margins is the use of Blephex, a mechanical device designed to scrub off biofilms, similar to removing plaque from teeth at the dentist.

[0070] Certain α-amylase strains have shown some in vitro effectiveness in inhibiting and reducing Staphylococcus aureus biofilms, including α-amylase from Aspergillus oryzae (Sigma-Aldrich, St. Louis, MO, catalog #10065), α-amylase from Bacillus subtilis (Sigma, catalog #10070), α-amylase from human saliva (Sigma, catalog #A1031), and β-amylase from sweet potato. Notably, existing data indicate that most tested human α-amylases are effective in preventing biofilm growth but not in breaking down existing biofilms.

[0071] The present invention relates to a composition and a method for using the composition to enzymatically break down biofilms in the eyelids by using the enzyme amylase or by combining the use of the enzyme amylase and lysozyme. The relevant properties of the enzyme amylase are described above, and lysozyme is a natural antibiotic. Although amylase and lysozyme can naturally occur in the tears of healthy individuals, the present disclosure hypothesizes that individuals suffering from dry eye disease produce insufficient amounts of these enzymes to effectively prevent the formation of biofilms that lead to the inflammatory cycle. In addition, the present disclosure shows that the use of these enzymes in a suitable formulation for ocular administration significantly reduces biofilm formation and bacterial accumulation on the eyelids.

[0072] II. Ophthalmic formulations containing enzymes

[0073] The present disclosure contemplates enzyme classes that can remove one or more layers of biofilm from a subject's eye, thereby treating conditions caused or exacerbated by the presence of the biofilm. In some aspects, the present disclosure contemplates enzyme classes that can remove biofilms by, for example, enzymatic degradation of polysaccharides resulting in biofilm removal. In some aspects, the present disclosure contemplates enzyme classes that can lyse microbial cells by degrading cell membrane components and destabilizing their anchoring to solid surfaces. In a preferred aspect, the enzymes contemplated by the present disclosure are α-amylase and / or a combination of α-amylase and lysozyme.

[0074] Amylases are a group of important enzymes, which are classified into α-subtype, β-subtype, γ-subtype, isoamylase, glucoamylase, and others. Amylases can be found in both plant sources and microbial sources. Based on the mode of action, amylases can be classified into exoamylases and endoamylases. Exoamylases hydrolyze the substrate from the non-reducing end, producing shorter end products, while endoamylases act randomly on the internal glycosidic bonds within the starch molecule, producing oligosaccharides of different lengths. α-Amylase and β-amylase have the potential to catalyze the hydrolysis of chitosan and reduce its molecular weight, which makes chitosan more soluble and suitable for formulation in liquid ophthalmic compositions. Multiple amylases present in, for example, Legionella pneumophila are necessary for hydrolyzing polysaccharides into glucose and for assisting intracellular proliferation. Amylases also help trigger a pro-inflammatory response, which further helps prevent bacterial replication.

[0075] α-Amylase, which acts mainly on starch (polysaccharide) as the main substrate, consists of two glucose polymers - amylose and amylopectin. α-Amylase helps with the hydrolysis of α-1,4-glycosidic bonds and α-1,6-glycosidic bonds, which results in the formation of small glucose (monosaccharide) and maltose (disaccharide). To function, α-amylase is essentially a metalloenzyme, which usually requires a metal such as Ca 2+ , for maintaining the stability of the enzyme molecule. Sequence alignment studies have found that α-amylase has four conserved regions that are also present within the β-strand. α-Amylase can be widely present in plants, microorganisms, and higher animals. The metabolites obtained through the enzymatic action of α-amylase are oligosaccharides of various lengths, including branched maltooligosaccharides with 6-8 glucose units having -1,6 bonds and -1,4 bonds, maltose, and maltotriose. These amylases can bind to the substrate via catalytic groups that catalyze the cleavage of glycosidic bonds.

[0076] This disclosure anticipates compositions containing a suitable amount of a metal (preferably Ca 2+)A composition of a composite α - amylase for maintaining the stability of the enzyme. The amylase of the present disclosure can be prepared, for example, by recombinant techniques, and it can have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35% or at least 30% homology with the α - amylase peptide sequence from Aspergillus oryzae. The α - amylase of the present disclosure can be prepared by expressing a suitable nucleic acid molecule from a recombinant organism, and the nucleic acid molecule can have at least 99.99%, at least 99.9%, at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35% or at least 30% homology with the nucleic acid sequence encoding the α - amylase peptide sequence from Aspergillus oryzae. The suitable nucleic acid sequence for preparing the α - amylase of the present invention can be a degenerate sequence. The percentage of homology between sequences can be calculated using more than one algorithm, including the algorithms described in the definitions.

[0077] Table 1 lists non - limiting examples of the amylases contemplated by the present disclosure and a preliminary assessment of their activity against biofilms:

[0078]

[0079]

[0080] The enzymes of the present disclosure (such as α - amylase) can be purchased from commercial sources and combined with other components to produce the ophthalmic compositions described herein. Alternatively, the enzymes of the present disclosure can be prepared, for example, by peptide synthesis or the expression of a suitable nucleic acid molecule. Non - limiting examples of peptide sequencing methods include: a) liquid - phase peptide synthesis; b) solid - phase peptide synthesis using a combination of polystyrene resin, polyamide resin, PEG - hybrid polystyrene resin, PEG - based resin, and / or any solid - phase support; and c) synthetic biology. Non - limiting examples of methods for expressing a suitable nucleic acid molecule include molecular cloning and recombinant DNA techniques.

[0081] III. Ophthalmic Compositions

[0082] The ophthalmic compositions of the present disclosure can be a combination of one or more of the enzymes described herein with other chemical components, such as metal components (e.g., Ca 2+and α - amylase), a carrier, a stabilizer, a diluent, a dispersant, a suspending agent, a thickening agent, and / or an excipient. The ophthalmic composition facilitates the administration of α - amylase in a functional and active form to the eye of a subject. The ophthalmic composition can be administered in a therapeutically effective amount, for example, as eye drops (e.g., a solution, gel, or cream directly applied to the eyelid) via an optical or topical route, via an eye wash with a solution, gel, or cream, or via a wipe (e.g., a wet wipe) containing a dose of the enzyme.

[0083] Active compounds, including, for example, α - amylase, can be formulated into a variety of liquid and topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments. Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0084] Active compounds, including, for example, α - amylase, can be formulated into wipes, such as single - use, individually wrapped wipes or packages containing more than one wipe.

[0085] In practicing the methods of treatment or use provided herein, a therapeutically effective amount of the enzymes described herein is administered in an ophthalmic composition to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal, such as a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. In a preferred case, the disease or condition is blepharitis, dry eye, or another related inflammatory condition.

[0086] The enzymes described herein, such as α - amylase, can be present in liquid compositions in the range from about 0.1 mg / mL to about 2000 mg / mL; from about 0.1 mg / mL to about 1000 mg / mL; from about 0.1 mg / mL to about 500 mg / mL; from about 0.1 mg / mL to about 100 mg / mL; from about 0.1 mg / mL to about 50 mg / mL; from about 0.1 mg / mL to about 25 mg / mL; from about 0.1 mg / mL to about 10 mg / mL; from about 1 mg / mL to about 2000 mg / mL; from about 1 mg / mL to about 1000 mg / mL; from about 1 mg / mL to about 500 mg / mL; from about 1 mg / mL to about 100 mg / mL; from about 1 mg / mL to about 50 mg / mL; from about 1 mg / mL to about 25 mg / mL; from about 1 mg / mL to about 10 mg / mL.

[0087] The enzymes described herein can be present in a liquid composition, such as alpha-amylase, in a range from about 5 mg / mL to about 1000 mg / mL, from about 5 mg / mL to about 500 mg / mL, from about 5 mg / mL to about 100 mg / mL, from about 5 mg / mL to about 50 mg / mL, from about 5 mg / mL to about 25 mg / mL, or from about 5 mg / mL to about 20 mg / mL. The foregoing ranges are merely illustrative. The dosage can vary depending on a number of variables, including, for example, the activity of the enzyme being used, the disease or condition being treated, the mode of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0088] Each enzyme described herein, such as alpha-amylase, can be present in a single-use vial or wipe in an amount of about 0.1 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, for example, in powder form or in solution form.

[0089] Each enzyme described herein, such as α-amylase, can be present, for example, in a single-use tube or swab in powder form or in solution form in an amount providing the following activities: greater than 15.0 U / mg, greater than 16.0 U / mg, greater than 17 U / mg, greater than 18 U / mg, greater than 19 U / mg, greater than 20 U / mg, greater than 21 U / mg, greater than 22 U / mg, greater than 23 U / mg, greater than 24 U / mg, greater than 25 U / mg, greater than 26 U / mg, greater than 27 U / mg, greater than 28 U / mg, greater than 29 U / mg, greater than 30 U / mg, greater than 31 U / mg, greater than 32 U / mg, greater than 33 U / mg, greater than 34 U / mg, greater than 35 U / mg, greater than 36 U / mg, greater than 37 U / mg, greater than 38 U / mg, greater than 39 U / mg, 40 U / mg, greater than 41 U / mg, greater than 42 U / mg, greater than 43 U / mg, greater than 44 U / mg, greater than 45 U / mg, greater than 46 U / mg, greater than 47 U / mg, greater than 48 U / mg, greater than 49 U / mg, greater than 50 U / mg, greater than 51 U / mg, greater than 52 U / mg, greater than 53 U / mg, greater than 54 U / mg, greater than 55 U / mg, greater than 56 U / mg, greater than 57 U / mg, greater than 58 U / mg, greater than 59 U / mg, greater than 60 U / mg, greater than 61 U / mg, greater than 62 U / mg, greater than 63 U / mg, greater than 64 U / mg, greater than 65 U / mg, greater than 66 U / mg, greater than 67 U / mg, greater than 68 U / mg, greater than 69 U / mg, greater than 70 U / mg, greater than 71 U / mg, greater than 72 U / mg, greater than 73 U / mg, greater than 74 U / mg, greater than 75 U / mg, greater than 76 U / mg, greater than 77 U / mg, greater than 78 U / mg, greater than 79 U / mg, greater than 80 U / mg, greater than 81 U / mg, greater than 82 U / mg, greater than 83 U / mg, greater than 84 U / mg, greater than 85 U / mg, greater than 86 U / mg, greater than 87 U / mg, greater than 88 U / mg, greater than 89 U / mg, greater than 90 U / mg, greater than 91 U / mg, greater than 92 U / mg, greater than 93 U / mg, greater than 94 U / mg, greater than 95 U / mg, greater than 96 U / mg, greater than 97 U / mg, greater than 98 U / mg, greater than 99 U / mg or greater than 100 U / mg, where the activity is defined as the amount of one unit that will release 1 mg of maltose from starch in 3 minutes at pH 6.9 and 20°C.

[0090] Each enzyme described herein, such as α - amylase, can be present in a single - use tube or a disposable wipe in powder form or in solution form in an amount providing an activity between 15.0 U / mg and 40 U / mg, between 15.0 U / mg and 50 U / mg, between 15.0 U / mg and 60 U / mg, between 15.0 U / mg and 70 U / mg, between 15.0 U / mg and 80 U / mg, between 15.0 U / mg and 90 U / mg, between 15.0 U / mg and 100 U / mg, between 15.0 U / mg and 110 U / mg, between 15.0 U / mg and 120 U / mg, between 15.0 U / mg and 130 U / mg, between 15.0 U / mg and 140 U / mg, between 15.0 U / mg and 150 U / mg, between 15.0 U / mg and 160 U / mg, between 15.0 U / mg and 170 U / mg, between 15.0 U / mg and 180 U / mg, between 15.0 U / mg and 190 U / mg or between 15.0 U / mg and 200 U / mg, where the activity is defined as one unit releasing 1 mg of maltose from starch in 3 minutes at pH 6.9 and 20 °C.

[0091] The co - factors described herein, such as CaCl 2 , can be present at a concentration in the range from 0.01% (w / v) to 1.5% (w / v), 0.1% (w / v) to 1.5% (w / v), 0.2% (w / v) to 1.5% (w / v), 0.3% (w / v) to 1.5% (w / v), 0.4% (w / v) to 1.5% (w / v), 0.5% (w / v) to 1.5% (w / v), 0.6% (w / v) to 1.5% (w / v), 0.01% (w / v) to 1.0% (w / v), 0.1% (w / v) to 1.0% (w / v), 0.2% (w / v) to 1.0% (w / v), 0.3% (w / v) to 1.0% (w / v), 0.4% (w / v) to 1.0% (w / v), 0.2% (w / v) to 0.8% (w / v), 0.3% (w / v) to 0.8% (w / v), 0.4% (w / v) to 0.8% (w / v) or in another suitable range.

