Dry Eye Treatment

Abstract

Ophthalmic compositions for removing biofilms from the eye and for treating ocular inflammation, blepharitis, and dry eye are disclosed, comprising α-amylase, and optionally other ingredients.

Description

[Technical Field]

[0001] In the following discussion, certain articles and methods will be described for background and introductory purposes. Nothing contained herein should be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referred to herein do not constitute prior art under applicable statutory provisions. [Background technology]

[0002] Blepharitis is inflammation of the eyelids and surrounding areas. It usually affects both eyes, including the area along the eyelid margin. Blepharitis is thought to occur when small oil glands near the base of the eyelashes become clogged, causing irritation and redness. Several diseases and conditions can be associated with blepharitis. In addition to being uncomfortable and unsightly, blepharitis often occurs as a chronic condition that is difficult to treat because existing treatments, including artificial tears, immunosuppressants (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 treatment, or mechanically pushing meibum out of the meibomian glands, do not address the underlying cause of the disease. Summary of the Invention [Problem to be solved by the invention]

[0003] There remains a significant gap in the therapeutic strategies for treating blepharitis, and this is largely due to the limitations of existing treatments in addressing the underlying cause.The present disclosure addresses this need by first identifying eyelid biofilm formation as a critical factor influencing the prognosis of blepharitis.Second, the present disclosure provides a composition and its use method that provides effective therapeutic treatment for blepharitis and dry eye. [Means for solving the problem]

[0004] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended for use in limiting the scope of the claimed subject matter. Other features, details, applications, and advantages of the claimed subject matter will become apparent from the following Detailed Description, including the embodiments illustrated in the accompanying drawings and defined in the appended claims.

[0005] In some embodiments, the present disclosure provides 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 enzyme or a functional fragment thereof, optionally stabilized with a concentration of CaCl2, and one or more pharmaceutically acceptable excipients. In some cases, administration promotes the removal of biofilm in the subject's eye or in the area surrounding the subject's eye. In some cases, the 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 at least 1 month.

[0006] In some embodiments, the present disclosure provides an ophthalmic composition comprising a therapeutically effective amount of a functionally active α-amylase enzyme for treating blepharitis or a dry eye condition, CaCl in a concentration that stabilizes the α-amylase enzyme, and one or more pharmaceutically acceptable excipients.

[0007] In some embodiments, the present disclosure provides compositions and methods comprising an α-amylase enzyme. In some embodiments, the present disclosure provides 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 enzyme or a functional fragment thereof stabilized with a concentration of CaCl2 and one or more pharmaceutically acceptable excipients. In some formulations, the composition is present in a wipe, for example, a pre-moistened wipe or a wipe containing dry ingredients. In many cases, the composition is applied to the eyelid or used as a facial wipe. In some cases, administration promotes the removal of biofilm in the subject's eye or in the area surrounding the subject's eye. The composition can be administered daily for a time sufficient to provide contact with the biofilm, for example, at least 1 second, at least 5 seconds, or at least 10 seconds. The composition can also be administered daily for a period of at least one week. In some embodiments, the α-amylase enzyme shares at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity with an α-amylase peptide sequence from Aspergillus oryzae. In some embodiments, the composition comprises 1 mg / mL (w / v) to 20 mg / mL (w / v) of active α-amylase enzyme, 1 mg / mL (w / v) to 10 mg / mL (w / v) of active α-amylase enzyme, or another suitable amount. In some embodiments, the therapeutically effective amount comprises one or more droplets of a formulation having between 1 IU / mg and 3000 IU / mg of active α-amylase enzyme. In some embodiments, the concentration of CaCl2 ranges from 0.2% to 0.8% (w / v). In some embodiments, the composition further comprises a citrate buffer.

[0008] In some embodiments, the present disclosure provides an ophthalmic composition comprising a therapeutically effective amount of a functionally active α-amylase enzyme for treating blepharitis or a dry eye condition, a concentration of CaCl2 for stabilizing the α-amylase enzyme, and one or more pharmaceutically acceptable excipients. The α-amylase enzyme in the composition may share at least 90%, at least 95%, or at least 99% sequence identity with the α-amylase peptide sequence from Aspergillus oryzae. The composition may contain from 1 mg / ml (w / v) to 20 mg / ml (w / v) of functionally active α-amylase enzyme. The composition may contain between 1 IU / mg and 3000 IU / mg of active α-amylase enzyme. In some embodiments, the concentration of CaCl2 ranges from 0.2% 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), for example, 0.2% to 3.5% CMC. In some formulations, the demulcent may be polyvinyl alcohol (PVA), for example, 0.1% to 5% PVA. In some embodiments, the composition further comprises a citrate buffer.

[0009] In some formulations, the composition comprises between 0.2% (v / v) and 3.5% (v / v) hydroxyethylcellulose, between 0.2% (v / v) and 3.5% (v / v) hypromellose, between 0.2% (v / v) and 3.5% (v / v) methylcellulose, between 0.01% (v / v) and 1.0% (v / v) dextran, between 0.001% (v / v) and 0.1% (v / v) gelatin, between 0.01% (v / v) and 1.5% (v / v) glycerin, between 0.05% (v / v) and 1.5% (v / v) polyethylene glycol 300, between 0.05% (v / v) and 1 ... methylcellulose, between 0.01% (v / v) and 1.0% (v / v) methylcellulose, between 0.01% (v / v) and 1.0% (v / v) methylcellulose, between 0.01% (v / v) and 1.0% (v / v) methylcellulose, between 0.01% (v / v) and 1.0% (v / v) methylcellulose, between 0.01% Further containing between 1.5% (v / v) polyethylene glycol 400, between 0.2% (v / v) and 1.0% (v / v) polysorbate, between 0.2% (v / v) and 2.0% (v / v) propylene glycol, between 0.2% (v / v) and 6.0% (v / v) polyvinyl alcohol, between 0.1% (v / v) and 4.0% (v / v) povidone, between 0.001% (v / v) and 0.1% (v / v) benzalkonium chloride (BAK), and / or between 0.0001% (v / v) and 0.01% (v / v) polyquad (polyquaternium-1). In some formulations, the composition further comprises stabilized oxychloro complexes, sodium perborate, disodium edetate, and sorbic acid, borate, sorbitol, propylene glycol, and zinc ion buffer, polyhexanide (polyhexamethylene biguanide). In preferred cases, the composition can be stabilized in a citrate buffer.

[0010] The composition may be an ophthalmic composition, i.e., a composition that is administered to the eye. In some embodiments, the administration is topical administration to the outside of the eye. In some embodiments, the composition is formulated as a liquid. In others, the composition is formulated as a gel. Furthermore, in certain preferred cases, the composition is formulated into a wipe, such as a dry powder or a moistened wipe.

[0011] In some embodiments, the present disclosure provides a composition comprising a functionally active α-amylase enzyme at a concentration ranging from 1 mg / mL to 20 mg / mL, CaCl at a concentration ranging from 0.4 to 0.8% (w / v) to stabilize the α-amylase enzyme, polyvinyl alcohol (PVA) at a concentration ranging from 0.1% to 5%, and at least one pharmaceutically acceptable excipient. In some embodiments, the concentration of the functionally active α-amylase enzyme is 10 mg / mL, and / or the concentration of polyvinyl alcohol (PVA) is 4%, and / or the concentration of CaCl is 0.4%.

[0012] In some embodiments, the present disclosure provides a topical applicator, e.g., a wipe, comprising a functionally active α-amylase enzyme at a concentration ranging from 1 mg / mL to 20 mg / mL, CaCl at a concentration ranging from 0.4 to 0.8% (w / v) to stabilize the α-amylase enzyme, polyvinyl alcohol (PVA) at a concentration ranging from 0.1% to 5%, and at least one pharmaceutically acceptable excipient. In some formulations, the topical applicator, e.g., a wipe, comprises functionally 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 embodiments, the wipes are individually packaged.

[0013] These aspects, as well as other features and advantages of the present invention, are described in further detail below.

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided to the Patent and Trademark Office upon request and payment of the necessary fee.

[0015] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0016] [Figure 1] 1A and 1B are photographs showing two different areas of a subject's eye suffering from blocked meibomian glands, shiny biofilm at the base of the eyelashes, shiny biofilm on the eyelashes, and maloriented eyelash growth. After photography, the subject was treated with Blephax and intense pulsed light therapy to remove the biofilm and treat the inflammation. [Figure 2] Figures 2A and 2B are photographs showing two different areas of the subject's eye approximately six months after the photographs in Figures 1A and 1B were taken. After the photographs were taken, the subject underwent one treatment with Blephax and intense pulsed light therapy to remove biofilm and treat inflammation. The subject was then prescribed daily use of Avenova (0.01% hypochlorous acid (HOCL)). [Figure 3] Figures 3A and 3B are photographs showing two different areas of the subject's eye approximately six months after the photographs in Figures 2A and 2B were taken. After the photographs were taken, the subject underwent one treatment with Blephax and intense pulsed light therapy to remove biofilm and treat inflammation. The subject was then prescribed daily use of Avenova (0.01% hypochlorous acid (HOCL)). [Figure 4] Figures 4A and 4B are photographs showing two different areas of the subject's eye approximately four months after the photographs in Figures 3A and 3B were taken. After the photographs were taken, the subject received one treatment of Blephex and intense pulsed light therapy to remove biofilm and treat inflammation. The subject was then treated with daily use of Avenova (0.01% hypochlorous acid (HOCL)). [Figure 5] Figures 5A and 5B are photographs showing two different areas of a subject's eye approximately one month after the photographs in Figures 4A and 4B were taken, and after the subject received several applications of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. [Figure 6]Figures 6A and 6B are photographs showing two different areas of the subject's eye approximately six months after the photographs in Figures 5A and 5B were taken. After the photographs in Figures 5A and 5B were taken, the subject underwent routine daily use of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. As shown in Figures 6A and 6B, the biofilm had visibly disappeared, the eyelashes were cleaned of all biofilm, inflammation and redness in the eyelids had decreased, and the patient reported a significant improvement in her dry eye symptoms. [Figure 7] 7A and 7B are photographs showing two different areas of a subject's eye approximately six months after the photographs of FIGS. 5A and 5B were taken, and after approximately six months of routine daily use of a composition comprising 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid over a period of time. [Figure 8] Figures 8A and 8B are photographs showing two different areas of the subject's eye approximately four months after the photographs of Figures 6A and 6B were taken, and after routine daily use of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. [Figure 9] 9A and 9B are photographs showing the area of ​​a subject's eye after receiving a dose of 10 mg / ml of α-amylase in 0.01% hypochlorous acid. [Figure 10] 10A and 10B are photographs showing a direct comparison of an area of ​​the eye before and after treatment with a composition comprising 10 mg / ml α-amylase and Ca in 0.01% hypochlorous acid. [Figure 11A] 1 is a chart showing the results of testing various α-amylase compositions containing various concentrations of calcium cofactor (CaCl). [Figure 11B] 1 is a chart quantifying the percent of biofilm removed within 10 minutes by various compositions containing a range of CaCl2. [Figure 12A]This chart quantifies the analytical results of the effect on enzyme stability of various preservatives: BAK (benzalkonium chloride), oxychloro 0.1%, polyquad (polyquaternium-1) 0.001%, and a bleach control. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065), approximately 30 U / mg, supplemented with 0.4% CaCl2 and 0.56% NaCl. [Figure 12B] This chart quantifies the analytical results of the effect of various preservatives, namely BAK (benzalkonium chloride) and GenAqua / Dequest (sodium perborate), on enzyme stability. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065), approximately 30 U / mg, supplemented with 0.4% CaCl2 and 0.56% NaCl. [Figure 13] 1 is a chart quantifying the analytical results of enzyme activity of a 0.01% hypochlorous acid preservative. [Figure 14A] This chart shows the analytical results of various demulcents, including polyethylene glycol 400 (PEG400) 1%, carboxymethylcellulose sodium (CMC) 2.5%, glycerin 1%, povidone (PVP) 2%, propylene glycol 1%, polyvinyl alcohol (PVA) 4%, dextran 70 0.1% + hypromellose 0.3%. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065), approximately 30 U / mg, supplemented with 0.4% CaCl2 and 0.56% NaCl. [Figure 14B]This chart shows the analytical results of various demulcents, including polyvinyl alcohol (PVA), PVA + α-amylase, sodium carboxymethylcellulose (CMC), and sodium carboxymethylcellulose (CMC) + α-amylase. The α-amylase solution is approximately 30 U / mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) at 10 mg / mL, supplemented with 0.4% CaCl and 0.56% NaCl. [Figure 14C] This chart shows the results of a 36-day test using α-amylase stabilized in 4% PVA. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065), approximately 30 U / mg, supplemented with 0.4% CaCl and 0.56% NaCl. [Figure 15] Figure 1 shows a chart showing the comparative results of ophthalmic antibiotics for the reduction of Staphylococcus aureus biofilm. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065) approximately 30 U / mg, supplemented with 0.4% CaCl and 0.56% NaCl. [Figure 16] Figure 1 shows the results of various heat-stabilizing sugars and demulcents on biofilm reduction, including trehalose and sucrose in combination with demulcents. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065), approximately 30 U / mg, supplemented with 0.4% CaCl and 0.56% NaCl. [Figure 17] This chart shows test results using various concentrations of trehalose, including 3%, 6.8%, and 8%. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065), approximately 30 U / mg, supplemented with 0.4% CaCl and 0.56% NaCl. [Figure 18A]1 is a chart showing the results of testing a 50 mM citrate buffer containing a sugar stabilizer and demulcent. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065), approximately 30 U / mg, supplemented with 0.4% CaCl and 0.56% NaCl. [Figure 18B] 1 is a chart showing the results of testing a 100 mM citrate buffer containing a sugar stabilizer and demulcent. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065), approximately 30 U / mg, supplemented with 0.4% CaCl and 0.56% NaCl. [Figure 19] 1 is a chart showing the results of a test of the effect of hyaluronic acid and tea tree oil on a composition containing α-amylase. The α-amylase solution is 10 mg / mL of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065), approximately 30 U / mg, supplemented with 0.4% CaCl and 0.56% NaCl. DETAILED DESCRIPTION OF THE INVENTION