[0092] Ophthalmic compositions, such as wipes, drops, or another suitable composition, can be formulated using one or more physiologically acceptable carriers comprising excipients and adjuvants, which facilitate the processing of the active compound into a pharmaceutically useful article. The formulation can be modified depending on the chosen route of administration. Ophthalmic compositions comprising the compounds described herein can be manufactured in a conventional manner, such as by means of conventional mixing, dissolving, granulating, or emulsifying. The ophthalmic composition can include at least one pharmaceutically acceptable carrier, diluent, or excipient and the compounds described herein or in pharmaceutically acceptable salt form.

[0093] Methods for preparing compositions comprising the compounds described herein can include formulating the compound (e.g., α - amylase at a suitable concentration) with one or more inert, pharmaceutically acceptable excipients. Liquid compositions include, for example, solutions in which the compound is dissolved, emulsions comprising the compound, or solutions comprising liposomes, micelles, or nanoparticles containing the compounds as disclosed herein. Semi - solid compositions include, for example, gels, suspensions, and creams. The composition can be in the form of a liquid solution or suspension, a solid form suitable for dissolving or suspending in a liquid prior to use, or as an emulsion. The composition can be formulated in a wipe. These compositions can also contain small amounts of non - toxic auxiliary substances, such as wetting agents or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives. Non - limiting examples of dosage forms suitable for the present disclosure include liquids, elixirs, nanosuspensions, aqueous or oily suspensions, drops, syrups, and any combination thereof.

[0094] Non - limiting examples of pharmaceutically acceptable excipients suitable for the present disclosure include astringents, demulcents, emollients, granulating agents, binders, lubricants, disintegrants, sweeteners, glidants, anti - sticking agents, antistatic agents, surfactants, antioxidants, gums, coating agents, colorants, flavoring agents, plasticizers, preservatives, sugars, suspending agents, emulsifying agents, plant cellulose materials, and spheronization agents and any combination thereof.

[0095] In some cases, ophthalmic compositions of the present disclosure comprising one or more of α - amylase, lysozyme, and / or dextranase contain an astringent. A suitable astringent is zinc sulfate at the approximate concentrations disclosed below:

[0096] Table 2 Ophthalmic astringent Zinc sulfate, 0.25%

[0097] Ophthalmic emollient

[0098] The present disclosure demonstrates that α - amylase is effective in inhibiting and reducing Staphylococcus aureus biofilms in vivo (see Figures 6A - 9B)。The Staphylococcus aureus biofilm on the eyelids is considered a major cause of dry eye disease, and α-amylase has not previously been used to remove biofilms from the eyelids. The present disclosure anticipates ophthalmic preparations with certain ophthalmic emollients for removing biofilms from the eye, eyelids, and surrounding tissues to treat blepharitis and dry eye conditions.

[0099] Ophthalmic emollients can be used in eye drops to both thicken the drops, lubricate the epithelium, and relieve dryness and irritation. The ophthalmic compositions of the present disclosure can include α-amylase and one or more of the ophthalmic emollients of Table 3.

[0100] Table 3 Ophthalmic emollient 0.2% to 2.5% sodium carboxymethylcellulose 0.2% to 2.5% hydroxyethylcellulose 0.2% to 2.5% hypromellose 0.2% to 2.5% methylcellulose 0.1% dextran 70 when used with another polymeric emollient in this section 0.01% gelatin 0.2% to 1% glycerol 0.2% to 1% polyethylene glycol 300 0.2% to 1% polyethylene glycol 400 0.2% to 1% polysorbate 80 0.2% to 1% propylene glycol 0.1% to 4% polyvinyl alcohol 0.1% to 2% polyvinylpyrrolidone

[0101] Ophthalmic emollient

[0102] Ophthalmic emollients generally refer to components that can form a film on the eye or in the skin. Generally, ophthalmic emollients can relieve dryness, itching, and flaking, and potentially help maintain a moist layer of α-amylase on the surface of the skin or eye. The ophthalmic compositions of the present disclosure can include α-amylase and one or more of the ophthalmic emollients of Table 4.

[0103]

[0104]

[0105] Ophthalmic hyperosmotic agent

[0106] Ophthalmic hyperosmotic agents are used to reduce swelling of the cornea (the front surface of your eye) caused by surgery, infection, trauma, or other eye conditions. The ophthalmic compositions of the present disclosure can include α-amylase and one or more of the ophthalmic hyperosmotic agents of Table 5.

[0107] Table 5 Ophthalmic hyperosmotic agent 2% to 5% sodium chloride

[0108] Ophthalmic vasoconstrictor

[0109] The use of vasoconstrictors can help reduce inflammation on the eyelids caused by biofilms. The ophthalmic compositions of the present disclosure can include α-amylase and one or more of the ophthalmic vasoconstrictors of Table 6, which include: ephedrine hydrochloride, naphazoline hydrochloride, phenylephrine hydrochloride, tetrahydrozoline hydrochloride, benzalkonium chloride, oxymetazoline, brimonidine tartrate.

[0110] Table 6 Ophthalmic vasoconstrictor 0.123% ephedrine hydrochloride 0.01% to 0.03% naphazoline hydrochloride 0.08% to 0.2% phenylephrine hydrochloride 0.01% to 0.05% tetrahydrozoline hydrochloride 0.025% brimonidine tartrate.

[0111] The ophthalmic compositions of the present disclosure can include α-amylase and one or more additional components of Table 7:

[0112]

[0113]

[0114] The ophthalmic composition of the present disclosure may comprise α - amylase and one or more additional components of Table 8:

[0115]

[0116] Buffer system

[0117] A buffer system is used to obtain the pH of artificial tears for eye health and comfort. A pH of ∼7 is the most comfortable for dry - eye patients (normal tear pH is about 6.5 - 7.6). Although purified human pancreatic α - amylase (α - 1,4 - glucan - 4 - glucanohydrolase, EC 3.2.1.1) is stable over a wide range of pH values (5.0 to 10.5), the buffer system can also help maintain the optimal activity of α - amylase in the ophthalmic composition of the present disclosure. The present disclosure anticipates that the optimal pH for the enzymatic activity of α - amylase is 5.5, and it anticipates using one or more of the following components to achieve a pH between 5 - 7.5 for certain compositions.

[0118]

[0119]

[0120] Electrolytes (e.g., metals)

[0121] Electrolytes can be added to the ophthalmic composition to maintain or reduce the tear osmotic pressure, since hyperosmotic products draw water from epithelial cells and interfere with metabolism. Some of the added electrolytes are also important for corneal epithelial metabolism. Some electrolytes are part of the buffer system described above. In the ophthalmic composition of the present disclosure, some electrolytes may be required as metal stabilizers for one or more enzymes.

[0122]

[0123] The ophthalmic composition of the present disclosure may comprise one or more of α - amylase, lysozyme, and / or glucanase and one or more additional preservatives of Table 11:

[0124]

[0125]

[0126] IV. Ophthalmic Preparations

[0127] The ophthalmic compositions described herein can be formulated into eye drop containers (5 mL, 10 mL, 15 mL, 20 mL or another suitable volume) or into unit dosage forms suitable for precise single-dose administration. When formulated as eye drops, the preparation can be such that each "drop" contains a suitable amount of an enzyme with a certain activity range. The ophthalmic compositions described herein can be "embedded" in topical applicators such as cloths or wipes, which can have a dry powder containing the preparation or which can be wet.

[0128] In unit dosage forms, the preparation is divided into unit doses containing a suitable amount of one or more enzymes (such as α-amylase or α-amylase and lysozyme). The unit dose can be in the form of a package containing discrete amounts of the preparation. Non-limiting examples are liquids in vials or ampoules. An aqueous suspension composition can be packaged in a single-dose non-reclosable container. A multi-dose reclosable container can be used, for example, in combination with a preservative. A variety of preservatives and emollients can be added to the composition (described further above). For example, the container can have multiple dry or wet wipes with the preparation of the present disclosure.

[0129] Non-limiting examples of ophthalmic eye drops that can be formulated with the α-amylase, lysozyme or dextranase of the present disclosure include:

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] Non-limiting examples of ophthalmic eye drops that can be formulated with the α-amylase of the present disclosure include:

[0140]

[0141]

[0142] Examples

[0143] The following examples are presented in order to provide a complete disclosure and description of how to make and use the present invention to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments described below are all or the only experiments conducted. Those skilled in the art will understand that many variations and / or modifications can be made to the present invention as shown in the specific aspects without departing from the spirit or scope of the present invention as broadly described. Accordingly, the aspects of the present invention are considered to be illustrative rather than restrictive in all respects.

[0144] Example 1. Ophthalmic emollient composition

[0145] This example describes a procedure for testing a composition containing a therapeutically effective concentration of α - amylase from Aspergillus oryzae and various ophthalmic demulcents. The rationale for selecting the following ophthalmic demulcents is as follows:

[0146] Selection of emollient

[0147] Cellulose derivatives (demulcent compositions 1 - 4):

[0148] α - Amylase from Aspergillus oryzae (E.C. 3.2.1.1) catalyzes the endohydrolysis of 1,4 - α - D - glucosidic bonds in polysaccharides. Although cellulose is made of polysaccharides, since they are linked by β(1→4) glucan bonds, α - amylase is considered not to break such bonds and may become inhibited by binding to them. This study will evaluate whether cellulose derivatives are suitable as demulcents for compositions containing α - amylase, or whether α - amylase activity is inhibited.

[0149] Dextran:

[0150] Dextran is a branched - chain glucan polysaccharide containing α - 1,6 glycosidic bonds. It is noteworthy that dextran is different from dextrin, which is a polysaccharide that can contain α - 1,4 bonds or α - 1,6 bonds. This study will evaluate whether dextran is suitable as a demulcent in compositions containing α - amylase.

[0151] Gelatin:

[0152] Gelatin contains proteins and peptides and is not expected to interact functionally with α - amylase. This study will evaluate whether gelatin is suitable as a demulcent in compositions containing α - amylase.

[0153] Polyols:

[0154] It is hypothesized that adding polyols to an aqueous solution of α-amylase can increase the thermal stability of α-amylase in the solution. This study will evaluate whether polyols are suitable as moderators in compositions containing α-amylase.

[0155] Polyvinyl alcohol:

[0156] Polyvinyl alcohol is a biocompatible, water-soluble synthetic polymer and should not interact with α-amylase. This study will evaluate whether polyvinyl alcohol is suitable as a moderator in compositions containing α-amylase.

[0157] Povidone:

[0158] Povidone is a biocompatible, water-soluble polymer and should not interact with α-amylase. This study will evaluate whether povidone is suitable as a moderator in compositions containing α-amylase.

[0159] The compositions tested in this study are as follows:

[0160]

[0161]

[0162] 1.1 Composition development - Emollient characterization

[0163] Compositions of α-amylase combined with the ophthalmic moderators mentioned above are prepared by using a commercial eye drop without preservatives in a buffered saline solution. It should be noted that α-amylase is considered to require calcium ions to function, and the compositions of the present disclosure include calcium ions. Note: The concentration of CaCl 2 can be adjusted in the protocol based on the Ca cofactor test results. If excessive Ca causes enzyme inactivation, the concentration can be adjusted.

[0164] Compositions containing different preservatives are tested in different experiments, as described in Example 2.

[0165] 1.1.1 Composition development - α - amylase preparation with cofactors (without emollient):

[0166] Step 1: Prepare 10 ml of a 10 mg / ml α - amylase solution with cofactors. Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α - amylase is dissolved and evenly distributed.

[0167] Step 2: Dissolve 40 mg of calcium chloride and 56 mg of sodium chloride into the solution. Mix until all components are dissolved and evenly distributed.

[0168] 1.1.2 Composition development - α - amylase preparation + cellulose derivative emollient (Compositions 1 - 4):

[0169] Protocol for preparing a 10 ml saline solution of 10 mg / ml α-amylase with 0.5% sodium carboxymethylcellulose. Note: Sodium carboxymethylcellulose can be replaced by hydroxyethyl cellulose, hypromellose, or methyl cellulose within the ranges described in the table above.

[0170] Step 1: Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to the flask. Add 40 mg of calcium chloride and 56 mg of sodium chloride to the flask. Mix until all components are dissolved and evenly distributed.

[0171] Step 2: Add 50 mg of sodium carboxymethylcellulose (Sigma-Aldrich catalog #419273) (or another suitable cellulose derivative described herein) and mix until dissolved.