[0017] It should be understood that the drawings and photographs are not necessarily to scale.

[0018] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0019] definition The terms "protein," "polypeptide," and "peptide" are used interchangeably herein and 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 terms also include modified polymers, such as polypeptides with modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide that has a particular function or structure; for example, the catalytic domain of α-amylase or lysozyme refers to a domain that can break peptide bonds.

[0020] Proteins are said to have an "N-terminus" and a "C-terminus." The term "N-terminus" refers to the beginning of a protein or polypeptide terminated by an amino acid with a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH).

[0021] The term "biofilm," as used herein, refers to a population of microbial cells that adhere to the surface of the eyelid and are encapsulated within a matrix typically made of polysaccharide materials. The lower layer of the biofilm contains microorganisms bound together in a polysaccharide matrix along with DNA, proteins, and other organic components such as inorganic materials. The upper layer is a loose, amorphous layer that extends into the surrounding environment. The fluid layer adjacent to the biofilm typically has a static and a dynamic sublayer.

[0022] The term "conservative amino acid substitution" refers to the substitution of an amino acid normally occurring in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Furthermore, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another, are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue for a non-polar residue. Typical amino acid classifications are summarized below.

[0023] [Table 1]

[0024] "Sequence identity" or "identity," in the context of two polynucleotide or polypeptide sequences, refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison range. When sequence identity percentages are used in reference to proteins, non-identical residue positions often differ through conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), thus not altering the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upward 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 this adjustment are well known. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thus increasing the percentage sequence identity. Thus, for example, if identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions will receive a score between 0 and 1. Conservative substitution scoring is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0025] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned sequences over a comparison range (the highest number of perfectly matched residues), and portions of the polynucleotide sequence in the comparison range may contain additions or deletions (i.e., gaps) when compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison range, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence contains a linked non-homologous sequence), the comparison range is the full length of the shorter of the two sequences being compared.

[0026] Unless otherwise specified, sequence identity / similarity values ​​include values ​​obtained using 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 an equivalent program of any of these, using GAP version 10. "Equivalent program" includes any sequence comparison program that produces alignments that have identical nucleotide or amino acid residue matches and identical sequence percent identity for any two sequences in question when compared to the corresponding alignment produced by GAP version 10.

[0027] 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 organism or body) and processes or reactions that occur within a natural environment. The term "ex vivo" includes cells removed from an individual's body and processes or reactions that occur within such cells.

[0028] A composition or method "comprising" or "including" one or more recited elements may include other elements not specifically recited. For example, a composition "comprises" or "includes" a protein may include the protein alone or in combination with other components. The transitional phrase "consisting essentially of" means that the scope of the claim should be understood to include the specified elements recited in the claim as well as those that do not materially affect the basic novel characteristics of the claimed invention. Thus, the term "consisting essentially of" is not intended to be understood as the equivalent of "comprising" when used in the claims of the present invention.

[0029] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0030] The designations of various values ​​include all integers within or defining a range, and all subranges defined by integers within the range.

[0031] Unless otherwise clear from the context, the term "about" encompasses values ​​within the standard range of measurement error (eg, SEM) of the stated value.

[0032] The term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and when interpreted alternatively ("or") refers to and includes the absence of a combination.

[0033] The term "or" refers to any one member of a particular list and also includes any combination of members of that list.

[0034] The singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a protein" or "at least one protein" can include a plurality of proteins, including mixtures thereof.

[0035] Statistically significant means p≦0.05.

[0036] Detailed Description I. Overview It is intended that all of the functions described in connection with one embodiment of a method, composition, or formulation described herein can be compatible with additional embodiments of the methods, compositions, or formulations described herein, unless expressly stated otherwise or unless the feature or function is incompatible with the additional embodiment. For example, if 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 utilized and performed even when arranged in connection with the alternative embodiment, unless the feature or component is compatible with the alternative embodiment.

[0037] One of the primary causes of dry eye disease is thought to be inflammation within the tissues that produce and support tear production. Blepharitis refers to inflammation of the eyelid margin. It is usually an early symptom of dry eye disease and is commonly observed in patients presenting with dry eye disease. When patients present for blepharitis, it is usually because they have begun to experience symptoms of dry eye disease. Current treatments for dry eye disease include artificial tears, immunosuppressants (restasis), temporary antibiotics, temporary steroids, lymphocyte function-associated antigen-1 (LFA-1) antagonists (xiidra), warm compresses, intense pulsed light treatment, or mechanical removal of meibum from the meibomian glands.

[0038] A key driver of blepharitis is thought to be bacterial congestion of the meibomian glands in the eyelids. Increased bacterial levels, especially the presence of biofilms, on the eyelids, eyes, and surrounding tissues are common in patients with dry eye disease. The eyelid margin is ideal for biofilm formation because it provides moisture, oil, and nutrients for bacterial survival. Analysis of biofilms from the eyelids of patients with blepharitis showed that Staphylococcus spp. was the most prevalent bacterium. Further analysis showed that 46% to 51% of patients tested positive for Staphylococcus aureus in staphylococcal blepharitis tests. Staphylococcus aureus is thought to be a more pathogenic species than other Staphylococcus spp. found on the eyelids. Biofilms form as these bacteria grow and reproduce on the eyelids.

[0039] Bacterial biofilms encapsulate bacteria in a polysaccharide matrix, which can create biofilms that are more resistant to antibiotics and white blood cells. Bacteria in biofilms can also exchange genetic information to further survive in their environment. As the biofilm grows, the bacteria begin to release toxins and destroy eyelid tissue to obtain nutrients. This leads to an inflammatory response from the body. As bacteria become encapsulated in the biofilm, the body's immune response is virtually incapable of stopping them from attacking the eyelid tissue. This leads to inflammation in the tissue, interfering with the production of tear components, for example. Further complications arise because Staphylococcus aureus and Staphylococcus epidermidis release lipases from the biofilm into the meibomian glands. This converts the clear oil in the meibomian glands into a cloudy, muddy oil, which clogs the glands and leads to meibomian gland dysfunction (MGD) and poor tear quality. Current treatments for biofilm on the eyelid margin include mechanical scrubbing by an ophthalmologist using specialized tools and at-home eyelid cleansers and scrubs. The only FDA-approved method for removing biofilm from the eyelid margin is with Blephax, a mechanical device designed to scrape the biofilm, similar to removing plaque from teeth at the dentist's office.

[0040] Certain strains of α-amylases, including α-amylase from Aspergillus oryzae (Sigma-Aldrich, St. Louis, MO, catalog no. 10065), α-amylase from Bacillus subtilis (Sigma, catalog no. 10070), α-amylase from human saliva (Sigma, catalog no. A1031), and β-amylase from sweet potato, have shown some in vitro efficacy in inhibiting and reducing Staphylococcus aureus biofilms. Notably, existing data demonstrate that most human α-amylases tested were effective in preventing biofilm growth but not in disrupting existing biofilms.

[0041] The present invention relates to a composition for enzymatically destroying biofilms on eyelids by using amylase enzymes or by using amylase enzymes in combination with lysozyme enzymes, and a method for using the same. The relevant properties of amylase enzymes have been described above, and lysozyme enzymes are natural antibiotics. Although amylase and lysozyme can be naturally found in the tears of healthy patients, the present disclosure hypothesizes that patients with dry eye disease produce insufficient amounts of these enzymes to effectively prevent the formation of biofilms that trigger the inflammatory cycle. Furthermore, the present disclosure demonstrates that the use of these enzymes in a suitable formulation for ocular administration significantly reduces biofilm formation and bacterial accumulation on the eyelids.

[0042] II. Ophthalmic preparations containing enzymes The present disclosure contemplates a class of enzymes capable of removing one or more layers of biofilm from a subject's eye, thereby treating conditions caused or exacerbated by the presence of biofilm. In some embodiments, the present disclosure contemplates a class of enzymes capable of removing biofilm, for example, by enzymatically degrading polysaccharides, resulting in biofilm removal. In some embodiments, the present disclosure contemplates a class of enzymes capable of lysing microbial cells by degrading cell membrane components and destabilizing their attachment to solid surfaces. In preferred embodiments, the enzymes contemplated by the present disclosure are α-amylase and / or a combination of α-amylase and lysozyme.

[0043] Amylases are an important group of enzymes, classified into α, β, and γ subtypes, isoamylases, glucoamylases, etc. Amylases can be found in both plant and microbial sources. Based on their mode of action, amylases can be classified as exoamylases and endoamylases. Exoamylases hydrolyze substrates from the non-reducing end, resulting in short end products, while endoamylases act on internal glycosidic bonds in a random manner within starch molecules, resulting in oligosaccharides of various lengths. α- and β-amylases have the potential to catalyze the hydrolysis of chitosan, reducing its molecular weight, making it more soluble and adaptable for formulation as a liquid ophthalmic composition. For example, multiple amylases present in Legionella pneumophila are essential for hydrolyzing polysaccharides to glucose and supporting intracellular growth. Amylases also help induce a pro-inflammatory response, which further aids in inhibiting bacterial replication.

[0044] α-Amylases act primarily on starch (a polysaccharide) consisting of two glucose polymers - amylose and amylopectin - as their primary substrate. α-Amylases aid in the hydrolysis of α-1,4 and α-1,6-glycosidic bonds, which leads to the formation of the smaller glucose (monosaccharide) and maltose (disaccharide). To function, α-amylases are essentially metalloenzymes, which typically require the addition of Ca to maintain the stability of the enzyme molecule. 2+ They require metals such as α-amylases. Sequence alignment studies have revealed that α-amylases have four conserved regions, which are also present within the β-strands. α-Amylases are widely found in plants, microorganisms, and higher animals. The metabolic products obtained by the enzymatic action of α-amylases are oligosaccharides of various lengths, including branched maltooligosaccharides, maltose, and maltotriose, each containing six to eight glucose units with 1,6 and 1,4 linkages. These amylase enzymes can bind to substrates via catalytic groups that catalyze the cleavage of glycosidic bonds.

[0045] The present disclosure provides a suitable amount of metal, preferentially Ca, to maintain enzyme stability. 2+ Contemplated are compositions comprising an α-amylase complexed with an amylase of the present disclosure. The amylase of the present disclosure may be prepared, for example, by recombinant techniques, and may 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 to an α-amylase peptide sequence from Aspergillus oryzae. The α-amylases of the present disclosure can be prepared by expression of suitable nucleic acid molecules from recombinant organisms and 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% identity to a nucleic acid sequence encoding an α-amylase peptide sequence from Aspergillus oryzae. Suitable nucleic acid sequences for preparing the α-amylases of the present disclosure can be degenerate sequences. The percent identity between sequences can be calculated using several algorithms, including those described in the definitions.

[0046] Non-limiting examples of amylases contemplated by the present disclosure and a preliminary evaluation of their activity against biofilms are listed in Table 1:

[0047] [Table 2]

[0048] The enzyme of the present disclosure, for example, α-amylase, can be purchased from a commercial source and can be combined with other components to produce the ophthalmic composition described herein. Alternatively, the enzyme of the present disclosure can be prepared, for example, by peptide synthesis or the expression of suitable nucleic acid molecules. Non-limiting examples of methods for sequencing peptides include: a) liquid-phase peptide synthesis; b) solid-phase peptide synthesis using polystyrene resin, polyamide resin, PEG hybrid polystyrene resin, PEG-based resin, and / or any combination of solid-phase supports; and c) synthetic biology. Non-limiting examples of methods for expressing suitable nucleic acid molecules include molecular cloning and recombinant DNA technology.

[0049] III. Ophthalmic composition The ophthalmic compositions of the present disclosure may comprise one or more enzymes as described herein and a metal component (e.g., Ca) that stabilizes the enzyme and supports its activity. 2+ The ophthalmic composition may be a combination of the α-amylase (and α-amylase) with other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The ophthalmic composition facilitates administration of the α-amylase in a functional and active form to the eye of a subject. The ophthalmic composition may be administered in a therapeutically effective amount, for example, as eye drops via intraocular or topical routes (e.g., solutions, gels, or creams applied directly to the eyelids), via eye washes using solutions, gels, or creams, or via wipes (e.g., moistened wipes) containing a dosage of the enzyme.

[0050] For example, active compounds, including 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 include solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0051] For example, the active compounds, including α-amylase, may be formulated into wipes, eg, single-use individually wrapped wipes, or into a package having multiple wipes therein.

[0052] In carrying out the treatment methods or uses provided herein, a therapeutically effective amount of the enzyme described herein is administered as 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 may 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 preferred cases, the disease or condition is blepharitis, dry eye, or another related inflammatory condition.

[0053] Enzymes described herein, such as α-amylase, may be present in the liquid composition in ranges 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.

[0054] The enzymes described herein, such as α-amylase, may be present in the liquid composition in a range of about 5 mg / mL to about 1000 mg / mL, about 5 mg / mL to about 500 mg / mL, about 5 mg / mL to about 100 mg / mL, about 5 mg / mL to about 50 mg / mL, about 5 mg / mL to about 25 mg / mL, or about 5 mg / mL to about 20 mg / mL. The foregoing ranges are merely suggestive. Dosages may vary depending on numerous variables, including, for example, the activity of the enzyme used, the disease or condition being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0055] Each enzyme described herein, e.g., α-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.

[0056] Each enzyme described herein, e.g., α-amylase, is present in a single-use vial or wipe, e.g., in powder form or in solution form, at greater than 15.0 U / mg, e.g., 16.>0U / mg, >17U / mg, >18U / mg, >19U / mg, >20U / mg, >21U / mg, >22U / mg, >23U / mg, >24U / mg, >25U / mg, >26U / mg, >27U / mg, >28U / mg, >29U / mg, >30U / mg, >31U / mg, >32U / mg, >33U / mg, >34U / mg, >35U / mg, >36U / mg, >37U / mg, >38U / mg >3940U / mg, >41U / mg, >42U / mg, >43U / mg, >44U / mg, >45U / mg, >46U / mg, >47U / mg, >48U / mg, >49U / mg, >50U / mg, >51U / mg, >52U / mg, >53U / mg, >54U / mg, >55U / mg, >56U / mg, >57U / mg, >58U / mg, >59U / mg, >60U / mg, >61U / mg, >62U / mg >63U / mg, >64U / mg, >65U / mg, >66U / mg, >67U / mg, >68U / mg, >69U / mg, >70U / mg, >71U / mg, >72U / mg, >73U / mg, >74U / mg, >75U / mg, >76U / mg, >77U / mg, >78U / mg, >79U / mg, >80U / mg, >81U / mg, >82U / mg, >83U / mg, >84U / mg, It may be present in an amount to provide greater than 5 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 of activity, where one unit is defined as liberating 1 mg of maltose from starch within 3 minutes at pH 6.9 and 20°C.

[0057] Each enzyme described herein, e.g., α-amylase, may be present in a single-use vial or disposable wipe, e.g., in powder form or in solution form, at a concentration of 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 1 It may be present in an amount to provide an activity of between 20 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 one unit is defined as liberating 1 mg of maltose from starch within 3 minutes at pH 6.9 at 20°C.

[0058] Cofactors described herein, e.g., CaCl2, may be present in concentrations of 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. It may be present at a concentration ranging from 0.1 (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% to 0.8% (w / v), 0.3% to 0.8% (w / v), 0.4% to 0.8% (w / v), or another suitable range.

[0059] Ophthalmic compositions, such as wipes, drops, or other suitable compositions, can be formulated using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate the processing of active compounds into pharmaceutically usable preparations.Formulations can be modified according to the selected route of administration.Ophthalmic compositions containing the compounds described herein can be prepared in a conventional manner, for example, by conventional mixing, dissolving, granulating, or emulsifying.Ophthalmic compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and the compounds described herein or a pharmaceutically acceptable salt form.

[0060] Methods for preparing compositions containing 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 containing the compound, or solutions containing liposomes, micelles, or nanoparticles containing the compounds described herein. Semisolid compositions include, for example, gels, suspensions, and creams. The compositions can be liquid solutions or suspensions, solid forms suitable for solution or suspension as a liquid before use, or emulsions. The compositions can also be formulated into wipes. These compositions can also contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffers, and other pharmaceutically acceptable additives. Non-limiting examples of dosage forms suitable for use in the present disclosure include solutions, elixirs, nanosuspensions, aqueous or oily suspensions, drops, syrups, and any combination thereof.

[0061] Non-limiting examples of pharmaceutically acceptable excipients suitable for use in the present disclosure include astringents, demulcents, softeners, granulating agents, binders, lubricants, disintegrants, sweeteners, glidants, anti-adherents, antistatic agents, surfactants, antioxidants, gums, coating agents, colorants, flavoring agents, coating agents, plasticizers, preservatives, sugars, suspending agents, emulsifiers, plant cellulose materials, and spheronizing agents, and any combination thereof.

[0062] In some cases, the ophthalmic compositions of the present disclosure, which include one or more of α-amylase, lysozyme, and / or glucanase, include an astringent. A suitable astringent is zinc sulfate in the approximate concentrations disclosed below:

[0063] [Table 3]

[0064] Ophthalmic demulcent The present disclosure demonstrates that α-amylase is effective in inhibiting and reducing Staphylococcus aureus biofilms in vivo (see Figures 6A-9B). Staphylococcus aureus biofilms on the eyelids are believed to be a major cause of dry eye disease, and α-amylase has not previously been utilized in removing biofilms from the eyelids. The present disclosure contemplates ophthalmic formulations containing certain ophthalmic demulcents for use in removing biofilms from the eye, eyelids, and surrounding tissues, thus treating blepharitis and dry eye conditions.

[0065] Ophthalmic demulcents may be used in eye drops to thicken the drops, lubricate the epithelium, and relieve dryness and irritation. The ophthalmic compositions of the present disclosure may include α-amylase and one or more of the ophthalmic demulcents in Table 3.

[0066] [Table 4]

[0067] Ophthalmic softener Ophthalmic emollients generally refer to components that can form a film on the eye or on the skin. Generally, ophthalmic emollients can relieve dryness, itching, and scaling, and may help maintain a moist layer of α-amylase on the skin or ocular surface. The ophthalmic compositions of the present disclosure may include α-amylase and one or more of the ophthalmic emollients in Table 4.

[0068] [Table 5]

[0069] Ophthalmic hypertonic agent Ophthalmic tonicity agents are used to reduce swelling of the cornea (the front surface of the eye) caused by surgery, infection, trauma, or other eye conditions. The ophthalmic compositions of the present disclosure may include α-amylase and one or more ophthalmic emollients of Table 4.

[0070] [Table 6]

[0071] Ophthalmic vasoconstrictors The use of a vasoconstrictor can help reduce inflammation on the eyelid caused by biofilms. The ophthalmic compositions of the present disclosure include α-amylase and one or more ophthalmic vasoconstrictors from Table 6, including ephedrine hydrochloride, naphazoline hydrochloride, phenylephrine hydrochloride, tetrahydrozoline hydrochloride, benzalkonium chloride, oxymetazoline, and brimonidine tartrate.

[0072] [Table 7]

[0073] The ophthalmic compositions of the present disclosure may include α-amylase and one or more additional components from Table 7:

[0074] [Table 8]

[0075] The ophthalmic compositions of the present disclosure may include α-amylase and one or more additional components of Table 8:

[0076] [Table 9]

[0077] buffer system A buffer system was used to achieve a pH for artificial tears that is healthy and comfortable for the eye. A pH of about 7 is most comfortable for dry eye patients (normal tear pH is about 6.5-7.6). Purified human pancreatic alpha-amylase (alpha-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) has been found to be stable over a wide range of pH values ​​(5.0-10.5), and a buffer system may also help maintain optimal activity of alpha-amylase in the ophthalmic compositions of the present disclosure. The present disclosure contemplates that the optimal pH for alpha-amylase enzymatic activity is 5.5, and certain compositions employ one or more of the following components to achieve a pH between 5 and 7.5:

[0078] [Table 10]

[0079] Electrolytes (e.g., metals) Electrolytes can be added to the ophthalmic composition to maintain or reduce the osmolality of tears, because high osmolality products draw water from epithelial cells and interfere with metabolism.Some of the added electrolytes are also important for the metabolism of the corneal epithelium.Some electrolytes are part of the buffer system mentioned above.Some electrolytes may be required as metal stabilizers for one or more enzymes in the ophthalmic composition of the present disclosure.

[0080] [Table 11]

[0081] The ophthalmic compositions of the present disclosure may include one or more of α-amylase, lysozyme, and / or glucanase and one or more additional preservatives from Table 11:

[0082] [Table 12]

[0083] IV. Ophthalmic Preparations The ophthalmic compositions described herein can be formulated as eye drop containers (5 mL, 10 mL, 15 mL, 20 mL, or another suitable volume) or in unit dosage forms suitable for single administration of precise dosages. When formulated as eye drops, the formulation can be such that each "drop" contains a suitable amount of enzyme in a particular activity range. The ophthalmic compositions described herein can be "embedded" into a topical applicator, such as a cloth or wipe, which may have a dry powder containing the formulation or may be moistened.

[0084] In unit dosage form, the formulation is divided into unit doses containing appropriate amounts of one or more enzymes, for example, α-amylase or α-amylase and lysozyme. The unit dosages may be in the form of packages containing discrete amounts of the formulation. Non-limiting examples are solutions in vials or ampoules. Aqueous suspension compositions may be packaged in single-dose non-reclosable containers. Multi-dose reclosable containers may be used, for example, in combination with preservatives. Various preservatives and demulcents may be added to the composition (as detailed above). For example, the container may contain multiple dry or moistened wipes bearing the formulation of the present disclosure.