[0172] 1.1.3 Composition development - α - amylase preparation + dextran emollient (Composition 5):

[0173] Protocol for preparing a 10 ml saline solution of 10 mg / ml α-amylase with 0.1% dextran 70 and 0.3% hypromellose

[0174] Step 1: Add 20 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to a 2 ml ALCON GenTeal Tears preservative-free disposable vial in the flask. Mix until all components are dissolved and evenly distributed.

[0175] Step 2: Dissolve 0.006 g of calcium chloride into the solution and mix until dissolved.

[0176] 1.1.4 Composition development - α - amylase preparation + gelatin (Composition 6):

[0177] Protocol for preparing a 2 ml saline solution of 10 mg / ml α-amylase with 0.01% gelatin:

[0178] Step 1: Add 0.2 mg of pure gelatin powder to 2 ml of room temperature saline in the flask without mixing.

[0179] Step 2: Allow the gelatin to bloom for 5 min.

[0180] Step 3: Warm the flask to 50 °C and mix the gelatin until dissolved.

[0181] Step 4: Cool the flask to room temperature before proceeding.

[0182] Step 5: Add 20 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich Catalog #10065) to the flask. Mix until the α-amylase is dissolved and evenly distributed.

[0183] Dissolve 0.006 g of calcium chloride in the solution.

[0184] 1.1.5 Composition development - α - amylase preparation + glycerol (Composition 7):

[0185] Protocol for preparing 10 ml of a saline solution of 10 mg / ml α-amylase with 0.5% glycerol:

[0186] Step 1: Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich Catalog #10065) to the flask. Add 40 mg of calcium chloride and 56 mg of sodium chloride to the flask. Mix until the α-amylase is dissolved and evenly distributed.

[0187] Step 2: Add 100 mg of glycerol to the flask. Fill the flask to the 10 ml mark with sterile DI water. Mix until all components are dissolved and evenly distributed.

[0188] 1.1.6 Composition development - α - amylase preparation in saline + polyol (Compositions 8 - 11):

[0189] Polyethylene glycol 400 - 1.0%

[0190] Step 1: Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich Catalog #10065) to the flask. Add 40 mg of calcium chloride and 56 mg of sodium chloride to the flask.

[0191] Mix until the α-amylase is dissolved and evenly distributed.

[0192] Step 2: Add 100 mg of polyethylene glycol 400 (Sigma-Aldrich Catalog #8074850050) and mix until dissolved. Fill the flask to the 10 ml mark with sterile DI water. Mix until all components are dissolved and evenly distributed.

[0193] Note: Polyethylene glycol 400 can be replaced by polyethylene glycol 300 and other suitable polyols.

[0194] Propylene glycol 1.0%

[0195] Step 1: Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich Catalog #10065) to the flask. Add 40 mg of calcium chloride and 56 mg of sodium chloride to the flask.

[0196] Step 2: Add 100 mg of propylene glycol to the flask. Fill the flask with sterile DI water up to the 10 ml mark. Mix until all components are dissolved and evenly distributed.

[0197] Polyvinyl alcohol 4% (PVA)

[0198] Step 1: A 4% PVA solution (4 g PVA per 100 ml of DI water) should be prepared in advance as PVA must be added to room temperature DI water in a gently capped culture bottle with a magnetic stirrer, gently heated to 90 °C while stirring continuously, held at 90 °C for 1 hour, and allowed to return to room temperature.

[0199] Step 2: Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of 4% PVA solution. Mix until the α-amylase is dissolved and evenly distributed.

[0200] Step 3: Dissolve 40 mg of calcium chloride and 56 mg of sodium chloride into the solution. Mix until all components are evenly dissolved.

[0201] 1.2 Composition evaluation - Well plate setup for in vitro testing of biofilm formation inhibition

[0202] Prepare Staphylococcus aureus using the protocol listed below and plate it for the biofilm formation protocol.

[0203] 1.2.1 Thaw Staphylococcus aureus

[0204] Using gentle agitation in a 25 °C to 30 °C water bath, vertically thaw the bacterial strain. The thawing will be rapid; approximately 2 - 3 minutes or until all ice crystals have melted.

[0205] After thawing, immediately wipe down the cryovial with 70% ethanol and aseptically inoculate a vented sterile culture tube of aerobic bacteria with 10 mL of tryptic soy broth (30 g / 1000 ml DI H 2 O) using a sterile inoculation loop. Incubate at 37 °C in an orbital incubator for 24 hours.

[0206] 1.2.2 Prepare the culture for plating

[0207] Step 1. Place a new CuveTip on the Photopette

[0208] Step 2. Turn on the Photopette and ensure that Bluetooth is connected to the iPad.

[0209] Step 3. Open the App and set the measurement to collect at 600 nm (using the ecoli setting for brevity).

[0210] Step 4. Zero the Photopette in DI water and clean the tip with a chemwipe.

[0211] Step 5. Transfer 100 ml of TSB citrate medium (TSBC) aseptically to a flask.

[0212] Step 6. Using a pipette, aseptically transfer an overnight culture of Staphylococcus aureus subsp. aureus Rosenbach (ATCC 29213) in tryptic soy broth to the TSB citrate medium (TSBC) until the culture is diluted to an A600 of 0.02 (~2x10^8 CFU / ml) measured with the Photopette.

[0213] Step 7. Remove a sterile 96-well plate from the sterile packaging in the hood and place it on the bench.

[0214] Step 8. Set an 8-channel pipette to 100 μl and attach a sterile tip.

[0215] Step 9. Plate the desired number of wells with 100 μl of the diluted culture.

[0216] Step 10. Cover the well plate and incubate overnight at 37 °C for 18 hours.

[0217] 1.2.3 Plate

[0218] Plate Staphylococcus aureus in all wells of a 96-well tissue culture-treated plate. The well plate should be incubated at 37 °C for 18 hours before measuring biofilm formation.

[0219] 1.3 Composition testing

[0220] 1.3.1 Test composition on day 1

[0221] Carefully tilt the plate and remove all liquid from the wells by pipetting, inserting the pipette tip into the bottom corner of the well, to remove non-adherent cells without disturbing the biofilm growing on the bottom of the well.

[0222] When treating the first row with an 8-channel pipette, start a timer for 10 minutes. Each row should be treated every 30 seconds, ensuring gentle release of the composition in the corner of the well so as not to mechanically disturb the biofilm.

[0223] For row 1: Add 50 μl of 10 mg / ml α-amylase with cofactor to all wells.

[0224] For row 2: Add 50 μl of 10 mg / ml α - amylase prepared with 1.0% polyethylene glycol 400 to all wells.

[0225] For row 3: Add 50 μl of 10 mg / ml α - amylase prepared with 2.5% sodium carboxymethylcellulose to all wells.

[0226] For row 4: Add 50 μl of 10 mg / ml α - amylase prepared with 1.0% glycerol to all wells.

[0227] For row 5: Add 50 μl of 10 mg / ml α - amylase prepared with 2.0% povidone to all wells.

[0228] For row 6: Add 50 μl of 10 mg / ml α - amylase prepared with 1.0% propylene glycol to all wells.

[0229] For row 7: Add 50 μl of 10 mg / ml α - amylase prepared with 2.7% polyvinyl alcohol to all wells.

[0230] For row 8: Add 50 μl of 10 mg / ml α - amylase in saline with 0.1% dextran 70 and 0.3% hypromellose 2910 to all wells.

[0231] For row 9: Add 50 DI water. Do not process rows 11 - 12. Incubate the plate at 37 °C for 10 minutes.

[0232] 1.4 Evaluation of biofilm reduction

[0233] After 10 min, starting from row 1, all fluids should be removed row - by - row every 30 seconds. The fluids must be removed with gentle pipetting, with the plate at an angle and the pipette at the corner, to remove non - adherent cells without disturbing the biofilm growing on the bottom of the wells.

[0234] Add 50 μl of 0.1% crystal violet (CV) to stain the adherent cells in all wells.

[0235] Carefully rinse all wells by dipping the plate into a DI water bath to remove all remaining crystal violet.

[0236] Dry the well plate and measure the optical density of the biofilm at 600 nm using a plate reader.

[0237] Once all measurements have been made, the well plate should be transferred to a bleach solution for disinfection and disposed of in a bio - waste bin.

[0238] 1.5 Evaluation of the antiseptic effect of multiple compositions

[0239] For the scheduled test days at 7, 14, 21, 28, and 35 days, repeat the steps outlined in 1.2 and 1.3. For each day of testing scheduled at 7, 14, 21, 28, and 35 days, the biofilm microtiter plates should be prepared the day before.

[0240] Once all measurements have been made, the microtiter plates should be transferred to a bleach solution for disinfection and disposed of in a biohazard bin. All experiments were conducted using aseptic techniques and sterile equipment in a clean environment.

[0241] Example 2. Ophthalmic antiseptic composition testing

[0242] This example describes a procedure for testing the compatibility of a composition of α-amylase from Aspergillus oryzae with ophthalmic preservatives. These preservatives being tested are intended to prevent microbial growth rather than extend the shelf life of α-amylase.

[0243] 2.1 Composition development - Evaluation of the effect of preservatives on α - amylase function

[0244] This disclosure assumes that a Staphylococcus aureus biofilm on the eyelid is a major cause of dry eye disease. α-Amylase has not previously been used to remove biofilms from the eyelid. This disclosure describes the testing and characterization of the efficacy of various combinations of ophthalmic solution ingredients with α-amylase in removing biofilms in cell culture, and then testing these solutions on tissue. This experiment was designed to evaluate whether ophthalmic preservatives assist or hinder the effectiveness of α-amylase in reducing Staphylococcus aureus biofilms. The following preservatives are evaluated in this example:

[0245] A) BAK usually acts as a detergent for dissolving cell walls and cell membranes. However, it is not clear whether it affects the stability / function of α-amylase. B) Polyquaternium-1 is thought to act on cell membranes. Similarly, it is not clear whether it affects the stability / function of α-amylase. C) Stabilized oxy-chloride complexes are thought to act as preservatives via the oxidation of intracellular lipids and glutathione. Similarly, it is not clear whether it affects the stability / function of α-amylase. D) Sodium perborate is thought to act by forming hydrogen peroxide and oxidation. However, it is unknown whether it affects the stability / function of α-amylase. E) Disodium edetate and sorbic acid, or disodium edetate, or ethylenediaminetetraacetic acid (EDTA) are thought to act by binding to heavy metals such as iron or calcium. Since α-amylase uses calcium as a cofactor, this protocol will test whether these preservatives affect the stability / function of α-amylase. F) Polixetonium (Polyquaternium-42) is not commonly used as a preservative. It is not clear whether it affects the stability / function of α-amylase. G) Borate, sorbitol, propylene glycol, and zinc ion buffer can act via multiple potential methods of action. This protocol will test whether these preservatives affect the stability / function of α-amylase. H) Polyhexamethylene biguanide is thought to act by impairing bacterial cell membrane activity. This protocol will test whether these preservatives affect the stability / function of α-amylase.

[0246]

[0247] 2.1.1 Composition development - α - amylase preparation with cofactor (preservative - free - control composition):

[0248] Step 1: Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0249] Step 2: Dissolve 40 mg of calcium chloride and 56 mg of sodium chloride into the solution. Mix until all components are dissolved and evenly distributed.

[0250] 2.1.2 Composition development - α - amylase preparation + benzalkonium chloride (Composition 14):

[0251] Protocol for preparing 10 ml of 10 mg / ml α-amylase solution with 0.01% benzalkonium chloride:

[0252] Step 1: Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of benzalkonium chloride (Sigma-Aldrich catalog #PHR1371) diluted to 0.01% with DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0253] Step 2: Dissolve 40 mg of calcium chloride and 56 mg of sodium chloride into the solution.

[0254] 2.1.3 Composition development - α - amylase preparation + Polyquad (polyquaternium - 1) (Composition 15):

[0255] Protocol for preparing 10 ml of 10 mg / ml α - amylase solution with 0.001% Polyquad:

[0256] Step 1: Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to 2 ml of Systane original in a flask. Mix until the α - amylase is dissolved and evenly distributed.

[0257] Prepare a saline solution of 10 mg / ml α - amylase with 0.01% sodium perborate in 10 ml.

[0258] Step 2: Dissolve in 1 mg of Polyquaternium - 1 (Santa Cruise Biochemicals catalog #sc - 476677). Dissolve 40 mg of calcium chloride and 56 mg of sodium chloride into the solution.

[0259] 2.1.4 Composition development - α - amylase preparation + stabilized oxy - chlorine complex (Composition 16):

[0260] Protocol for preparing 10 ml of a saline solution of 10 mg / ml α - amylase with stabilized oxychlorocomplex:

[0261] Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to 10 ml of Blink Tears lubricating eye drops in a flask, which already contains a suitable amount of stabilized oxychlorocomplex. Mix until the α - amylase is dissolved and evenly distributed.

[0262] 2.1.5 Composition development - α - amylase preparation + GenAqua / Dequest (sodium perborate) (Composition 17):

[0263] Protocol for preparing 2 ml of a saline solution of 10 mg / ml α - amylase with 0.01% GenAqua / Dequest (sodium perborate):

[0264] Step 1: Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to DI in a flask. Mix until the α - amylase is dissolved and evenly distributed.

[0265] Step 2: Dissolve 1 mg of sodium perborate into the solution.

[0266] Step 3: Dissolve in 30 mg of calcium chloride and 60 mg of sodium chloride.

[0267] 2.1.6 Composition development - α - amylase preparation + 0.1% disodium edetate and 0.1% sorbic acid (Composition 18):

[0268] Protocol for preparing sodium hypochlorite solution:

[0269] Step 1: Prepare 10 ml of 5% bleach (sodium hypochlorite) solution.

[0270] Step 2: Add 6.66 ml of commercially available bleach 7.5% sodium hypochlorite to a 10 ml flask and fill to 10 ml with DI water.

[0271] 2.2 Composition evaluation - Well plate setup for in vitro testing of the compatibility of α - amylase from Aspergillus oryzae with ophthalmic preservatives Well plate setup

[0272] Using the protocol listed in Example 1 (1.2.1), prepare plates for plating Staphylococcus aureus for the preservative compatibility protocol test. Briefly, plate Staphylococcus aureus on rolls 1-10, 1-8 as described in Example 1, leaving rows 11-12 for DI water for spectrophotometric comparison. Incubate the microtiter plate at 37 °C for 18 hours before determining biofilm formation.

[0273] 2.3 Composition testing

[0274] 1.3.1 Test composition, Day 1

[0275] Carefully tilt the plate and remove all liquid from the wells by pipetting from the bottom corners of the wells, inserting the pipette tip into the bottom corners of the wells, to remove non-adherent cells without disturbing the biofilm growing on the bottom of the wells.

[0276] When processing the first row using an 8-channel pipette, start the timer for 10 minutes. Each row should be processed every 30 seconds, ensuring gentle release of the composition in the corners of the wells so as not to mechanically disturb the biofilm.

[0277] For row 1: Add 50 μl of 10 mg / ml α-amylase with cofactor to row 1.

[0278] For row 2: Add 50 μl of 10 mg / ml α-amylase with 0.01% benzalkonium chloride to row 2.

[0279] For row 3: Add 50 μl of 10 mg / ml α-amylase with 0.001% Polyquad to row 3.

[0280] For row 4: Add 50 μl of 10 mg / ml α-amylase with 0.01% sodium perborate to row 4.

[0281] For row 5: Add 50 μl of 10 mg / ml α-amylase with stabilized oxychlorine complex to row 5.

[0282] For row 6: Add 50 μl of 7.5% bleach (sodium hypochlorite) solution to row 6.

[0283] For row 7: Add 50 μl of DI water to row 7.

[0284] Incubate the plate at 37 °C for the remaining 10 minutes.

[0285] 2.4 Evaluation of biofilm reduction

[0286] After 10 min, starting from row 1, remove all the fluid row by row every 30 seconds. The fluid must be removed with gentle pipetting, with the plate at an angle and the pipette at the corner, to remove non-adherent cells without disturbing the biofilm growing on the bottom of the wells.

[0287] Add 50 μl of 0.1% crystal violet (CV) to stain the adherent cells in all the wells.

[0288] Carefully rinse all the wells by dipping the plate into a DI water bath to remove all the remaining crystal violet.

[0289] Dry the well plate and measure the optical density of the biofilm at 600 nm using a plate reader.

[0290] Once all the measurements have been made, transfer the well plate to a bleach solution for disinfection and dispose of it in a biohazard bin.

[0291] Example 3. Biofilm formation

[0292] Overview of the protocol for co-culture studies of human conjunctival epithelial cells with Staphylococcus aureus.

[0293] Using corneal epithelial cell medium, grow human conjunctival epithelial cells (HConEpiC) from ScienCell Research Laboratories catalog #6630 as a monolayer on poly-L-lysine-coated 24-well plates.

[0294] 3.1 Start culturing:

[0295] ScienCell primary cells are cultured in a 37 °C, 5% CO 2 incubator as follows:

[0296] 1. Prepare poly-L-lysine-coated culture vessels (2 μg / cm 2 ; T-75 flasks are recommended). To obtain 2 μg / cm2 A poly-L-lysine-coated culture vessel. Add 10 ml of sterile water to a T-75 flask, and then add 15 μl of poly-L-lysine stock solution (10 mg / ml, catalog #0413). Leave the vessel in an incubator at 37 °C overnight (or for at least one hour).

[0297] 2. Prepare complete medium. Decontaminate the outer surfaces of the medium bottle and the medium supplement tube with 70% ethanol and transfer them to a sterile area. Transfer the supplement aseptically to the basal medium using a pipette. Rinse the supplement tube with medium to recover the full volume.

[0298] 3. Rinse the poly-L-lysine-coated vessel twice with sterile water, and then add 20 ml of complete medium. Leave the vessel in the sterile area and continue to thaw the cryopreserved cells.

[0299] 4. Place the cryovial in a 37 °C water bath. Hold and gently rotate the vial until the contents are completely thawed. Immediately remove the vial from the water bath, wipe it down with 70% ethanol, and transfer it to a sterile area.

[0300] 5. Carefully remove the cap without touching the internal threads. Gently resuspend the contents of the vial and dispense them into the equilibrated poly-L-lysine-coated culture vessel.

[0301] Note: Diluting and centrifuging the cells after thawing is not recommended as these actions may be harmful to the cells. It is also important to plate the cells in a vessel such as a poly-L-lysine-coated culture vessel under conditions favorable for cell attachment to promote cell attachment.

[0302] 6. Replace the cap or lid of the culture vessel and gently shake the vessel to distribute the cells evenly. If necessary, loosen the cap to allow gas exchange.

[0303] 7. Return the culture vessel to the incubator.

[0304] 8. Do not disturb the culture for at least 16 hours after starting. Replace the medium the next day to remove residual DMSO and non-attached cells.

[0305] 3.2 Maintain culturing:

[0306] 1. After establishing a culture from cryopreserved cells, replace the supplemented medium the next morning.

[0307] 2. Thereafter, replace the medium every three days until the culture is approximately 70% confluent.

[0308] 3. Once the culture reaches 70% confluence, replace the medium every other day until the culture is approximately 90% confluent.

[0309] 3.3 Sub - culturing:

[0310] 1. Passage the culture when it reaches 90% confluence.

[0311] 2. Prepare poly-L-lysine-coated culture vessels (2 μg / cm 2 ) one day before passage.

[0312] 3. Warm the complete medium, 0.05% trypsin / EDTA solution (T / E, Catalog #0183), T / E neutralization solution (TNS, Catalog #0113), and DPBS (without Ca++ and Mg++, Catalog #0303) to room temperature. We do not recommend warming the reagents and media in a 37°C water bath before use.

[0313] 4. Rinse the cells with DPBS.

[0314] 5. Add 10 ml of 0.05% T / E solution (Catalog #0183) to the flask (in the case of a T-75 flask). Gently rock the flask to ensure that the T / E solution completely covers the cells. Use a microscope to monitor the morphological changes of the cells.

[0315] Note: It is recommended to use ScienCell 0.05% T / E solution, which is optimized to minimize cell damage due to over-trypsinization, although other solutions can also be used. If using 0.25% T / E solution (Catalog #0103), 8 ml of DPBS and 2 ml of 0.25% T / E solution should be used.

[0316] 6. During incubation, prepare a 50 ml conical centrifuge tube with 5 ml of fetal bovine serum (FBS, Catalog #0500).

[0317] 7. Once the cells are completely detached, transfer the T / E solution from the flask to the 50 ml centrifuge tube (a small fraction of cells may detach), and incubate the flask at 37°C for 2 - 3 minutes (there is no solution in the flask at this time).

[0318] 8. At the end of the incubation, tap the side of the flask to dislodge the cells from the surface. Check under the microscope to ensure that all cells are detached.

[0319] 9. Add 5 ml of TNS solution to the flask and transfer the detached cells to the 50 ml centrifuge tube. Rinse the flask with another 5 ml of TNS to collect the remaining cells.

[0320] 10. Check under the microscope if the flask has been successfully harvested by observing the number of cells left; it should be less than 5%.

[0321] 11. Centrifuge 50 ml centrifuge tubes at 1000 rpm for 5 minutes. Gently resuspend the cells in the medium.

[0322] 12. Count and plate the cells at the recommended cell density in a new poly-L-lysine coated culture vessel. A seeding density of 5,000 cells / cm 2 is recommended.

[0323] 3.4 Co - culture development

[0324] After establishing a monolayer of human conjunctival epithelial cells, a layer of Staphylococcus aureus subsp. aureus (ATCC 29213) will be established on top of the epithelial cells.

[0325] 1. Add 650 μL of Staphylococcus aureus subsp. aureus (ATCC 29213) in tryptic soy broth to the TSB citrate medium (TSBC) diluted to an A600 of 0.01 (~1x10^8 CFU / ml) in each well of rows A - C, leaving row D untreated.

[0326] 2. Cover the well plate and incubate at 37 °C for 4 hours.

[0327] 3.5 Testing

[0328] Test the α - amylase compositions in this co - culture, preferably compositions that have been shown to have the ability to remove biofilms in cell culture as described in Example 1, to study their potential toxicity to the underlying monolayer of human conjunctival epithelial cells. The hypothesis is that by treating and eliminating Staphylococcus aureus that damages the human body, the inflammatory response from the epithelial cells will return to normal at the mRNA and protein levels.

[0329] Add 1 ml of each test composition to each well of row A.

[0330] Test row B with 1 ml of negative control (saline).

[0331] Test row C with 1 ml of positive control (gentamicin).

[0332] The test compositions should be incubated at 37 °C for 10 min.

[0333] Before lysis, all layers should be washed three times. The plate was gently lysed with 1 ml of trypsin to remove cells. The lysed cells were stained with trypan blue and the cell count was evaluated under a microscope. The presence of blue cells indicates cell death. The cell number and visibility curve of the samples were evaluated. Samples were also collected and frozen before and after the challenge for mRNA, DNA, and protein analysis for future expression analysis.

[0334] Example 4. Treatment of Staphylococcus aureus biofilm with antibiotics + α - amylase

[0335] Staphylococcus aureus produces extracellular capsular polysaccharides that link to produce biofilms. Aspergillus oryzae α-amylase (E.C. 3.2.1.1) catalyzes the endohydrolysis of 1,4-α-D-glucosidic linkages in polysaccharides containing three or more 1,4-α-linked D-glucose units. α-Amylase does not have antibacterial properties, but it can inhibit and reduce Staphylococcus aureus biofilms by disrupting the extracellular polysaccharide bonds of the biofilm. Antibiotics have been shown to have some effect in reducing biofilms, but are less effective against established biofilms. In addition, long-term use of antibiotics can lead to antibiotic-resistant bacteria. α-Amylase does not have antibacterial properties and does not have the known risk of producing antibiotic-resistant bacteria.

[0336] The external application of α-amylase described in this example has never been used to reduce or prevent biofilm formation on the eyelids. This experiment aimed to characterize the use of α-amylase and α-amylase compositions with other antibacterial or antimicrobial agents against Staphylococcus aureus biofilms grown in a well plate for 24 hours. It was hypothesized that the addition of α-amylase would improve the biofilm removal of all compounds.

[0337] This study describes the testing of compositions of 0.01% hypochlorous acid solution with and without α-amylase from Aspergillus oryzae on in vitro-formed biofilms. This study also describes the testing of compositions of common ophthalmic antibiotics with and without α-amylase from Aspergillus oryzae. These antibiotics include: erythromycin, moxifloxacin, and gentamicin sulfate. This study used sterile saline as a negative control and a 5% bleach solution as a positive control.

[0338] 4.1 Composition development - α - amylase preparation in saline:

[0339] α-Amylase requires calcium ions to function. A saline solution was prepared with 0.4% calcium chloride and 0.56% sodium chloride. 0.4% calcium chloride was added to the α-amylase solution to ensure the presence of calcium ions.