[0085] Non-limiting examples of ophthalmic eye drops that may be formulated with the α-amylase, lysozyme, or glucanase of the present disclosure include the following:

[0086] [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6] [Table 13-7] [Table 13-8] [Table 13-9] [Table 13-10]

[0087] Non-limiting examples of ophthalmic eye drops that may be formulated with the α-amylases of the present disclosure include:

[0088] [Table 14-1] [Table 14-2] [Example]

[0089] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors themselves regard as their invention, nor are they intended to represent or imply that the following examples represent all or the only experiments performed. Those skilled in the art will recognize that numerous variations and / or modifications as shown in the specific embodiments can be made to the invention without departing from the spirit or scope of the invention as broadly described. The present embodiments, therefore, are to be considered in all respects as illustrative and not restrictive. [Example]

[0090] Ophthalmic mucosal protective composition This example describes a procedure for testing compositions containing therapeutically effective concentrations of α-amylase enzyme from Aspergillus oryzae and various ophthalmic demulcents. The following ophthalmic demulcents were selected for the following reasons:

[0091] Selection of demulcent Cellulose derivatives (mucosal protective compositions 1 to 4): Aspergillus oryzae α-amylase (EC 3.2.1.1) catalyzes the endohydrolysis of 1,4-alpha-D-glucosidic bonds in polysaccharides. Because cellulose is made up of polysaccharides, which are held together by β(1→4) glucan bonds, α-amylase is not thought to cleave such bonds and may become inhibited by binding thereto. This study will evaluate whether cellulose derivatives are suitable for use as demulcents for compositions containing α-amylase or whether α-amylase activity is inhibited.

[0092] Dextran: Dextrans are branched glucan polysaccharides containing α-1,6 glycosidic linkages. Notably, dextrans differ from dextrins, which are polysaccharides that may contain either α-1,4 or α-1,6 linkages. This study will evaluate whether dextrans are suitable for use as demulcents in compositions containing α-amylase.

[0093] gelatin: Gelatin is composed of proteins and peptides and is not expected to functionally interact with α-amylase. This study will evaluate whether gelatin is suitable for use as a demulcent in compositions containing α-amylase.

[0094] Polyol: It is hypothesized that the addition of polyols to aqueous α-amylase solutions may increase the thermal stability of α-amylase in solution. This study will evaluate whether polyols are suitable for use as demulcents in compositions containing α-amylase.

[0095] Polyvinyl alcohol: Polyvinyl alcohol is a biocompatible, water-soluble synthetic polymer that should not interact with α-amylase. This study will evaluate whether polyvinyl alcohol is suitable for use as a demulcent in compositions containing α-amylase.

[0096] Povidone: Povidone is a biocompatible water-soluble polymer that should not interact with α-amylase. This study will evaluate whether povidone is suitable for use as a demulcent in compositions containing α-amylase.

[0097] The compositions tested in this study were as follows:

[0098] [Table 15]

[0099] 1.1 Composition development - Demulcent property evaluation Compositions of α-amylase in combination with the aforementioned ophthalmic demulcent were made by using commercially available preservative-free eye drops in a buffered saline solution. Notably, α-amylase is believed to require calcium ions to function, and the compositions of the present disclosure include calcium ions. Note: The CaCl2 concentration may be adjusted in the protocol based on the results of Ca cofactor testing. If excess Ca causes enzyme inactivation, the concentration may be adjusted.

[0100] Compositions containing different preservatives were tested in different experiments described in Example 2.

[0101] 1.1.1 Composition Development - α-Amylase Formulation with Cofactors (Demulcent-Free): Step 1: Prepare 10 ml of a 10 mg / ml solution of α-amylase and cofactors. Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0102] Step 2: 40 mg of calcium chloride and 56 mg of sodium chloride were dissolved in the solution. Mix until all components were dissolved and evenly distributed.

[0103] 1.1.2 Composition Development—α-Amylase Preparation + Cellulose Derivative Demulcent (Compositions 1 to 4): Protocol for preparing 10 ml of a 10 mg / ml solution of α-amylase in saline containing 0.5% sodium carboxymethylcellulose. Note: Sodium carboxymethylcellulose can be replaced with hydroxyethylcellulose, hydroxyethylcellulose, hypromellose, or methylcellulose within the ranges listed in the table above. Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to a flask. 40 mg of calcium chloride and 56 mg of sodium chloride were added to the flask. Mix until all components were dissolved and evenly distributed.

[0104] Step 2: 50 mg of sodium carboxymethylcellulose (Sigma-Aldrich catalog number 419273) (or another suitable cellulose derivative described herein) was added and mixed until dissolved.

[0105] 1.1.3 Composition Development—α-Amylase Preparation + Dextran Demulcent (Composition 5): Protocol for preparing 10 ml of a 10 mg / ml solution of α-amylase in saline containing 0.1% dextran 70 and 0.3% hypromellose Step 1: 20 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to a 2 ml ALCON GenTeal Tears Preservative Free disposable vial in a flask. Mix until all components were dissolved and evenly distributed.

[0106] Step 2: 0.006g of calcium chloride was dissolved in the solution and mixed until dissolved.

[0107] 1.1.4 Development of the composition - α-amylase preparation + gelatin (composition 6): Protocol for preparing 2 ml of a 10 mg / ml solution of α-amylase in saline containing 0.01% gelatin: Step 1: 0.2 mg of pure gelatin powder was added to 2 ml of room temperature saline in a flask without mixing.

[0108] Step 2: The gelatin was allowed to soften for 5 minutes.

[0109] Step 3: The flask was warmed to 50°C and mixed until the gelatin was dissolved.

[0110] Step 4: The flask was cooled to room temperature before continuing further.

[0111] Step 5: 20 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to the flask. Mixing was continued until the α-amylase was dissolved and evenly distributed.

[0112] 0.006 g of calcium chloride was dissolved in the solution.

[0113] 1.1.5 Development of composition - α-amylase preparation + glycerin (composition 7): Protocol for preparing 10 ml of a 10 mg / ml α-amylase solution in saline containing 0.5% glycerol Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to a flask. 40 mg of calcium chloride and 56 mg of sodium chloride were added to the flask. Mixing was performed until the α-amylase was dissolved and evenly distributed.

[0114] Step 2: 100 mg of glycerin was added to the flask. The flask was filled to the 10 ml mark with sterile DI water. Mixed until all components were dissolved and evenly distributed.

[0115] 1.1.6 Composition development - α-amylase preparation in saline + polyol (compositions 8-11): Polyethylene glycol 400-1.0% Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to a flask. 40 mg of calcium chloride and 56 mg of sodium chloride were added to the flask.

[0116] The α-amylase was mixed until dissolved and evenly distributed.

[0117] Step 2: 100 mg of polyethylene glycol 400 (Sigma-Aldrich catalog number 8074850050) was added and mixed until dissolved. The flask was filled to the 10 ml mark with sterile DI water. Mixed until all components were dissolved and evenly distributed.

[0118] Note: Polyethylene glycol 400 can be replaced with polyethylene glycol 300 and other suitable polyols.

[0119] Propylene glycol 1.0% Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to a flask. 40 mg of calcium chloride and 56 mg of sodium chloride were added to the flask.

[0120] Step 2: 100 mg of propylene glycol was added to the flask. The flask was filled to the 10 ml mark with sterile DI water. Mixed until all components were dissolved and evenly distributed.

[0121] Polyvinyl alcohol 4% (PVA) Step 1: A 4% PVA solution (4g PVA per 100ml DI water) should be made in advance because the PVA must be added to room temperature DI water in a loosely capped media bottle equipped with a magnetic stir bar, gradually heated and constantly stirred to 90°C, held at 90°C for 1 hour, and allowed to return to room temperature.

[0122] Step 2: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of 4% PVA solution. Mix until the α-amylase was dissolved and evenly distributed.

[0123] Step 3: 40 mg of calcium chloride and 56 mg of sodium chloride were dissolved in the solution. Mix until all components were uniformly dissolved.

[0124] 1.2 Composition Evaluation - Well Plate Setup for In-vitro Testing of Inhibition of Biofilm Formation The biofilm formation protocol was performed by preparing and plating S. aureus using the protocol listed below.

[0125] 1.2.1 Thawing of Staphylococcus aureus Bacterial strains were thawed upright in a water bath at 25-30°C using gentle agitation. Thawing should be rapid; approximately 2-3 minutes, or until all ice crystals have melted.

[0126] Immediately after thawing, the mini-cryovials were wiped with 70% ethanol and aseptically inoculated into breathable sterile aerobic culture tubes with 10 mL of tryptic soy broth (30 g per 1000 mL of DI H2O) using a sterile inoculating loop. The tubes were incubated at 37°C in a shaking incubator for 24 hours.

[0127] 1.2.2 Preparation of cultures for plating Step 1: A new CuveTip was attached to the Photopette.

[0128] Step 2: Turn on the Photopette and make sure the Bluetooth is connected to the iPad.

[0129] Step 3. Opened the app and set the measurement to collect at 600 nm (I used the ecoli setting for simplicity).

[0130] Step 4. Zero the Photopette in DI water and wipe the tip with a chemical wipe.

[0131] Step 5. 100 ml of TSB Citrate Broth (TSBC) was aseptically transferred to the flask.

[0132] Step 6. Prepare an overnight culture of Staphylococcus aureus subsp. Rosenbach (ATCC 29213) in tryptic soy broth until the A600 of the culture is 0.02 (~2 x 10), as measured by Photopette. 8 The cells were aseptically transferred to TSB citrate broth (TSBC) using a pipette until diluted to 100 CFU / ml.

[0133] Step 7. A sterile 96 well plate was removed from its sterile packaging in the hood and placed on the table.

[0134] Step 8. Set an 8 channel pipette to 100 μl and attach a sterile tip.

[0135] Step 9. 100 μl of the diluted culture was plated into the desired number of wells.

[0136] Step 10. The well plate was covered and incubated at 37°C overnight for 18 hours.

[0137] 1.2.3 Plating S. aureus was plated into all wells of tissue culture treated 96 well plates. The well plates should be incubated at 37° C. for 18 hours before assaying for biofilm formation.

[0138] 1.3 Composition testing 1.3.1 Test Composition Day 1 All liquid was pipetted from the wells by carefully tilting the plate and inserting the pipette tip into the corner of the bottom of the well to remove non-adherent cells without disturbing the biofilm growth on the bottom of the well.

[0139] A 10-minute timer was started when the first row was treated using an 8-channel pipette. Each row should be treated every 30 seconds, ensuring that the composition at the corners of the wells is released gradually to avoid mechanically disturbing the biofilm.

[0140] For column 1: 50 μl of 10 mg / ml α-amylase with cofactors was added to all wells.

[0141] For column 2: 50 μl of 10 mg / ml α-amylase formulated with 1.0% polyethylene glycol 400 was added to all wells.

[0142] For column 3: 50 μl of 10 mg / ml α-amylase formulated with 2.5% sodium carboxymethylcellulose was added to all wells.

[0143] For column 4: 50 μl of formulated 10 mg / ml α-amylase with 1.0% glycerol was added to all wells.

[0144] For column 5: 50 μl of 10 mg / ml formulated α-amylase with 2.0% povidone was added to all wells.

[0145] For column 6: 50 μl of 10 mg / ml α-amylase formulated with 1.0% propylene glycol was added to all wells.

[0146] For column 7: 50 μl of 10 mg / ml α-amylase formulated with 2.7% polyvinyl alcohol was added to all wells.

[0147] For column 8: 50 μl of 10 mg / ml α-amylase in saline containing 0.1% dextran 70, 0.3% hypromellose 2910 was added to all wells.

[0148] For column 9: 50 DI water was added to all wells. Columns 11-12 were not treated. The plate was incubated at 37°C for 10 minutes.

[0149] 1.4 Evaluation of biofilm reduction After 10 minutes, all fluid should be removed column by column every 30 seconds, starting with column 1. Fluid should be removed by tilting the plate, placing the pipette in the corner, and gently pipetting to dislodge non-adherent cells without disturbing biofilm growth on the well bottom.

[0150] 50 μl of 0.1% crystal violet (CV) was added to all wells to stain adherent cells.

[0151] All wells were carefully rinsed by immersing the plate in a DI water bath to remove any remaining crystal violet.

[0152] The well plates were dried and the optical density of the biofilms was measured at 600 nm using a plate reader.

[0153] Once all measurements are complete, the well plates should be disinfected by transferring them to a bleach solution and disposed of in a biological waste bin.

[0154] 1.5 Evaluation of the preservative effectiveness of various compositions The steps described in 1.2 and 1.3 were repeated for scheduled test days 7, 14, 21, 28, and 35. For each scheduled test day 7, 14, 21, 28, and 35, biofilm well plates should be prepared the day before.