[0340] Step 1: Prepare a 10 mg / ml α-amylase solution with cofactors in 10 ml. Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0341] Step 2: Dissolve 40 mg of calcium chloride and 56 mg of sodium chloride into the solution. Mix until all components are evenly distributed.

[0342] 4.1.2 Composition development - α - amylase in 0.01% hypochlorous acid solution

[0343] Protocol for preparing a 2 ml solution of 10 mg / ml α-amylase in 0.01% hypochlorous acid solution:

[0344] Step 1: Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of 0.01% hypochlorous acid (Avenova OTC antimicrobial spray solution) in saline in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0345] Step 2: Dissolve 40 mg of calcium chloride into the solution. Mix until the α-amylase is dissolved and evenly distributed.

[0346] 4.1.3 Composition development - α - amylase in ofloxacin solution

[0347] Protocol for preparing 2 ml of 10 mg / ml α-amylase in 0.3% ofloxacin ophthalmic solution:

[0348] Briefly: Add 20 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 1 ml of 0.5% erythromycin ophthalmic solution in a flask and mix until evenly distributed. Dissolve 8 mg of calcium chloride into the solution.

[0349] 4.1.4 Composition development - α - amylase in 0.01% moxifloxacin solution

[0350] Protocol for preparing 1 ml of 10 mg / ml α-amylase in 0.5% moxifloxacin ophthalmic solution:

[0351] Briefly: Add 10 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 1 ml of 0.5% moxifloxacin ophthalmic solution in a flask and mix until evenly distributed. Dissolve 3 mg of calcium chloride into the solution.

[0352] 4.1.5 Composition development - α - amylase in gentamicin sulfate

[0353] Protocol for preparing 2 ml of 10 mg / ml α-amylase in 0.5% gentamicin sulfate ophthalmic solution:

[0354] Briefly: Add 20 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 1 ml of 0.5% gentamicin sulfate ophthalmic solution in a flask and mix until evenly distributed. Dissolve 8 mg of calcium chloride into the solution.

[0355] 4.1.6 Composition development - α - amylase in bleach

[0356] Protocol for preparing 10 ml of 5% bleach (sodium hypochlorite) solution:

[0357] Briefly: Add 6.66 ml of commercially available bleach 7.5% sodium hypochlorite to a 10 ml flask and fill to 10 ml with DI water.

[0358] 4.2 Well plate setup

[0359] Using the protocol listed in Example 1 (1.2.1), prepare plates for plating Staphylococcus aureus for the preservative compatibility protocol test. Briefly, plate Staphylococcus aureus in all wells of a 96-well tissue culture-treated plate as described in Example 1. The plate should be incubated at 37 °C for 18 hours before measuring biofilm formation.

[0360] 4.3 Test composition

[0361] Carefully tilt the plate and remove all liquid from the wells by pipetting from the bottom corners of the wells using a pipette tip to remove non-adherent cells without disturbing the biofilm growing on the bottom of the wells.

[0362] When treating the first row using an 8-channel pipette, start the timer for 10 minutes. Each row should be treated every 30 seconds, ensuring gentle release of the composition in the corners of the wells so as not to mechanically disturb the biofilm.

[0363] For row 1: Add 50 μl of 10 mg / ml α-amylase in cofactor solution to all wells.

[0364] For row 2: Add 50 μl of 10 mg / ml α-amylase in 0.01% hypochlorous acid solution to all wells.

[0365] For row 3: Add 50 μl of 0.01% hypochlorous acid in saline (Avenova OTC antimicrobial spray solution) to all wells.

[0366] For row 4: Add 50 μl of 10 mg / ml α-amylase in 0.3% ofloxacin ophthalmic solution to all wells.

[0367] For row 5: Add 50 μl of 0.3% ofloxacin ophthalmic solution to all wells.

[0368] For row 6: Add 50 μl of 10 mg / ml α-amylase in 0.5% gentamicin sulfate ophthalmic solution to all wells.

[0369] For row 7: Add 50 μl of 0.5% gentamicin sulfate ophthalmic solution to all wells.

[0370] For row 8: Add 50 μl of 5% bleach (sodium hypochlorite) solution to all wells.

[0371] For row 9: Add 50 μl of DI water to all wells.

[0372] Do not process rows 10 - 12. Incubate the plate at 37 °C for the remaining 10 minutes.

[0373] 4.4. Test for biofilm reduction

[0374] After 10 min, starting from row 1, remove all the fluid row by row every 30 seconds. The fluid must be removed with gentle pipetting, with the plate at an angle and the pipette at the corner, to remove non-adherent cells without disturbing the biofilm growing on the bottom of the wells.

[0375] Add 50 μl of 0.1% crystal violet (CV) to stain the adherent cells in all wells.

[0376] Carefully rinse all wells by dipping the plate into a DI water bath to remove all the remaining crystal violet.

[0377] Dry the microplate and measure the optical density of the biofilm at 600 nm using a plate reader. Once all the measurements have been made, transfer the microplate to a bleach solution for disinfection and dispose of it in a biohazard bin.

[0378] Example 5. Range of treatment of Staphylococcus aureus biofilm with cofactor + α - amylase

[0379] This protocol was designed to evaluate the optimal range of the cofactor calcium for α-amylase from Aspergillus oryzae when used for removing Staphylococcus aureus biofilms.

[0380] Ca 2+ has been identified as a necessary cofactor for α-amylase, however previous studies on α-amylase on Staphylococcus aureus biofilms did not include Ca 2+Introduced into the composition to remove or inhibit biofilm growth in cell cultures. α-Amylase has two binding sites for Ca ions. 1 mg of α-amylase contains 1.075 * 10^16 enzymes, and the present disclosure provides that it can consume at least 0.0039 mg of CaCl 2 to provide two calcium ions per enzyme. It is not clear how much Ca 2+ is required to provide optimal function for biofilm removal, and some literature suggests that excessive calcium may have a negative impact. Although some electrolytes, including Ca 2+ , are naturally present in the eye, this study was designed to characterize the effectiveness of α-amylase from Aspergillus oryzae in removing Staphylococcus aureus biofilms in cell cultures at different concentrations of Ca 2+ cofactor.

[0381] Normal electrolyte concentrations (mMol / liter) in human tears

[0382]

[0383] 5.1 Composition development

[0384] 5.1.1 - Prepare 10 ml of a 10 mg / ml aqueous DI solution of α - amylase

[0385] Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 2 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0386] 5.1.2 - Preparation of 10 ml of DI water solution of 10 mg / ml α - amylase with 0.05% CaCl 2 0.85% NaCl Solution.

[0387] Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed. Add 5 mg of CaCl 2 . Add 85 mg of NaCl. Mix until dissolved.

[0388] 5.1.3 - Preparation of 10 ml of DI aqueous solution of 10 mg / ml α - amylase with 0.1% CaCl 2 and 0.8% NaCl.

[0389] Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed. Add 10 mg of CaCl 2 . Add 80 mg of NaCl. Mix until dissolved.

[0390] 5.1.4 - Preparation of 10 ml of DI aqueous solution of 10 mg / ml α - amylase with 0.2% CaCl 2 and 0.7% NaCl.

[0391] Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α - amylase is dissolved and evenly distributed. Add 20 mg of CaCl 2 . Add 70 mg of NaCl. Mix until dissolved.

[0392] 5.1.5 - Preparation of 10 ml of DI aqueous solution of 10 mg / ml α - amylase with 0.3% CaCl 2 and 0.6% NaCl.

[0393] Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α - amylase is dissolved and evenly distributed. Add 30 mg of CaCl 2 . Add 60 mg of NaCl. Mix until dissolved.

[0394] 5.1.6 - Preparation of 10 ml of DI aqueous solution of 10 mg / ml α - amylase with 0.4% CaCl 2 and 0.5% NaCl.

[0395] Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α - amylase is dissolved and evenly distributed. Add 40 mg of CaCl 2 . Add 50 mg of NaCl. Mix until dissolved.

[0396] 5.1.7 - Preparation of 10 ml of DI aqueous solution of 10 mg / ml α - amylase with 0.5% CaCl 2 and 0.4% NaCl.

[0397] Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α - amylase is dissolved and evenly distributed. Add 50 mg of CaCl 2 . Add 40 mg of NaCl. Mix until dissolved.

[0398] 5.1.8 - Preparation of 10 ml of DI aqueous solution of 10 mg / ml α - amylase with 0.6% CaCl 2 and 0.3% NaCl.

[0399] Add 100 mg of α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α - amylase is dissolved and evenly distributed. Add 60 mg of CaCl 2 . Add 30 mg of NaCl. Mix until dissolved.

[0400] 5.1.9 - Preparation of 10 ml of DI aqueous solution of 10 mg / ml α - amylase with 0.8% CaCl 2 and 0.1% NaCl.

[0401] Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed. Add 80 mg of CaCl 2 . Add 10 mg of NaCl. Mix until dissolved.

[0402] 5.2 Test composition

[0403] Carefully tilt the plate and remove all the liquid from the wells by pipetting from the bottom corners of the wells, inserting the pipette tip into the bottom corners of the wells, to remove non-adherent cells without disturbing the biofilm growing on the bottom of the wells.

[0404] When processing the first row using an 8-channel pipette, start the timer for 10 minutes. Each row should be processed every 30 seconds, ensuring gentle release of the composition in the corners of the wells so as not to mechanically disturb the biofilm.

[0405] For row 1: Add 50 μl of 10 mg / ml α-amylase in DI water to row 1.

[0406] For row 2: Add 50 μl of an aqueous DI solution of 10 mg / ml α-amylase with 0.05% CaCl 2 0.85% NaCl to row 2.

[0407] For row 3: Add 50 μl of an aqueous DI solution of 10 mg / ml α-amylase with 0.1% CaCl 2 0.8% NaCl to row 3.

[0408] For row 4: Add 50 μl of an aqueous DI solution of 10 mg / ml α-amylase with 0.2% CaCl 2 0.7% NaCl to row 4.

[0409] For row 5: Add 50 μl of an aqueous DI solution of 10 mg / ml α-amylase with 0.3% CaCl 2 0.6% NaCl to row 5.

[0410] For row 6: Add 50 μl of an aqueous DI solution of 10 mg / ml α-amylase with 0.4% CaCl 2 0.5% NaCl to row 6.

[0411] For row 7: Add 50 μl of an aqueous DI solution of 10 mg / ml α-amylase with 0.5% CaCl 2 0.4% NaCl to row 7.

[0412] For row 8: Add 50 μl of a DI aqueous solution of 10 mg / ml α - amylase with 0.6% CaCl 2 0.3% NaCl.

[0413] For row 9: Add 50 μl of a DI aqueous solution of 10 mg / ml α - amylase with 0.8% CaCl 2 0.1% NaCl.

[0414] For row 10: Add 50 μl of a 7.5% bleach (sodium hypochlorite) solution.

[0415] For row 11: Add 50 μl of DI water.

[0416] Do not treat row 12. The plate should be placed in an incubator at 37 °C for the remaining time of the 10 - min period.

[0417] 5.3 Test for biofilm

[0418] After 10 min, starting from row 1, all fluids should be removed row - by - row every 30 seconds. The fluids must be removed with gentle pipetting, with the plate at an angle and the pipette at the corner, to remove non - adherent cells without disturbing the biofilm growing on the bottom of the wells.

[0419] Add 50 μl of 0.1% crystal violet (CV) to stain the adherent cells in all wells.

[0420] Carefully rinse all wells by immersing the plate in a DI water bath to remove all remaining crystal violet. Dry the well plate and measure the optical density of the biofilm at 600 nm using a plate reader.

[0421] Once all measurements have been made, the well plate should be transferred to a bleach solution for disinfection and disposed of in a biohazard waste bin.

[0422] Example 6. containing a calcium cofactor (CaCl 2 in a volume ranging from 0.4% CaCl 2 to 0.8% CaCl 2 ) of α- Amylase composition

[0423] The following experiment summarizes the results observed in the presence of different compositions of various additives containing α - amylase from Aspergillus oryzae to determine whether any such additive enhances, has no effect on, or inhibits the ability of the enzyme to remove biofilms growing in cell cultures. The remaining biofilm density was evaluated at 600 nm using a cell plate reader, referred to as optical density 600 (OD 600 ) measurement.

[0424] Example 6.1. An α-amylase composition containing a calcium cofactor (CaCl 2 ).

[0425] Calcium cofactor optimization:

[0426] Calcium has been identified as an essential cofactor for α - amylase. However, previous studies on α - amylase on Staphylococcus aureus biofilms did not introduce calcium into the composition to remove or inhibit biofilm growth in cell cultures. This study aimed to determine how much calcium was needed to provide optimal function for biofilm removal. Some literature indicates that excessive calcium may have negative effects. Therefore, considering the concentration range of Ca 2+ that supports α - amylase activity without inhibiting it is important.

[0427] General method:

[0428] Staphylococcus aureus biofilms were grown in 96 - well (Corning Falcon) plates for cell culture treatment with TSB broth supplemented with yeast extract, glucose, and sodium citrate to induce biofilm formation and incubated for 18 hours.

[0429] Non - adherent cells were removed and the biofilms were treated with different solutions to test the ability to degrade Staphylococcus aureus biofilms. α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) at ~30 U / mg was used to produce a solution with α - amylase at 10 mg / mL.

[0430] After incubation for 10 minutes, non - adherent cells were removed and the remaining biofilms were stained with crystal violet. The density of the remaining biofilms was measured at 600 nm using a Byonoy Absorbance 96 - well plate reader.

[0431] Figure 11A is a graph showing the experimental results described above. The optical density of the biofilms is shown on the Y - axis. The X - axis shows different concentrations of CaCl 2 cofactor and two controls: deionized water (negative control) and bleach (positive control for biofilm removal). The concentrations tested included 0.05% CaCl 2 cofactor, 0.1% CaCl 2 cofactor, 0.2% CaCl 2 cofactor, 0.3% CaCl 2 cofactor, 0.4CaCl 2 cofactor, 0.5% CaCl 2 cofactor, 0.6% CaCl 2 cofactor, or 0.8% CaCl 2 cofactor.

[0432] The following table summarizes the results of the Tukey multiple comparison test analysis:

[0433]

[0434]

[0435]

[0436] A decrease in biofilm density indicates the removal of biofilm by the enzyme. The results showed that the biofilm was continuously removed at CaCl 2 concentrations from 0.4% to 0.8%. See Figure 11A . Figure 11B is a graph quantifying the percentage of biofilm removed by each tested composition within 10 min. Bleach was used as the Figure 11B positive control in

[0437] Example 7. Ophthalmic preservative

[0438] In configurations where the composition is delivered in individual sterile packages, aseptic filtration and packaging can be used to avoid adding preservatives. In cases where the composition is delivered in multi-dose packages, preservatives may be present. This example describes general preservatives that were tested and found not to inhibit enzyme effectiveness:

[0439] General method:

[0440] Staphylococcus aureus biofilms were grown in 96-well (Corning Falcon) cell culture-treated plates using TSB broth supplemented with yeast extract, glucose, and sodium citrate to induce biofilm formation and incubated for 18 hours.

[0441] Non-adherent cells were removed and the biofilms were treated with different solutions to test the ability to degrade Staphylococcus aureus biofilms. A solution with α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) at ~30 U / mg was produced at 10 mg / mL.

[0442] After incubation for 10 minutes, non-adherent cells were removed and the remaining biofilms were stained with crystal violet. The density of the remaining biofilms was measured at 600 nm using a Byonoy Absorbance 96-well plate reader.

[0443] Figure 12A is a graph showing the experimental results of α-amylase, BAK + α-amylase, sodium perborate + α-amylase and their comparison with the negative control (DI water) and the positive control (bleach). Figure 12AThe preservatives tested were: Preservative 1: Purite / OcuPure (stabilized oxy-chlorine complex) - no reduction in enzyme effectiveness; Preservative 2: Polyquad (polyquaternium-1) 0.001% - no reduction in enzyme effectiveness.

[0444] Figure 12B The preservatives tested were: Preservative 1: BAK (benzalkonium chloride) 0.01% - no reduction in enzyme effectiveness; and Preservative 2: GenAqua / Dequest (sodium perborate) - no reduction in enzyme effectiveness.

[0445] The above-mentioned tested compositions at the specified concentrations were found not to inhibit enzyme activity.

[0446] Another preservative tested for treating dry eye conditions was 0.01% hypochlorous acid. Dilute hypochlorous acid is used in eyelid cleansers in a percentage range from 0.01% to 0.02%. In our study, 0.01% hypochlorous acid did not inhibit the enzyme's removal of biofilm. There was no statistical difference in biofilm reduction between 0.01% hypochlorous acid and α-amylase plus cofactor mixed immediately before treatment, 30 minutes, or 1 hour. Although 0.01% hypochlorous acid did slightly reduce the biofilm, there was no additional benefit of increased biofilm removal when combined with α-amylase and cofactor. See Figure 13 。

[0447] Example 8. Ophthalmic emollient

[0448] This protocol studied the efficacy of α-amylase, an enzyme that effectively inhibits and reduces Staphylococcus aureus biofilm, in combination with ophthalmic emollients. These emollients are used in eye drops to relieve the symptoms of dry eye and may potentially prolong the contact of α-amylase with the biofilm on the eye. Some emollients may also have a stabilizing effect on the enzyme. This study evaluated the effects of various emollients on α-amylase activity.

[0449] The following emollients were considered:

[0450] (a) Cellulose derivatives: (1) sodium carboxymethylcellulose from 0.2% to 2.5%; (2) hydroxyethylcellulose from 0.2% to 2.5%; (3) hypromellose from 0.2% to 2.5%; (4) methylcellulose from 0.2% to 2.5%.

[0451] (b) Dextran 70 at 0.1% when used with another polymeric emollient in this section

[0452] (c) Gelatin at 0.01%.

[0453] (d) Liquid polyols: (1) 0.2% to 1% glycerol; (2) 0.2% to 1% polyethylene glycol 300; (3) 0.2% to 1% polyethylene glycol 400; (4) 0.2% to 1% polysorbate 80; (5) 0.2% to 1% propylene glycol.

[0454] (e) 0.1% to 4% polyvinyl alcohol.

[0455] (f) 0.1% to 2% povidone.

[0456] All test solutions were prepared with 10 mg / mL α - amylase from Aspergillus oryzae (Sigma - Aldrich catalog #10065) at ~30 U / mg, 0.4% CaCl 2 and 0.5% NaCl, and tested using the protocol described in the above - mentioned examples. The concentrations tested in this study were as follows:

[0457]

[0458] Figure 14A is a graph showing the analysis results of various tested emollients, including 1% polyethylene glycol 400 (PEG 400), 2.5% sodium carboxymethyl cellulose (CMC), 1% glycerol, 2% povidone (PVP), 1% propylene glycol, 4% polyvinyl alcohol (PVA), 0.1% dextran 700 + 0.3% hypromellose. Figure 14B is a graph showing the analysis results of various tested emollients, including polyvinyl alcohol (PVA), PVA + α - amylase, sodium carboxymethyl cellulose (CMC), and sodium carboxymethyl cellulose (CMC) + α - amylase. As Figure 14A and Figure 14B shown in, CMC and PVA showed beneficial effects on the removal of biofilms. Based on these results, it is hypothesized that PVA may contribute to the stabilization of α - amylase in certain formulations.

[0459] Figure 14C is a graph showing the results of testing with α - amylase stabilized in 4% PVA after 36 days. It was found that PVA and CMC independently helped in the removal of biofilms and also acted together with α - amylase to remove biofilms. PVA is preferred over CMC for filter sterilization and lack of sensitivity to bacterial growth. PVA provides a pH of 5.8, which is ideal for enzyme activation. The literature indicates that a pH between 5 - 6 is ideal for α - amylase from Aspergillus oryzae.

[0460] The PVA solution is a better candidate for sterile filtration than the CMC solution. The 4% PVA solution was filter-sterilized using a 2-μm sterile syringe filter and kept refrigerated for 36 days before the repeated biofilm removal tests. On day 36, the 4% PVA solution performed better than a fresh solution of α-amylase with cofactors. This indicates that PVA can stabilize α-amylase in an aqueous solution refrigerated at 4 °C for at least 36 days.

[0461] Example 9. Antibiotic testing

[0462] This protocol investigated the efficacy of α-amylase from Aspergillus oryzae in combination with commonly prescribed ophthalmic antibiotics to see if this would help in biofilm removal. Generally, it is believed that antibiotics do not penetrate biofilms on their own. Biofilms can also allow bacteria to become antibiotic-resistant as bacteria can transfer DNA within the biofilm.

[0463] Figure 15 is a graph showing the results of the comparison of ophthalmic antibiotics for Staphylococcus aureus biofilm reduction. 10 mg / mL α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) formulated at 30 U / mg with 0.4% CaCl 2 and 0.56% NaCl as cofactors was used in formulations with gentamicin and ofloxacin. Both antibiotics were also tested alone.

[0464] Figure 15 The study results shown in indicate that the ability of α-amylase from Aspergillus oryzae to remove biofilms is not inhibited or enhanced by topical antibiotics. The combination of ophthalmic antibiotics and α-amylase can help patients with chronic infections in which bacteria are encapsulated in biofilms.

[0465] Example 10. Stabilized sugar

[0466] Previously, it has been reported that sugars, especially sucrose and trehalose, are used to increase the thermal stability of α-amylase from Aspergillus oryzae in aqueous solutions. Trehalose is used in eye drops for patients with dry eye due to its bioprotective properties for the epithelial cells of the eye. Solutions of up to 200 mM (6.8%) of trehalose have been used in commercial eye drops. This experiment tested sucrose and trehalose in 10% solutions with and without the moderators carboxymethyl cellulose and polyvinyl alcohol to see if they affected the ability of the enzyme to reduce biofilms. This effect of these formulations on biofilm reduction is shown in Figure 16 shown above.

[0467] These data indicate that the two stabilizing sugars do not have any statistically significant (α=0.05) effect on the ability of the enzyme to reduce biofilm. When the stabilizing sugar is combined with carboxymethyl cellulose, the ability of the enzyme to reduce biofilm is limited. This indicates that carboxymethyl cellulose is not an ideal moderator in combination with the stabilizing sugars trehalose or sucrose. The study indicates that the stabilizing sugar combined with PVA still has a strong ability to remove biofilm. Since trehalose is an ideal stabilizing sugar for use around the eye, the combination of PVA and trehalose will be examined in the buffer study below. Trehalose is also studied as an independent variable below.

[0468] Trehalose

[0469] The ability of α-amylase to break down biofilm was tested with 3%, 6.8%, and 8% trehalose solutions. There were no significant differences in enzyme activity at these concentrations (α=0.05). 6.8% trehalose is the highest concentration reported in commercially available eye drops and is considered well tolerated. Higher concentrations of trehalose may be considered because the solution is applied to intact skin rather than the eye itself. See Figure 17 .

[0470] Example 11. pH

[0471] The literature shows that the optimal activity of alpha-amylase from Aspergillus oryzae is between a pH of 5-6. Tears have a pH of 6.5 to 7.6, normal skin has a pH of 4.7 to 5.75, and oily skin has a pH of 5.7-7. Application of the enzymatic composition will be on the eyelid, not in the eye. However, due to the proximity to the eye, a pH of 5.8-6 will be used in the final composition to balance enzymatic activity and user comfort. In testing, the present disclosure determined that a 4% PVA solution had a pH of 5.8 and a 2% PVA solution had a pH of 5.9.

[0472] The following buffers are contemplated for pH stabilization:

[0473]

[0474] Phosphate buffer and citrate buffer were further analyzed for use in compositions to maintain a pH of 5.8 as they are ideal candidates for this pH range. 2 During the test, calcium reacts with phosphate and precipitates. If CaCl 2When used as a cofactor, the reaction eliminates phosphate buffer as a candidate. This indicates that the optimal ophthalmic buffer candidate is a citrate buffer. Based on the literature, solutions were prepared and tested for their ability to remove biofilms at citrate buffer concentrations of 50 mM and 100 mM. Trehalose, used as a thermal stabilizer for the enzyme, was added at 200 mM as it was reported in the literature to be well tolerated for use in eye drops. When citrate buffer was added directly to the 4% PVA solution, sodium citrate formed a gel and did not dissolve. To test the citrate buffer with PVA, sodium citrate and citric acid were dissolved in DI water and mixed with the 4% PVA solution in a 50:50 ratio to prepare a 2% PVA solution for testing. All citrate buffer solutions were brought to pH 5.8 using NaOH and HCl. Figure 18A and Figure 18B is a graph showing the results of testing 50 mM - 100 mM citrate buffers in the presence of sugar stabilizers and moderators.

[0475] The study indicates that citrate buffers interact with PVA to inhibit the biofilm removal function of the composition at both 50 mM and 100 mM concentrations. Citrate buffers used alone with NaOH and HCl to bring the composition pH to 5.8 enhanced the performance of the enzyme. See Figure 18A and Figure 18B . The optimal performance of the enzyme was in the absence of citrate buffer, in 2% PVA or 4% PVA with or without trehalose.