[0155] Once all measurements are complete, the well plates should be disinfected by transferring them to a bleach solution and disposed of in a biological waste bin. All experiments were performed using sterile techniques with sterile equipment in a clean environment. [Example]

[0156] Testing of Ophthalmic Preservative Compositions This example describes a procedure for testing the compatibility of α-amylase compositions from Aspergillus oryzae with ophthalmic preservatives. These preservatives are intended to prevent microbial growth and do not extend the shelf life of the α-amylase.

[0157] 2.1 Formulation Development - Evaluation of Preservatives on α-Amylase Function This disclosure hypothesizes that Staphylococcus aureus biofilms on the eyelids are a major cause of dry eye disease. α-Amylase has not previously been used to remove biofilms from the eyelids. This disclosure describes the testing and characterization of various combinations of ophthalmic solution components and α-amylase for their effectiveness in removing biofilms in cell culture before testing these solutions on tissue. The goal of this experiment is to evaluate whether ophthalmic preservatives aid or hinder the effectiveness of α-amylase in reducing Staphylococcus aureus biofilms. The following preservatives were evaluated in this example:

[0158] A) BAK generally functions as a detergent to lyse cell walls and membranes, but it is unknown whether it affects α-amylase stability / function. B) Polyquaternium-1 is thought to act on cell membranes, and it is unknown whether it similarly affects α-amylase stability / function. C) Stabilized oxychloro complexes are thought to function as preservatives via the oxidation of intracellular lipids and glutathione, and it is unknown whether it similarly affects α-amylase stability / function. D) Sodium perborate is thought to function through the formation of hydrogen peroxide, an oxidative action, but it is unknown whether it affects α-amylase stability / function. E) Disodium edetate and sorbic acid, or disodium edetate, or ethylenediaminetetraacetic acid (EDTA), are thought to function by binding to heavy metals such as iron or calcium. Because α-amylase uses calcium as a cofactor, this protocol will test whether these preservatives affect α-amylase stability / function. F) Polixetonium (polyquaternium-42) is rarely used as a preservative and it is unknown whether it affects α-amylase stability / function. G) Borate buffer, sorbitol buffer, propylene glycol buffer, and zinc ion buffer may function through multiple potential modes of action; this protocol will test whether these preservatives affect α-amylase stability / function. H) Polyhexanide is thought to act by damaging bacterial cell membrane activity; this protocol will test whether these preservatives affect α-amylase stability / function.

[0159] [Table 16]

[0160] 2.1.1 Composition Development—α-Amylase Formulation with Cofactors (Preservative-Free—Control Composition): Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mix until the α-amylase was dissolved and evenly distributed.

[0161] Step 2: 40 mg of calcium chloride and 56 mg of sodium chloride were dissolved in the solution. Mix until all ingredients were dissolved and evenly distributed.

[0162] 2.1.2 Composition development - α-amylase preparation + benzalkonium chloride (Composition 14): Protocol for preparing 10 ml of a 10 mg / ml α-amylase solution containing 0.01% benzalkonium chloride:

[0163] Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of benzalkonium chloride (Sigma-Aldrich catalog number PHR1371) diluted to 0.01% with DI water in a flask. Mix until the α-amylase was dissolved and evenly distributed.

[0164] Step 2: 40 mg of calcium chloride and 56 mg of sodium chloride were dissolved in the solution.

[0165] 2.1.3 Composition Development—α-Amylase Formulation + Polyquad (Polyquaternium-1) (Composition 15): Protocol for preparing 10 ml of a 10 mg / ml α-amylase solution containing 0.001% Polyquad:

[0166] Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 2 ml of Systane original in a flask. The mixture was mixed until the α-amylase was dissolved and evenly distributed. This prepared 10 ml of a 10 mg / ml solution of α-amylase in saline containing 0.01% sodium perborate.

[0167] Step 2: 1 mg of Polyquaternium 1 (Santa Cruise Biochemicals catalog number sc-476677) was dissolved in 40 mg of calcium chloride and 56 mg of sodium chloride.

[0168] 2.1.4 Composition Development—α-Amylase Preparation + Stabilized Oxychloro Complex (Composition 16): Protocol for preparing 10 ml of a 10 mg / ml solution of α-amylase in saline containing a stabilized oxychloro complex:

[0169] 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of Blink Tears Lubricating Eye Drops in a flask, which already contained the appropriate amount of stabilized oxychloro complex. Mixing was performed until the α-amylase was dissolved and uniformly distributed.

[0170] 2.1.5 Composition Development—α-Amylase Formulation + GenAqua / Dequest (Sodium Perborate) (Composition 17): Protocol for preparing 2 ml of a 10 mg / ml α-amylase solution in saline and 0.01% GenAqua / Dequest (sodium perborate): Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to the DI solution in a flask. Mix until the α-amylase was dissolved and evenly distributed.

[0171] Step 2: 1 mg of sodium perborate was dissolved in the solution.

[0172] Step 3: Dissolved in 30 mg of calcium chloride and 60 mg of sodium chloride.

[0173] 2.1.6 Composition Development—α-Amylase Formulation + 0.1% Edetate Disodium and 0.1% Sorbic Acid (Composition 18): Protocol for preparing sodium hypochlorite solution: Step 1: Prepare 10 ml of 5% bleach (sodium hypochlorite) solution.

[0174] Step 2: 6.66 ml of 7.5% sodium hypochlorite, a commercially available bleaching agent, was added to a 10 ml flask and filled up to 10 ml with DI water.

[0175] 2.2 Composition Evaluation - Well Plate Setup for Performing In-Vitro Testing of the Compatibility of α-Amylase from Aspergillus oryzae with Ophthalmic Preservatives Plates were prepared for plating S. aureus for preservative compatibility protocol testing using the protocol set forth in Example 1 (1.2.1). Briefly, columns 1-10, rows 1-8 were plated with S. aureus as described in Example 1, while the remaining columns, 11-12, were plated with DI water for spectrophotometric comparison. The well plates were incubated at 37°C for 18 hours before assaying for biofilm formation.

[0176] 2.3 Composition testing 2.3.1 Test Composition Day 1 All liquid was pipetted from the wells by carefully tilting the plate and inserting the pipette tip into the corner of the bottom of the well to remove non-adherent cells without disturbing the biofilm growth on the bottom of the well.

[0177] A 10-minute timer was started when the first row was treated using an 8-channel pipette. Each row should be treated every 30 seconds, ensuring that the composition at the corners of the wells is released gradually to avoid mechanically disturbing the biofilm.

[0178] For column 1: 50 μl of 10 mg / ml α-amylase with cofactors was added to column 1.

[0179] For row 2: 50 μl of 10 mg / ml α-amylase containing 0.01% benzalkonium chloride was added to row 2.

[0180] For column 3: 50 μl of 10 mg / ml α-amylase with 0.001% Polyquad was added to column 3.

[0181] For column 4: 50 μl of 10 mg / ml α-amylase with 0.01% sodium perborate was added to column 4.

[0182] For column 5: 50 μl of 10 mg / ml α-amylase with stabilized oxychloro complex was added to column 5.

[0183] For column 6: 50 μl of 7.5% bleach (sodium hypochlorite) solution was added to column 6.

[0184] For column 7: 50 μl of DI water was added to column 7.

[0185] The plate was incubated at 37°C for the remaining 10 minutes.

[0186] 2.4 Evaluation of biofilm reduction After 10 minutes, all fluid should be removed column by column every 30 seconds, starting with column 1. Fluid should be removed by tilting the plate, placing the pipette in the corner, and gently pipetting to dislodge non-adherent cells without disturbing biofilm growth on the well bottom.

[0187] 50 μl of 0.1% crystal violet (CV) was added to all wells to stain adherent cells.

[0188] All wells were carefully rinsed by immersing the plate in a DI water bath to remove any remaining crystal violet.

[0189] The well plates were dried and the optical density of the biofilms was measured at 600 nm using a plate reader.

[0190] Once all measurements are complete, the well plates should be disinfected by transferring them to a bleach solution and disposed of in a biological waste bin. [Example]

[0191] Biofilm formation Overview of the protocol for studying co-cultures of human conjunctival epithelial cells and Staphylococcus aureus. Human conjunctival epithelial cells (HConEpiC) from ScienCell Research Laboratories catalog number 6630 were grown in monolayer on poly-L-lysine-coated 24-well plates using corneal epithelial cell medium.

[0192] 3.1 Initiation of cultures: ScienCell primary cells were cultured at 37°C in a 5% CO2 incubator as follows:

[0193] 1. Poly-L-lysine-coated culture vessel (2 μg / cm 2 A T-75 flask is recommended. 2 To obtain a poly-L-lysine-coated culture vessel, 10 ml of sterile water was added to a T-75 flask, followed by 15 μl of poly-L-lysine stock solution (10 mg / ml, catalog number 0413). The vessel was placed in a 37°C incubator overnight (or for a minimum of 1 hour).

[0194] 2. Complete medium was prepared. The outer surfaces of the medium bottle and medium supply tube were decontaminated with 70% ethanol and transferred to a sterile area. The supplement was aseptically transferred to the basal medium using a pipette. The supply tube was rinsed with medium to recover the entire volume.

[0195] 3. The poly-L-lysine-coated container was rinsed twice with sterile water, and then 20 ml of complete medium was added. The container was placed in a sterile area and the cryopreserved cells were allowed to thaw.

[0196] 4. The frozen vial was placed in a 37°C water bath. The vial was held and gently rotated until the contents were completely thawed. The vial was quickly removed from the water bath, wiped with 70% ethanol, and transferred to a sterile field.

[0197] 5. The cap was carefully removed without touching the internal threads. The contents of the vial were gently resuspended and dispensed into equilibrated poly-L-lysine-coated culture vessels.

[0198] NOTE: Diluting and centrifuging cells after thawing is not recommended, as these actions may be harmful to the cells. It is also important to plate the cells under conditions favorable for cell attachment, for example, in poly-L-lysine-coated culture vessels, which promotes cell attachment.

[0199] 6. Replace the cap or lid on the culture vessel and gently rock the vessel to evenly distribute the cells. Loosen the cap as needed to allow gas exchange.

[0200] 7. The culture vessel was returned to the incubator.

[0201] 8. After initiation, do not disturb the cultures for at least 16 hours. The next day, replace the culture medium with fresh medium to remove any remaining DMSO and unattached cells.

[0202] 3.2 Maintenance of cultures: 1. The morning after establishing cultures from cryopreserved cells, the supplemented culture medium was replaced with fresh medium.

[0203] 2. The medium was then changed every 3 days until the cultures were approximately 70% confluent.

[0204] 3. Once the cultures reached 70% confluency, the medium was changed every other day until the cultures were approximately 90% confluent.

[0205] 3.3 Subculture: 1. Subculture was performed when the culture reached 90% confluency.

[0206] 2. Poly-L-lysine-coated culture vessels (2 μg / cm 2 ) was prepared the day before subculture.

[0207] 3. Complete medium, Trypsin / EDTA solution, 0.05% (T / E, Cat. No. 0183), T / E Neutralizing Solution (TNS, Cat. No. 0113), and DPBS (Ca++- and Mg++-free, Cat. No. 0303) were warmed to room temperature. It is not recommended to warm reagents and media in a 37°C water bath before use.

[0208] 4. Cells were rinsed with DPBS.

[0209] 5. 10 ml of 0.05% T / E solution (Cat. No. 0183) was added to the flask (for a T-75 flask). The flask was gently rocked to ensure the cells were completely covered by the T / E solution. A microscope was used to monitor changes in cell morphology.

[0210] NOTE: Although other solutions may be used, we recommend using ScienCell 0.05% T / E solution, which has been optimized to minimize cell damage caused by excessive trypsinization. If using 0.25% T / E solution (Cat. No. 0103), 8 ml of DPBS and 2 ml of 0.25% T / E solution should be used.

[0211] 6. While incubating, prepare a 50 ml conical centrifuge tube containing 5 ml of fetal bovine serum (FBS, Catalog No. 0500).

[0212] 7. Once the cells were completely rounded, the T / E solution was transferred from the flask to a 50 ml centrifuge tube (a small percentage of cells may have detached) and the flask was incubated at 37°C for 2-3 minutes (no solution was left in the flask at this point).

[0213] 8. At the end of the incubation, gently tap the side of the flask to dislodge the cells from the surface. Check under a microscope to ensure all cells have detached.

[0214] 9. 5 ml of TNS solution was added to the flask and the detached cells were transferred to a 50 ml centrifuge tube. The flask was rinsed with another 5 ml of TNS to collect any remaining cells.

[0215] 10. The flasks are examined under a microscope for successful cell harvesting, as judged by the number of cells left; the number of cells left should be less than 5%.

[0216] 11. The 50 ml centrifuge tube was centrifuged at 1000 rpm for 5 minutes. The cells were gently resuspended in culture medium.

[0217] 12. Cells were counted and plated into new poly-L-lysine-coated culture vessels at the recommended cell density: 5,000 cells / cm. 2 A seeding density of 10 ...

[0218] 3.4 Co-culture development After establishing a monolayer of human conjunctival epithelial cells, a layer of Staphylococcus aureus subsp. Rosenbachii (ATCC 29213) will be established on top of the epithelial cells.

[0219] 1. Add 650 μl of Staphylococcus aureus subsp. Rosenbach (ATCC 29213) in tryptic soy broth to a 0.01 (approximately 1 × 10) 8 CFU / ml) was added to each well of rows A to C in TSB citrate medium (TSBC), while row D was left untreated.

[0220] 2. The well plate was covered and incubated at 37°C for 4 hours.

[0221] 3.5 Testing Alpha-amylase compositions, preferentially those with demonstrated ability to eliminate biofilms in cell cultures as described in Example 1, were tested in this co-culture to study and characterize their potential toxicity to the basal monolayer of human conjunctival epithelial cells. The hypothesis is that with treatment and clearance of the inflicting S. aureus, the inflammatory response from the epithelial cells will return to normal at the mRNA and protein levels.

[0222] 1 ml of each test composition was added to each well in row A.

[0223] Row B will be tested with 1 ml of negative control (saline).

[0224] Row C was tested with 1 ml of positive control (gentamicin).

[0225] The test composition should be incubated at 37°C for 10 minutes.

[0226] All layers should be washed three times before lysis. The plates were gently lysed with 1 ml of trypsin to remove the cells. Lysed cells were stained with trypan blue and evaluated under a microscope to count the cells. The presence of blue cells indicates cell death. Samples were evaluated for cell number and visibility curves. Samples were also collected and frozen before and after challenge to analyze mRNA, DNA, and protein for future expression. [Example]

[0227] Treatment of Staphylococcus aureus biofilms with antibiotics plus α-amylase Staphylococcus aureus produces extracellular capsular polysaccharides, which bind to form biofilms. Aspergillus oryzae α-amylase (EC 3.2.1.1) catalyzes the endohydrolysis of 1,4-alpha-D-glucosidic bonds in polysaccharides containing three or more 1,4-alpha-linked D-glucose units. While α-amylase does not possess antibacterial properties, it can inhibit and reduce S. aureus biofilms by disrupting the extracellular polysaccharide bonds of the biofilm. Antibiotics have been shown to have some effect in reducing biofilms, but their effectiveness against established biofilms is low. Furthermore, long-term use of antibiotics can lead to antibiotic-resistant bacteria. α-amylase does not possess antibacterial properties, and there is no known risk of producing antibiotic-resistant bacteria.

[0228] The topical application of α-amylase described in this example has not been used to reduce or prevent biofilm formation on the eyelids. This experiment aims to characterize the use of α-amylase compositions containing α-amylase and other antibacterial or antimicrobial agents against Staphylococcus aureus biofilms grown in well plates for 24 hours. It is hypothesized that the addition of α-amylase will improve biofilm removal for all compounds.

[0229] This study describes the testing of 0.01% hypochlorous acid solutions containing and without α-amylase from Aspergillus oryzae against biofilms formed in vitro. This study also describes the testing of common ophthalmic antibiotic compositions containing and without α-amylase from Aspergillus oryzae. These antibiotics include erythromycin, moxifloxacin, and gentamicin sulfate. This study uses sterile saline as a negative control and a 5% bleach solution as a positive control.

[0230] 4.1 Composition Development - Alpha-Amylase Preparation in Saline: Alpha-amylase requires calcium ions to function. A saline solution was prepared using 0.4% calcium chloride and 0.56% sodium chloride. The 0.4% calcium chloride was added to the alpha-amylase solution to ensure the presence of calcium ions.

[0231] Step 1: Prepare 10 ml of a 10 mg / ml solution of α-amylase and cofactors. Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0232] Step 2: 40 mg of calcium chloride and 56 mg of sodium chloride were dissolved in the solution. All ingredients were mixed until evenly distributed.

[0233] 4.1.2 Composition Development - α-Amylase in 0.01% Hypochlorous Acid Solution Protocol for preparing 2 ml of a 10 mg / ml solution. 2 ml of a 10 mg / ml solution of α-amylase in 0.01% hypochlorous acid solution was prepared:

[0234] Step 1: 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of 0.01% hypochlorous acid in saline (Avenova OTC antibacterial spray solution) in a flask. Mix until the α-amylase was dissolved and evenly distributed.

[0235] Step 2: 40 mg of calcium chloride was dissolved in the solution. Mix until the α-amylase was dissolved and evenly distributed.

[0236] 4.1.3 Composition Development - α-Amylase in Ofloxacin Solution Protocol for preparing 2 ml of 10 mg / ml α-amylase in 0.3% ofloxacin ophthalmic solution: Briefly: 20 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065) was added to 1 ml of 0.5% erythromycin ophthalmic solution in a flask and mixed until evenly distributed. 8 mg of calcium chloride was dissolved in the solution.

[0237] 4.1.4 Composition Development - α-Amylase in 0.01% Moxifloxacin Solution Protocol for preparing 1 ml of 10 mg / ml α-amylase in 0.5% moxifloxacin ophthalmic solution:

[0238] Briefly: 10 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065) was added to 1 ml of 0.5% moxifloxacin ophthalmic solution in a flask and mixed until evenly distributed. 3 mg of calcium chloride was dissolved in the solution.

[0239] 4.1.5 Development of Composition - Alpha-Amylase in Gentamicin Sulfate Protocol for preparing 2 ml of 10 mg / ml alpha-amylase in 0.5% gentamicin sulfate ophthalmic solution: Briefly: 20 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065) was added to 1 ml of 0.5% moxifloxacin ophthalmic solution in a flask and mixed until evenly distributed. 8 mg of calcium chloride was dissolved in the solution.

[0240] 4.1.6 Composition Development - α-Amylase in Bleaching Agents Protocol for preparing 10 ml of 5% bleach (sodium hypochlorite) solution: Briefly: 6.66 ml of 7.5% sodium hypochlorite, a commercially available bleaching agent, was added to a 10 ml flask and filled to 10 ml with DI water.

[0241] 4.2 Well plate setup The protocol set forth in Example 1 (1.2.1) was used to prepare plates for plating S. aureus for preservative compatibility protocol testing. Briefly, S. aureus was plated into all wells of a tissue culture-treated 96-well plate as described in Example 1. The well plates should be incubated at 37° C. for 18 hours before assaying for biofilm formation.

[0242] 4.3 Test Compositions All liquid was pipetted from the wells by carefully tilting the plate and inserting the pipette tip into the corner of the bottom of the well to remove non-adherent cells without disturbing the biofilm growth on the bottom of the well.

[0243] A 10-minute timer was started when the first row was treated using an 8-channel pipette. Each row should be treated every 30 seconds, ensuring that the composition at the corners of the wells is released gradually to avoid mechanically disturbing the biofilm.

[0244] For column 1: 50 μl of 10 mg / ml α-amylase in cofactor solution was added to all wells.

[0245] For column 2: 50 μl of 10 mg / ml α-amylase in 0.01% hypochlorous acid solution was added to all wells.

[0246] For column 3: 50 μl of 0.01% hypochlorous acid in saline (Avenova OTC antibacterial spray solution) was added to all wells.

[0247] For column 4: 50 μl of 10 mg / ml α-amylase in 0.3% ofloxacin ophthalmic solution was added to all wells.

[0248] For column 5: 50 μl of 0.3% ofloxacin ophthalmic solution was added to all wells.

[0249] For column 6: 50 μl of 10 mg / ml α-amylase in 0.5% gentamicin sulfate ophthalmic solution was added to all wells.

[0250] For column 7: 50 μl of 0.5% gentamicin sulfate ophthalmic solution was added to all wells.

[0251] For column 8: 50 μl of 5% bleach (sodium hypochlorite) solution was added to all wells.

[0252] For column 9: 50 μl of DI water was added to all wells.

[0253] Columns 10 to 12 were not treated. The plate was incubated at 37°C for the remaining 10 minutes.

[0254] 4.4. Biofilm reduction test After 10 minutes, all fluid should be removed every 30 seconds, column by column, starting with column 1. Fluid should be removed by tilting the plate, placing the pipette in the corner, and gently pipetting to dislodge non-adherent cells without disturbing biofilm growth on the well bottom.

[0255] 50 μl of 0.1% crystal violet (CV) was added to all wells to stain adherent cells.

[0256] All wells were carefully rinsed by immersing the plate in a DI water bath to remove any remaining crystal violet.

[0257] The well plates were dried and the optical density of the biofilms was measured at 600 nm using a plate reader. Once all measurements were completed, the well plates should be disinfected by transferring them to a bleach solution and disposed of in a biological waste bin. [Example]

[0258] Treatment of Staphylococcus aureus biofilms with a range of cofactors plus α-amylase This protocol was designed to evaluate the optimal range of cofactor calcium for α-amylase from Aspergillus oryzae when used to remove Staphylococcus aureus biofilms.

[0259] Ca 2+ Although Ca has been identified as a necessary cofactor for α-amylase, previous studies of α-amylase against S. aureus biofilms have shown that Ca is required to eliminate or inhibit biofilm growth in cell culture. 2+ was not introduced into the composition. α-Amylase has two binding sites for Ca ions. 1 mg of α-Amylase contains 1.075*10 16 enzymes, and the present disclosure suggests that at least 0.0039 mg of CaCl2 may be required to provide two calcium ions per enzyme. 2+ It is unclear whether calcium is required, and some literature suggests that excess calcium may have adverse effects. 2+ Although some electrolytes, including Ca, are naturally present in the eye, this study was designed to characterize the effectiveness of α-amylase from Aspergillus oryzae in removing S. aureus biofilms in cell cultures with different concentrations of the Ca2+ cofactor.

[0260] Normal electrolyte concentrations in human tears (mMol / liter)

[0261] [Table 17]

[0262] 5.1 Composition Development 5.1.1 - Preparation of 10 ml of a 10 mg / ml α-amylase solution in DI water 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 2 ml of sterile DI water in a flask. Mix until the α-amylase was dissolved and evenly distributed.

[0263] 5.1.2 - Preparation of 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.05% CaCl2, 0.85% NaCl. 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mixed until the α-amylase was dissolved and evenly distributed. 25 mg of CaCl was added. 85 mg of NaCl was added. Mixed until dissolved.

[0264] 5.1.3 - Preparation of 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.1% CaCl2, 0.8% NaCl. 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mixed until the α-amylase was dissolved and evenly distributed. 10 mg of CaCl2 was added. 80 mg of NaCl was added. Mixed until dissolved.

[0265] 5.1.4-Preparation of 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.2% CaCl2, 0.7% NaCl. 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mixed until the α-amylase was dissolved and evenly distributed. 20 mg of CaCl2 was added. 70 mg of NaCl was added. Mixed until dissolved.

[0266] 5.1.5-Preparation of 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.3% CaCl2, 0.6% NaCl. 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mixed until the α-amylase was dissolved and evenly distributed. 30 mg of CaCl2 was added. 60 mg of NaCl was added. Mixed until dissolved.

[0267] 5.1.6-Preparation of 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.4% CaCl2, 0.5% NaCl. 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mixed until the α-amylase was dissolved and evenly distributed. 40 mg of CaCl2 was added. 50 mg of NaCl was added. Mixed until dissolved.

[0268] 5.1.7-Preparation of 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.5% CaCl2, 0.4% NaCl. 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mixed until the α-amylase was dissolved and evenly distributed. 250 mg of CaCl was added. 40 mg of NaCl was added. Mixed until dissolved.