[0476] Example 12. Hyaluronic acid

[0477] Hyaluronic acid and tea tree oil are common additives in facial cleansers and eyelid cleansers. In this study, 0.3% hyaluronic acid and 5% tea tree oil were added to α - amylase with and without trehalose to quantify the effect on biofilm removal. Figure 19 is a graph showing the results of testing the effects of hyaluronic acid and tea tree oil on compositions containing α - amylase. As Figure 19 shown, 0.3% hyaluronic acid slightly increased the ability of the enzyme to remove biofilms. Tea tree oil did not significantly enhance or reduce the performance of the enzyme. This indicates that hyaluronic acid may have a beneficial effect in compositions for biofilm removal. In eye drop formulations, hyaluronic acid is commonly combined with trehalose to obtain their combined hydrating benefits.

[0478] Example 13. Treat human subjects with the compositions of the present disclosure

[0479] All images are from the same subject at the Watson Dry Eye Center in Raleigh, North Carolina, at the specified times. The images were reviewed retrospectively to determine whether visual signs of biofilm on the eyelids were removed by a composition of α - amylase from Aspergillus oryzae and 0.01% hypochlorous acid. It should be noted that from May 23, 2018 - November 20, 2020, the patient washed his eyes twice a day with only 0.01% hypochlorous acid in saline. Thereafter, the patient used a novel composition of α - amylase from Aspergillus oryzae and 0.01% hypochlorous acid. The first composition was prepared from powdered α - amylase from Aspergillus oryzae and liquid 0.01% hypochlorous acid in saline and then immediately applied to the eyelids with a cotton swab. The second composition includes Ca 2+ and has superior performance. The dosage information is written on the timeline below.

[0480] Treatment timeline - Before using the composition containing the following

[0481] The subject had the first treatment session for dry eye symptoms and recurrent ocular inflammation on May 23, 2018. Prior to this day, the subject received a combination of Azasite and oral antibiotics (100 mg doxycycline / day), but there seemed to be no relief of symptoms. Figure 1A and Figure 1B are two different area photos of the subject's eyes taken on May 23, 2018 (time 0), depicting meibomian gland obstruction, shiny biofilm at the base of the eyelashes, shiny biofilm on the eyelashes, and misdirected eyelash growth. After the photos were taken, the subject was treated with Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation.

[0482] The subject had a follow - up treatment session on December 4, 2018. Figure 2A and Figure 2B are photos depicting two different areas of the subject's eyes on December 4, 2018, approximately 6 months (time 1) after the photos of Figure 1A and Figure 1B were taken. After the photos were taken, the subject received 1 treatment of Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation. Subsequently, the subject received a daily dose of Avenova (0.01% hypochlorous acid (HOCL)). Minimal improvement in symptoms was reported during this session.

[0483] The subject had a follow - up treatment session on June 18, 2019, using Avenova (0.01% hypochlorous acid (HOCl)) daily. Figure 3A and Figure 3B are photos depicting two different areas of the subject's eyes on June 18, 2019, after the photos of Figure 2A and Figure 2BPhotographs of two different regions of the subject's eyes approximately 6 months (Time 2) after the Figure 3A photographs were taken. After the photographs were taken, the subject received 1 treatment of Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation. Subsequently, the subject was prescribed daily use of Avenova (0.01% hypochlorous acid (HOCl)). As

[0484] shown, the biofilm continued to grow at the base of the eyelashes. Figure 4A and Figure 4B are photographs of two different regions of the subject's eyes approximately 4 months (Time 3) after the Figure 3A photographs were taken. After the photographs were taken, the subject received 1 treatment of Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation. Subsequently, the subject continued treatment with daily use of Avenova (0.01% hypochlorous acid (HOCl)). As Figure 3B shown, a shiny biofilm remained at the eyelashes and inside the eyelids, indicating that the acid treatment alone was not sufficient to remove the biofilm. Figure 4A and Figure 4B In October 2020, a first composition was prepared with 1 mg / ml of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid.

[0485] and Figure 5A are photographs of two different regions of the subject's eyes approximately on November 2, 2020 after several applications of 1 mg / ml of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. This time frame is approximately 1 month (Time 4) after the Figure 5B photographs were taken. Within the 1-month treatment time frame, the biofilm was still visibly present after several low-dose applications, as Figure 4A shown, and Figure 4B shown. Figure 5A and Figure 5B The subject received daily doses of a composition containing 1 mg / ml of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid between Time 4 and May 26, 2021 (Time 5), where the subject underwent regular daily use of the composition.

[0486] and Figure 6A are photographs of two different regions of the subject's eyes approximately 6 months after the Figure 6B photographs were taken. After the Figure 5A photographs were taken, and Figure 5B shown, and Figure 5A and Figure 5BAfter photographs were taken, the subject received a conventional daily application of a composition containing 1 mg / ml of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. As Figure 6A and Figure 6B shown, the biofilm was significantly eliminated, the eyelashes were free of all biofilm, the inflammation and redness in the eyelids were reduced, and the patient reported a significant improvement in dry eye symptoms.

[0487] On October 5, 2021, approximately 5 months after the conventional daily application of a composition containing 1 mg / ml of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid, the subject reported significantly improved symptoms, and new eye images were taken at the Watson Dry Eye Center in Raleigh NC. As Figure 7A and Figure 7B shown, biofilm growth was blocked and the eyelid tissue was no longer inflamed.

[0488] The subject was continued to be monitored. On February 3, 2022, approximately 4 months (time 7) after the conventional daily application of a composition containing 1 mg / ml of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid, biofilm growth on the subject's eyes was blocked, see Figure 8A and Figure 8B . In addition, no inflammation was observed within this time frame. Between time 7 and a period of approximately 6 months (August 4, 2022), a tolerance test of a higher concentration dose (i.e., 10 mg / ml of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid) was conducted on the subject at a twice / week usage. No adverse effects were observed for the higher dose of 10 mg / ml α-amylase. See Figure 9A and Figure 9B .

[0489] Example 14. Treatment of human subjects with the compositions of the present disclosure

[0490] A composition containing approximately 3% α-amylase, 0.06% sodium chloride, and 0.02% hypochlorous acid solution was prepared in ionized water and applied directly to the lid margins of patients presenting elevated levels of eyelid biofilm once every two weeks. Observations within a 6-month period indicated a significant reduction in biofilm on the lid margins, an increase in tear production, and a reduction in signs of ocular surface stress. Pilot studies indicated that a composition containing approximately 3% α-amylase, 1% lysozyme, 0.06% sodium chloride, and 0.02% hypochlorous acid solution in ionized water had similar improvements. The compositions in the pilot studies were freshly prepared before each use. Further composition development is to study combinations of the above-mentioned components with preservatives, astringents, emollients, emollients, hyperosmotic agents, and vasoconstrictors described throughout this disclosure to improve the long-term stability of the composition. Figure 10A andFigure 10B is a photograph depicting a direct comparison of the area of the eye before and after treatment with a composition containing 10 mg / ml α-amylase and Ca in 0.01% hypochlorous acid 2+ of the composition.

[0491] Example 15. Formulation

[0492] This protocol outlines the procedure for testing the compatibility of a composition of α-amylase from Aspergillus oryzae with 0.01% hypochlorous acid and its ability to remove biofilms from Staphylococcus aureus. 0.01% hypochlorous acid is intended to prevent microbial growth and not to extend the shelf life of α-amylase. This experiment will also test the effect of hypochlorous acid on the enzyme by using the composition of α-amylase and hypochlorous acid at different time points after the composition is prepared. The ability of the composition to remove biofilms will be tested immediately after the composition is prepared and at the 30-minute and 1-hour time points. This test will indicate whether it is beneficial to combine aqueous hypochlorous acid with dry α-amylase before biofilm application.

[0493] Formulation

[0494] This experiment uses the off-the-shelf eyelid cleanser Avenova (0.01% hypochlorous acid in saline). 0.4% calcium chloride will be added to the hypochlorous acid solution to ensure the presence of calcium ions in all the tested solutions.

[0495] Prepare 10 ml of a 10 mg / ml α-amylase solution with cofactors.

[0496] Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0497] Dissolve 40 mg of calcium chloride and 56 mg of sodium chloride into the solution

[0498] One hour before the test:

[0499] Prepare 5 ml of a 10 mg / ml α-amylase solution with 0.01% hypochlorous acid

[0500] Add 5 mL of Avenova (0.01% hypochlorous acid) to a flask.

[0501] Dissolve 20 mg of calcium chloride into the solution.

[0502] Add 50 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065). Mix until the α-amylase is dissolved and evenly distributed.

[0503] Thirty minutes before the test:

[0504] Prepare 5 ml of a 10 mg / ml α-amylase solution with 0.01% hypochlorous acid

[0505] Add 5 mL of Avenova (0.01% hypochlorous acid) to the flask.

[0506] Dissolve 20 mg of calcium chloride in the solution.

[0507] Add 50 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065). Mix until the α-amylase is dissolved and evenly distributed.

[0508] Immediately before plating:

[0509] Prepare 5 ml of a 10 mg / ml α-amylase solution with 0.01% hypochlorous acid

[0510] Add 5 mL of Avenova (0.01% hypochlorous acid) to the flask.

[0511] Dissolve 20 mg of calcium chloride in the solution.

[0512] Add 50 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog #10065). Mix until the α-amylase is dissolved and evenly distributed.

[0513] Test composition

[0514] Carefully tilt the plate and remove all the liquid from the wells by pipetting from the bottom corners of the wells with the pipette tip inserted, to remove non-adherent cells without disturbing the biofilm growing on the bottom of the wells.

[0515] When processing the first row using an 8-channel pipette, start the timer for 10 minutes. Each row should be processed every 30 seconds, ensuring that the composition in the corners of the wells is gently released so as not to mechanically disturb the biofilm.

[0516] For row 1: Add 50 μl of DI water to row 1.

[0517] For row 2: Add 50 μl of 10 mg / ml α-amylase with cofactor to row 2.

[0518] For row 3: Add 50 μl of 10 mg / ml α-amylase with cofactor and hypochlorous acid to row 3 and incubate for 30 minutes.

[0519] For row 4: Add 50 μl of 10 mg / ml α-amylase with cofactor and hypochlorous acid and incubate for 1 hour.

[0520] For row 5: Combine 5 ml of 10 mg / ml α-amylase solution with hypochlorous acid. Add 50 μl of 10 mg / ml α-amylase with cofactor and hypochlorous acid to row 5.

[0521] For row 6: Add 50 μl of 0.018% hypochlorous acid solution to row 7.

[0522] For row 7: Add 50 μl of 7.5% bleach (sodium hypochlorite) solution to row 7.

[0523] Incubate the plate at 37 °C for the remaining 10 minutes.

[0524] Test for biofilm reduction

[0525] After 10 min, starting from row 1, remove all the fluid row by row every 30 seconds. The fluid must be removed with gentle pipetting, with the plate at an angle and the pipette at the corner, to remove non-adherent cells without disturbing the biofilm growing on the bottom of the wells. Add 50 μl of 0.1% crystal violet (CV) to stain the adherent cells in all the wells. Carefully rinse all the wells by dipping the plate into a DI water bath to remove all the remaining crystal violet. Dry the well plate and measure the optical density of the biofilm at 600 nm using a plate reader. Once all the measurements have been made, transfer the well plate to a bleach solution for disinfection and dispose of it in a biohazard waste bin.

[0526] Results

[0527] The results showed that α-amylase with cofactor and α-amylase with cofactor combined with either hypochlorous acid spray had no statistical significance for biofilm reduction across all time points. This indicates that 0.01% hypochlorous acid does not inhibit the enzyme's removal of biofilm. Although 0.01% hypochlorous acid did slightly reduce the biofilm, there was no additional benefit of increased biofilm removal when combined with α-amylase and cofactor. See Figure 13 .

[0528] Example 16. Enzymatic facial and eyelid cleanser

[0529] The various compositions described herein have been developed for enzymatic facial and / or eyelid treatment.

[0530] The enzymatic facial and eyelid cleansing device utilizes α - amylase to break down the extracellular matrix of the biofilm formed on the eyelids. A biofilm is a structured community of microorganisms adhering to a surface, surrounded by a self - produced extracellular matrix (ECM). The ECM mainly contains polysaccharides, proteins, nucleic acids, and lipids. This matrix provides structural stability to the biofilm, protects the internal bacteria from external factors, and helps the biofilm adhere to the surface.