[0269] 5.1.8 - Preparation of 10 ml of a 10 mg / ml α-amylase solution in DI water containing 0.6% CaCl2, 0.3% NaCl 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mixed until the α-amylase was dissolved and evenly distributed. 60 mg of CaCl2 was added. 30 mg of NaCl was added. Mixed until dissolved.

[0270] 5.1.9 - Preparation of 10 ml of a 10 mg / ml α-amylase solution in DI water containing 0.8% CaCl2, 0.1% NaCl 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added to 10 ml of sterile DI water in a flask. Mixed until the α-amylase was dissolved and evenly distributed. 80 mg of CaCl2 was added. 10 mg of NaCl was added. Mixed until dissolved.

[0271] 5.2 Test Compositions All liquid was pipetted from the wells by carefully tilting the plate and inserting the pipette tip into the corner of the bottom of the well to remove non-adherent cells without disturbing the biofilm growth on the bottom of the well.

[0272] A 10-minute timer was started when the first row was treated using an 8-channel pipette. Each row should be treated every 30 seconds, ensuring that the composition at the corners of the wells is released gradually to avoid mechanically disturbing the biofilm.

[0273] For column 1: 50 μl of 10 mg / ml α-amylase in DI water was added to column 1.

[0274] For column 2: 50 μl of 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.05% CaCl 2 , 0.85% NaCl was added to column 2.

[0275] For column 3: 50 μl of the 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.1% CaCl 2 , 0.8% NaCl was added to column 3.

[0276] For column 4: 50 μl of 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.2% CaCl 2 , 0.7% NaCl was added to column 4.

[0277] For column 5: 50 μl of the 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.3% CaCl 2 , 0.6% NaCl was added to column 5.

[0278] For column 6: 50 μl of the 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.4% CaCl 2 , 0.5% NaCl was added to column 6.

[0279] For column 7: 50 μl of the 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.5% CaCl 2 , 0.4% NaCl was added to column 7.

[0280] For column 8: 50 μl of the 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.6% CaCl 2 , 0.3% NaCl was added to column 8.

[0281] For column 9: 50 μl of the 10 ml of a 10 mg / ml solution of α-amylase in DI water containing 0.8% CaCl 2 , 0.1% NaCl was added to column 9.

[0282] For column 10: 50 μl of 7.5% bleach (sodium hypochlorite) solution was added to column 10.

[0283] For column 11: 50 μl of DI water was added to column 11.

[0284] Column 12 was not treated. The plate should be placed in the 37°C incubator for the remaining 10 minutes.

[0285] 5.3 Biofilm Tests After 10 minutes, all fluid should be removed column by column every 30 seconds, starting with column 1. Fluid should be removed by tilting the plate, placing the pipette in the corner, and gently pipetting to dislodge non-adherent cells without disturbing biofilm growth on the well bottom.

[0286] 50 μl of 0.1% crystal violet (CV) was added to all wells to stain adherent cells.

[0287] All wells were carefully rinsed by immersing the plate in a DI water bath to remove any remaining crystal violet. The well plate was dried and the optical density of the biofilm was measured at 600 nm using a plate reader.

[0288] Once all measurements are complete, the well plates should be disinfected by transferring them to a bleach solution and disposed of in a biological waste bin. [Example]

[0289] 0.4% CaCl 2 to 0.8% CaCl 2 Calcium cofactor (CaCl) at concentrations ranging in volume 2 ) an α-amylase composition comprising The following experiments summarize the results observed with different compositions containing various additives to α-amylase from Aspergillus oryzae to determine whether any such additives enhance, do not affect, or inhibit the ability of the enzyme to remove biofilms grown in cell culture. The density of the remaining biofilm was measured at optical density 600 (OD 600 ) measurement was evaluated using a cell plate reader at 600 nm.

[0290] Example 6.1. Calcium Cofactor (CaCl 2 ) an α-amylase composition comprising Calcium cofactor optimization: Although calcium has been identified as a necessary cofactor for α-amylase, previous studies of α-amylase against Staphylococcus aureus biofilms did not incorporate calcium into the formulation to remove or inhibit biofilm growth in cell culture. This study was initiated to determine how much calcium is needed to provide optimal functionality for biofilm removal. Some literature suggests that excess calcium can have adverse effects. Therefore, we sought to determine how much calcium would support α-amylase activity without inhibiting it. 2+It was important to consider the concentration range of

[0291] Common methods: S. 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 h.

[0292] Non-adherent cells were removed, and the biofilms were treated with different solutions to test their ability to disrupt S. aureus biofilms. The α-amylase solution was prepared at 10 mg / mL using approximately 30 U / mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065).

[0293] After 10 min of incubation, non-adherent cells were removed and the remaining biofilm was stained with crystal violet. The density of the remaining biofilm was measured at 600 nm using a Byonoy Absorbance96 plate reader.

[0294] Figure 11A is a chart showing the results of the above-described experiment. The optical density of the biofilm is shown on the Y-axis. The X-axis shows various concentrations of CaCl2 cofactor and two controls: deionized water (negative control) and bleach (positive control for biofilm removal). The concentrations tested were 0.05% CaCl2 cofactor, 0.1% CaCl2 cofactor, 0.2% CaCl2 cofactor, 0.3% CaCl 2 Cofactor, 0.4CaCl 2 Cofactor, 0.5%CaCl 2 cofactor, 0.6% CaCl2 cofactor, or 0.8% CaCl2 cofactor.

[0295] The results of the Tukey's multiple comparison test analysis are summarized in the following table:

[0296] [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5]

[0297] A decrease in biofilm density indicates enzymatic biofilm removal. Results suggest that concentrations of 0.4% to 0.8% CaCl consistently removed biofilm. See Figure 11A. Figure 11B is a chart quantifying the percent of biofilm removed within 10 minutes by each test composition. In Figure 11B, bleach is used as a positive control. [Example]

[0298] Ophthalmic preservatives In configurations where the composition is delivered in individual sterile packages, sterile filtration and sterile packaging can be used to avoid the addition of preservatives. If the composition is delivered in multi-dose packages, preservatives may be present. In this example, common preservatives that have been tested and have not been found to inhibit the effectiveness of the enzyme are listed.

[0299] Common methods: S. 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 h.

[0300] Non-adherent cells were removed, and the biofilms were treated with different solutions to test their ability to disrupt S. aureus biofilms. The α-amylase solution was prepared at 10 mg / mL using approximately 30 U / mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065).

[0301] After 10 min of incubation, non-adherent cells were removed and the remaining biofilm was stained with crystal violet. The density of the remaining biofilm was measured at 600 nm using a Byonoy Absorbance96 plate reader.

[0302] Figure 12A is a chart showing experimental results for α-amylase, BAK + α-amylase, and sodium perborate + α-amylase, and their comparison to a negative control (DI water) and a positive control (bleach). The preservatives tested in Figure 12A were: Preservative 1: Purite / OcuPure (a stabilized oxychloro complex) - no reduction in enzyme effectiveness; Preservative 2: Polyquad (polyquaternium-1) 0.001% - no reduction in enzyme effectiveness.

[0303] The preservatives tested in Figure 12B are: Preservative 1: BAK (benzalkonium chloride) 0.01% - no reduction in enzyme effectiveness; and Preservative 2: GenAqua / Dequest (sodium perborate) - no reduction in enzyme effectiveness.

[0304] The aforementioned test compositions at the concentrations indicated were found not to inhibit enzyme activity.

[0305] Another preservative tested for use in treating dry eye conditions was 0.01% hypochlorous acid. Hypochlorous acid diluted at percentages ranging from 0.01% to 0.02% was used in eyelid cleansers. In our study, 0.01% hypochlorous acid did not inhibit enzymatic biofilm removal. No statistical difference was observed between biofilm reduction with 0.01% hypochlorous acid and biofilm reduction with α-amylase plus cofactors, whether mixed immediately before, 30 minutes before, or 1 hour before treatment. While 0.01% hypochlorous acid slightly reduced biofilm, no additive benefit of increased biofilm removal was observed when combined with α-amylase and cofactors. See Figure 13. [Example]

[0306] Ophthalmic demulcent This protocol investigated the efficacy of α-amylase, an enzyme effective in inhibiting and reducing Staphylococcus aureus biofilms, in combination with ophthalmic demulcents. These demulcents are used in eye drops to relieve dry eye symptoms and may potentially prolong the contact of α-amylase with biofilms on the eye. Some demulcents may have a stabilizing effect on the enzyme. The study evaluated the effect of various demulcents on α-amylase activity.

[0307] The following demulcents were considered:

[0308] (a) Cellulose derivatives: (1) sodium carboxymethylcellulose, 0.2 to 2.5 percent; (2) hydroxyethylcellulose, 0.2 to 2.5 percent; (3) hypromellose, 0.2 to 2.5 percent; (4) methylcellulose, 0.2 to 2.5 percent.

[0309] (b) Dextran 70, 0.1 percent when used with another polymeric demulcent in this section.

[0310] (c) Gelatin, 0.01 percent.

[0311] (d) Polyols, liquid: (1) glycerin, 0.2 to 1 percent; (2) polyethylene glycol 300, 0.2 to 1 percent; (3) polyethylene glycol 400, 0.2 to 1 percent; (4) polysorbate 80, 0.2 to 1 percent; (5) propylene glycol, 0.2 to 1 percent.

[0312] (e) Polyvinyl alcohol, 0.1 to 4 percent.

[0313] (f) Povidone, 0.1 to 2 percent.

[0314] All test solutions were prepared with 10 mg / mL α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) at approximately 30 U / mg, 0.4% CaCl2, and 0.5% NaCl and tested using the protocol described in the previous example. The test concentrations in this study were as follows:

[0315] [Table 19]

[0316] Figure 14A is a chart showing the analytical results of various test demulcents, including 1% polyethylene glycol 400 (PEG 400), 2.5% carboxymethylcellulose sodium (CMC), 1% glycerin, 2% povidone (PVP), 1% propylene glycol, 4% polyvinyl alcohol (PVA), and 0.1% dextran 700 + 0.3% hypromellose. Figure 14B is a chart showing the analytical results of various test demulcents, including polyvinyl alcohol (PVA), PVA + α-amylase, sodium carboxymethylcellulose (CMC), and sodium carboxymethylcellulose (CMC) + α-amylase. As shown in Figures 14A and 14B, CMC and PVA demonstrated beneficial effects on biofilm removal. Based on these results, we hypothesized that PVA may help stabilize α-amylase in certain packaging formats of the formulation.

[0317] Figure 14C is a chart showing the results of a 36-day test using α-amylase stabilized in 4% PVA. PVA and CMC were found to independently aid in biofilm removal and to work together with α-amylase to remove biofilm. PVA is preferable to CMC for filter sterilization and is not susceptible to bacterial growth. PVA provides a pH of 5.8, which is ideal for enzyme activation. Literature suggests that a pH between 5 and 6 is ideal for α-amylase from Aspergillus oryzae.

[0318] PVA solutions are better candidates for sterile filtration than CMC solutions. A 4% PVA solution was sterile filtered using a 2 μm sterile syringe filter and kept refrigerated for 36 days before repeating the biofilm removal test. The 4% PVA solution at 36 days performed better than a fresh solution of α-amylase and cofactors. This indicates that PVA can stabilize the α-amylase enzyme for at least 36 days in aqueous solution refrigerated at 4°C. [Example]

[0319] Antibiotic Testing This protocol investigated the effectiveness of α-amylase from Aspergillus oryzae in combination with a commonly prescribed ophthalmic antibiotic to determine whether the antibiotic aids in biofilm removal. Antibiotics are typically not thought to penetrate biofilms by themselves. Biofilms can also make bacteria resistant to antibiotics because bacteria can transfer DNA within biofilms.

[0320] Figure 15 is a chart showing the comparative results of ophthalmic antibiotics for reducing Staphylococcus aureus biofilm. 30 U / mg of 10 mg / mL α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog no. 10065) formulated with 0.4% CaCl and 0.56% NaCl as cofactors was used in the formulation along with gentamicin and ofloxacin. Both antibiotics were also tested by themselves.