[0531] Biofilms are aggregates of microorganisms such as bacteria, embedded in a self - produced matrix of proteins and polysaccharides on the eyelids. These biofilms can cause inflammation and infection of the eyelid margin, known as blepharitis, and can also lead to dry eye disease by disrupting the tear film and causing tear evaporation.

[0532] The α - amylase isoform in the composition acts as an enzyme that breaks down the polysaccharides in the biofilm matrix. By doing so, it destabilizes the structure of the biofilm, making it easier to remove from the eyelids. As the ECM becomes unstable, the biofilm becomes more easily mechanically removed (e.g., by wiping). Additionally, the bacteria within the biofilm lose their protective shield, making them more vulnerable to antimicrobials or the body's immune response.

[0533] In summary, the isoform α - amylase from Aspergillus oryzae targets the polysaccharide component of the extracellular matrix of the biofilm, breaks it down, and promotes the removal of the biofilm from the eyelids. This can be particularly helpful in managing conditions such as blepharitis and dry eye disease, in which the biofilm causes pathology.

[0534] The enzymatic composition can be used via different delivery mechanisms, such as sterile wipes, sprays, or single - dose or multi - dose sterile applicators. In some cases, the composition is a cleanser. The composition can be packaged as wipes, sprays, pads, or another suitable applicator infused with α - amylase powder or α - amylase solution derived from Aspergillus oryzae.

[0535] 16.1 Sterile wipes

[0536] The compositions of the present disclosure are packaged as sterile wipes pre - saturated with the composition. These wipes are individually packaged or packaged in sets. Such wipes can be further packaged in a container or as part of a kit together with other reagents.

[0537] Method of using the composition packaged as a sterile wipe: The subject gently closes their eyes and uses the sterile wipe to clean the eyelids from the inner corner to the outer corner, for example, with a horizontal stroke. The subject ensures that the composition is preferentially applied to the root of the eyelashes where biofilm may accumulate.

[0538] The wipes are individually packaged for single use, reducing the risk of contamination. The wipes are portable and convenient for travel or use outside.

[0539] 16.2 Sprays

[0540] The enzymatic composition can be formulated as a solution (e.g., spray or drops) in a bottle.

[0541] Method of using the composition packaged as a spray: A spray bottle containing the enzymatic composition can allow for direct application to the eyelids or spraying onto a clean cloth or cotton pad for application. The subject can close their eyes and gently spray the solution directly onto the eyelids. Alternatively, the subject can spray the solution onto a clean cloth or cotton pad. If using a cloth or pad, gently wipe the eyelids from the inner canthus to the outer canthus.

[0542] Spray bottles are easy to use and allow for quick application. They are also suitable for multiple uses without direct contact with the eyelids, which reduces the risk of contamination.

[0543] 16.3 Single-dose or multi-dose sterile containers:

[0544] Single-dose or multi-dose sterile containers are used to deliver the enzymatic solution to a sterile applicator such as a wipe or sponge in a controlled manner and then applied to the eyelids. The applicator can be attached to the sterile container or used separately.

[0545] Method of using the composition packaged in a single-dose or multi-dose sterile container: The subject closes their eyes and uses the applicator to apply the solution along the lid margin and lashes. The subject carefully cleans the eyelids with horizontal strokes, moving from the inner corner to the outer corner. The applicator supports the targeted application of the solution / gel, which may be beneficial for those who require more controlled application or have sensitivity issues.

[0546] While the present invention is satisfied by many different forms of embodiments as described in detail in connection with the preferred embodiments of the present invention, it should be understood that the present disclosure is considered an example of the principles of the present invention and is not intended to limit the present invention to the specific embodiments shown and described herein. Many variations can be made by those skilled in the art without departing from the spirit of the present invention. The scope of the present invention will be measured by the appended claims and their equivalents. The abstract and title should not be construed as limiting the scope of the present invention as their purpose is to enable the appropriate authorities as well as the general public to quickly determine the general nature of the present invention. In the appended claims, unless the term "means" is used, the features or elements recited therein should not be construed as a means-plus-function limitation under 35 U.S.C.§112, of the means-plus-function limitation.

Claims

1. A method for treating one or more of blepharitis and dry eye, comprising administering to a subject a therapeutically effective amount of a composition comprising an active α-amylase stabilized with a certain concentration of CaCl 2 or a functional fragment thereof and one or more pharmaceutically acceptable excipients.

2. The method according to claim 1, wherein the composition is in a topical applicator.

3. The method according to claim 2, wherein the topical applicator is a wipe.

4. The method according to claim 3, wherein the wipe is pre - moistened.

5. The method according to claim 3, wherein the wipe contains dry ingredients.

6. The method according to claim 1, wherein the composition is formulated as a spray or a liquid.

7. The method according to claim 1, wherein the composition is applied to the eyelids.

8. The method according to claim 1, wherein the administration promotes the removal of a biofilm in or in the area around the subject's eye.

9. The method according to claim 8, wherein the therapeutically effective amount of the composition is administered daily for a period of at least 10 seconds.

10. The method according to claim 9, wherein the therapeutically effective amount of the composition is administered daily for a period of at least 1 week.

11. The method according to claim 1, wherein the α - amylase shares 90% sequence homology with the α - amylase peptide sequence from Aspergillus oryzae.

12. The method according to claim 11, wherein the α - amylase shares 95% sequence homology with the α - amylase peptide sequence from Aspergillus oryzae.

13. The method according to claim 1, wherein the composition comprises from 1 mg / mL (w / v) of the active α - amylase to 20 mg / mL (w / v) of the active α - amylase.

14. The method according to claim 13, wherein the composition comprises from 1 mg / mL (w / v) of the active α - amylase to 10 mg / mL (w / v) of the active α - amylase.

15. The method according to claim 1, wherein the therapeutically effective amount comprises one or more drops of a formulation having the active α - amylase between 1 I.U. / mg and 3000 I.U. / mg.

16. The method according to claim 1, wherein the concentration of CaCl 2 is in the range from 0.2% (w / v) to 0.8% (w / v).

17. The method according to claim 1, wherein the composition further comprises a citrate buffer.

18. The method according to claim 1, wherein the administration is ocular administration.

19. The method according to claim 1, wherein the administration is topical administration to the external eye.

20. The method according to claim 1, wherein the composition further comprises a demulcent.

21. The method according to claim 20, wherein the demulcent is sodium carboxymethylcellulose (CMC).

22. The method according to claim 21, wherein the composition comprises from 0.2% to 2.5% of the CMC.

23. The method according to claim 20, wherein the demulcent is polyvinyl alcohol (PVA).

24. The method according to claim 23, wherein the composition comprises from 0.1% to 4% of the PVA.

25. The method according to claim 1, wherein the composition is an ophthalmic composition.

26. The method according to claim 25, wherein the composition further comprises one or more of an ophthalmic astringent, an ophthalmic emollient, an ophthalmic demulcent, an ophthalmic hyperosmotic agent, or an ophthalmic vasoconstrictor.

27. The method according to claim 25, wherein the composition is formulated as a liquid.

28. The method according to claim 25, wherein the composition is formulated as a gel.

29. The method according to claim 1, wherein the subject is a human.

30. An ophthalmic composition comprising a therapeutically effective amount of a functional active α - amylase for treating blepharitis or dry eye conditions, a certain concentration of CaCl for stabilizing the α - amylase 2 and one or more pharmaceutically acceptable excipients.

31. The ophthalmic composition according to claim 30, wherein the α - amylase shares 90% sequence homology with the α - amylase peptide sequence from Aspergillus oryzae.

32. The ophthalmic composition according to claim 31, wherein the α - amylase shares 95% sequence homology with the α - amylase peptide sequence from Aspergillus oryzae.

33. The ophthalmic composition according to claim 30, wherein the composition comprises from 1 mg / ml (w / v) of the functionally active α - amylase to 20 mg / ml (w / v) of the functionally active α - amylase.

34. The ophthalmic composition according to claim 30, wherein the concentration of CaCl 2 is in the range from 0.2% (w / v) to 0.8% (w / v).

35. The ophthalmic composition according to claim 30, wherein the composition further comprises one or more of an ophthalmic astringent, an ophthalmic emollient, an ophthalmic demulcent, an ophthalmic hyperosmotic agent, or an ophthalmic vasoconstrictor.

36. The ophthalmic composition according to claim 35, wherein the composition further comprises sodium carboxymethylcellulose between 0.2% (v / v) and 3.5% (v / v).

37. The ophthalmic composition according to claim 35, wherein the composition further comprises hydroxyethylcellulose between 0.2% (v / v) and 3.5% (v / v).

38. The ophthalmic composition according to claim 35, wherein the composition further comprises hypromellose between 0.2% (v / v) and 3.5% (v / v).

39. The ophthalmic composition according to claim 35, wherein the composition further comprises methylcellulose between 0.2% (v / v) and 3.5% (v / v).

40. The ophthalmic composition according to claim 35, wherein the composition further comprises dextran between 0.01% (v / v) and 1.0% (v / v).

41. The ophthalmic composition according to claim 35, wherein the composition further comprises gelatin between 0.001% (v / v) and 0.1% (v / v).

42. The ophthalmic composition according to claim 35, wherein the composition further comprises glycerol between 0.01% (v / v) and 1.5% (v / v).

43. The ophthalmic composition according to claim 35, wherein the composition further comprises polyethylene glycol 300 between 0.05% (v / v) and 1.5% (v / v).

44. The ophthalmic composition according to claim 35, wherein the composition further comprises polyethylene glycol 400 between 0.05% (v / v) and 1.5% (v / v).

45. The ophthalmic composition according to claim 35, wherein the composition further comprises polysorbate in the range of 0.2% (v / v) to 1.0% (v / v).

46. The ophthalmic composition according to claim 35, wherein the composition further comprises propylene glycol in the range of 0.2% (v / v) to 2.0% (v / v).

47. The ophthalmic composition according to claim 35, wherein the composition further comprises polyvinyl alcohol in the range of 0.2% (v / v) to 6.0% (v / v).

48. The ophthalmic composition according to claim 35, wherein the composition further comprises povidone in the range of 0.1% (v / v) to 4.0% (v / v).

49. The ophthalmic composition according to claim 35, wherein the composition further comprises benzalkonium chloride (BAK) in the range of 0.001% (v / v) to 0.1% (v / v).

50. The ophthalmic composition according to claim 35, wherein the composition further comprises polyquad (polyquaternium-1) in the range of 0.0001% (v / v) to 0.01% (v / v).

51. The ophthalmic composition according to claim 35, wherein the composition further comprises stabilized oxychloride complex.

52. The ophthalmic composition according to claim 35, wherein the composition further comprises sodium perborate.

53. The ophthalmic composition according to claim 35, wherein the composition further comprises disodium edetate and sorbic acid.

54. The ophthalmic composition according to claim 35, wherein the composition further comprises borate, sorbitol, propylene glycol and zinc ion buffer.

55. The ophthalmic composition according to claim 35, wherein the composition further comprises citrate buffer.

56. The ophthalmic composition according to claim 35, wherein the composition further comprises polyhexamethylene biguanide.

57. The ophthalmic composition according to claim 30, wherein the composition is formulated as a liquid.

58. The ophthalmic composition according to claim 30, wherein the composition is formulated as a gel.

59. A composition comprising any combination of the components described in claims 30 - 58.

60. A composition comprising a functional active α-amylase at a concentration in the range from 1 mg / mL to 20 mg / mL, CaCl at a concentration in the range from 0.4% (w / v) to 0.8% (w / v) for stabilizing the α-amylase 2 , polyvinyl alcohol (PVA) at a concentration in the range from 0.1% to 5% and at least one pharmaceutically acceptable excipient.

61. The composition according to claim 60, wherein the concentration of the functional active α - amylase is 10 mg / mL.

62. The composition according to claim 60, wherein the concentration of the polyvinyl alcohol (PVA) is 4%.

63. The composition according to claim 60, wherein the concentration of CaCl 2 is 0.4%.

64. A topical applicator comprising a functional active α-amylase having a concentration in the range from 1 mg / mL to 20 mg / mL, CaCl having a concentration in the range from 0.2% (w / v) to 0.8% (w / v) for stabilizing the α-amylase 2 , polyvinyl alcohol (PVA) having a concentration in the range from 0.1% to 5%, and at least one pharmaceutically acceptable excipient.

65. The topical applicator according to claim 64, comprising functional active α-amylase at 10 mg / mL, CaCl at a concentration of about 0.4% (w / v) 2 and polyvinyl alcohol (PVA) at a concentration of 4% (w / v).

66. The topical applicator according to claim 64, wherein the topical applicator is a wipe.

67. The topical applicator according to claim 64, wherein the wipe is individually wrapped.

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