[0321] The results of this study, shown in Figure 15, demonstrate that the ability of α-amylase from Aspergillus oryzae to clear biofilms is neither inhibited nor 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. [Example]

[0322] stabilizing sugar It has previously been reported that the use of sugars, particularly sucrose and trehalose, increases the thermal stability of α-amylase from Aspergillus oryzae in aqueous solutions. Trehalose is used in eye drops for dry eye patients due to its bioprotective properties for ocular epithelial cells. Trehalose solutions up to 200 mM (6.8%) have been used in commercially available eye drops. This experiment tested 10% solutions of sucrose and trehalose with and without the demulcents carboxymethylcellulose and polyvinyl alcohol to determine whether they affected the enzyme's ability to reduce biofilms. This effect of these formulations on biofilm reduction is shown in Figure 16.

[0323] These data indicated that neither stabilizing sugar had a statistically significant effect on the enzyme's ability to reduce biofilm (α = 0.05). When the stabilizing sugars were combined with carboxymethylcellulose, the enzyme's ability to reduce biofilm was limited. This indicates that carboxymethylcellulose is not an ideal demulcent to be combined with the stabilizing sugars trehalose or sucrose. The study shows that the stabilizing sugars combined with PVA still had a strong ability to remove biofilm. Because trehalose is an ideal stabilizing sugar for periocular use, the combination of PVA and trehalose will be explored in the buffer studies below. Trehalose was also studied independently as a variable below.

[0324] Trehalose The ability of α-amylase enzyme to disrupt biofilms was tested using 3%, 6.8%, and 8% trehalose solutions. There was no significant difference 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 could be considered, as the solution is applied to intact skin and not to the eye itself. See Figure 17. [Example]

[0325] pH Literature has shown that the optimal activity of α-amylase from Aspergillus oryzae is at a pH between 5 and 6. The pH of tear fluid is 6.5 to 7.6, the pH of normal skin is 4.7 and 5.75, and the pH of oily skin is 5.7 to 7. The enzyme composition will be applied to the eyelid, not the eye. However, due to its proximity to the eye, a pH of 5.8 to 6 will be used in the final composition to balance enzyme activity and user comfort. In testing, the present disclosure has determined that the pH of a 4% PVA solution is 5.8, and the pH of a 2% PVA solution is 5.9.

[0326] The following buffers were considered for pH stabilization:

[0327] [Table 20]

[0328] Phosphate and citrate buffers were ideal candidates due to their pH range, so they were further analyzed for use in the composition to maintain a pH of 5.8. When tested at a concentration of 0.4% CaCl, calcium reacted with phosphate and precipitated. This reaction ruled out phosphate buffer as a candidate when CaCl was used as a cofactor. This suggested citrate buffer as the optimal ophthalmic buffer candidate. Based on literature, solutions were made at 50 mM and 100 mM citrate buffer concentrations and tested for their ability to remove biofilms. Trehalose, used as a heat stabilizer for enzymes, was added at 200 mM because it has been reported in the literature to be well tolerated for use in eye drops. If citrate buffer was added directly to a 4% PVA solution, sodium citrate would form a gel and may not dissolve. To test citrate buffer and PVA, sodium citrate and citric acid were dissolved in DI water and mixed 50:50 with a 4% PVA solution to create a 2% PVA solution. All citrate buffer solutions were adjusted to pH 5.8 using NaOH and HCl. Figures 18A and 18B are charts showing the results of testing 50 mM to 100 mM citrate buffer with sugar stabilizers and demulcents.

[0329] Studies have shown that citrate buffer interacts with PVA and inhibits the biofilm removal function of the composition at both 50 mM and 100 mM concentrations. Simply using citrate buffer with NaOH and HCl to bring the composition to a pH of 5.8 enhanced the enzyme's performance. See Figures 18A and 18B. The enzyme's best performance was achieved without citrate buffer and with either 2% or 4% PVA, with or without trehalose. [Example]

[0330] hyaluronic acid Hyaluronic acid and tea tree oil are common additives in face washes 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 chart showing the results of a test of the effects of hyaluronic acid and tea tree oil on a composition containing α-amylase. As shown in Figure 19, 0.3% hyaluronic acid slightly increased the enzyme's ability to remove biofilms. Tea tree oil did not significantly enhance or decrease the enzyme's performance. This indicates that hyaluronic acid may have beneficial effects in compositions for removing biofilms. In eye drop formulations, hyaluronic acid is often combined with trehalose due to their combined hydration benefits. [Example]

[0331] Treatment of Human Subjects with Compositions of the Disclosure All images were from the same subject, obtained at the designated time at the Watson Dry Eye Center in Raleigh, North Carolina. Images were retrospectively reviewed 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 between May 23, 2018, and November 20, 2020, the patient washed her eyes twice daily with only 0.01% hypochlorous acid in saline. Following this, the patient used a novel composition of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. The first composition was made from α-amylase from Aspergillus oryzae powder and 0.01% hypochlorous acid liquid in saline immediately before application to the eyelids with a cotton swab. The second composition was made from Ca 2+ The drug contained 20 mg of acetaminophen and had excellent performance. The dosing information is listed in the timeline below.

[0332] Treatment timeline - before use of the composition The subject underwent his first treatment session to treat his dry eye symptoms and recurrent ocular inflammation on May 23, 2018. Prior to this date, the subject had received a combination of Azasite and oral antibiotics (doxycycline 100 mg / day), which did not appear to alleviate his symptoms. Figures 1A and 1B are photographs taken on May 23, 2018 (time 0) showing two different areas of the subject's eye suffering from blocked meibomian glands, a shiny biofilm at the base of the eyelashes, a shiny biofilm on the eyelashes, and maloriented eyelash growth. After photography, the subject was treated with Blephax and intense pulsed light therapy to remove the biofilm and treat the inflammation.

[0333] The subject underwent a subsequent treatment session on December 4, 2018. Figures 2A and 2B are photographs showing two different areas of the subject's eye on December 4, 2018, approximately six months after the photographs in Figures 1A and 1B were taken (Session 1). After the photographs were taken, the subject underwent one treatment with Blephex and intense pulsed light therapy to remove biofilm and treat inflammation. The subject then received a daily dose of Avenova (0.01% hypochlorous acid (HOCL)). Minimal improvement in symptoms was reported at this session.

[0334] The subject underwent a subsequent treatment session with daily use of Avenova (0.01% hypochlorous acid (HOCL)) on June 18, 2019. Figures 3A and 3B are photographs (second visit) showing two different areas of the subject's eye, approximately six months after the photographs in Figures 2A and 2B were taken. After the photographs were taken, the subject underwent one treatment with Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation. The subject was then prescribed daily use of Avenova (0.01% hypochlorous acid (HOCL)). As shown in Figure 3A, the biofilm continued to grow at the base of the eyelashes.

[0335] The subject underwent a subsequent treatment session with daily use of Avenova (0.01% hypochlorous acid (HOCL)) on October 15, 2019. Figures 4A and 4B are photographs (third visit) showing two different areas of the subject's eye, approximately four months after the photographs in Figures 3A and 3B were taken. After the photographs were taken, the subject underwent one treatment with Blephex and intense pulsed light therapy to remove the biofilm and treat the inflammation. The subject was then treated with daily use of Avenova (0.01% hypochlorous acid (HOCL)). As shown in Figures 4A and 4B, shiny biofilm remained on the eyelashes and inside the eyelids, suggesting that acid treatment alone was not sufficient to remove the biofilm.

[0336] In October 2020, a first composition was prepared using 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. Figures 5A and 5B are photographs showing two different areas of a subject's eye on or around November 2, 2020, after multiple applications of 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. This time frame is approximately one month after the photographs in Figures 4A and 4B were taken (the fourth time point). As shown in Figures 5A and 5B, biofilm was still visibly present after multiple applications of low doses within the one-month time frame for treatment.

[0337] The subject received a daily dose of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid between visit 4 and May 26, 2021 (visit 5), and the subject received routine daily use of the composition. Figures 6A and 6B are photographs showing two different areas of the subject's eye approximately 6 months after the photographs in Figures 5A and 5B were taken. After the photographs in Figures 5A and 5B were taken, the subject received routine daily use of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid. As shown in Figures 6A and 6B, the biofilm had visibly disappeared, the eyelashes were cleaned of all biofilm, inflammation and redness in the eyelids had decreased, and the patient reported a major improvement in her dry eye symptoms.

[0338] On October 5, 2021 (Visit 6), approximately 5 months after routine daily use of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid, the subject reported significant improvement in symptoms, and new eye images were taken at the Watson Dry Eye Center in Raleigh, North Carolina. As shown in Figures 7A and 7B, biofilm growth was inhibited and the eyelid tissue was no longer inflamed.

[0339] The subject continued to be monitored. On February 3, 2022 (Day 7), approximately four months after routine daily use of a composition containing 1 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid, biofilm growth on the subject's eye was inhibited. See Figures 8A and 8B. Furthermore, no inflammation was observed within that time frame. Between Day 7 and approximately six months (August 4, 2022), the subject was tested for tolerability of a higher dosage of 10 mg / ml α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid for biweekly (bi / weekly) use. No adverse effects were observed with the higher dose of 10 mg / ml α-amylase. See Figures 9A and 9B. [Example]

[0340] Treatment of Human Subjects with Compositions of the Disclosure 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 eyelid margins biweekly of patients exhibiting elevated levels of eyelid biofilm. Observations over a six-month period showed a significant reduction in biofilm on the eyelid margins, an increase in tear production, and a reduction in signs of ocular surface stress. In a pilot study, a composition containing approximately 3% α-amylase, 1% lysozyme, 0.06% sodium chloride, and 0.02% hypochlorous acid solution in ionized water showed similar improvements. Each composition in the pilot study was prepared fresh before use. Further composition development explored the combination of the aforementioned components described throughout this disclosure with preservatives, astringents, demulcents, emollients, tonicity agents, and vasoconstrictors to improve the long-term stability of the composition. Figures 10A and 10B show the results of 10 mg / ml α-amylase and Ca in 0.01% hypochlorous acid. 2+ 1 is a photograph showing a direct comparison of an area of ​​the eye before and after treatment with a composition comprising: [Example]

[0341] formulation This protocol describes a procedure for testing a composition of α-amylase from Aspergillus oryzae and 0.01% hypochlorous acid for its compatibility and ability to remove biofilm from Staphylococcus aureus. The 0.01% hypochlorous acid is intended to inhibit microbial growth and does not extend the shelf life of the α-amylase. This experiment will also test the effect of hypochlorous acid on the enzyme by using the α-amylase and hypochlorous acid composition at different times after the composition is made. The composition will be tested for its ability to remove biofilm immediately after the composition is made, as well as 30 minutes and 1 hour later. This test will indicate whether it is advantageous to combine aqueous hypochlorous acid with dry α-amylase before applying it to the biofilm.

[0342] formulation This experiment was conducted using the off-the-shelf eyelid cleanser Avenova (0.01% hypochlorous acid in saline). 0.4% calcium chloride was added to the hypochlorous acid solution to ensure calcium ions were present in all tested solutions.

[0343] A 10 ml solution of 10 mg / ml α-amylase and cofactors was prepared.

[0344] Add 100 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) to 10 ml of sterile DI water in a flask. Mix until the α-amylase is dissolved and evenly distributed.

[0345] 40 mg of calcium chloride and 56 mg of sodium chloride were dissolved in the solution.

[0346] 1 hour before trial 5 ml of a 10 mg / ml α-amylase solution containing 0.01% hypochlorous acid was prepared.

[0347] 5 mL of Avenova (0.01% hypochlorous acid) was added to the flask.

[0348] 20 mg of calcium chloride was dissolved in the solution.

[0349] 50 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added and mixed until the α-amylase was dissolved and evenly distributed.

[0350] 30 minutes before trial: Five ml of a 10 mg / ml α-amylase solution containing 0.01% hypochlorous acid was prepared.

[0351] 5 mL of Avenova (0.01% hypochlorous acid) was added to the flask.

[0352] 20 mg of calcium chloride was dissolved in the solution.

[0353] 50 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added and mixed until the α-amylase was dissolved and evenly distributed.

[0354] Immediately before plating: Five ml of a 10 mg / ml α-amylase solution containing 0.01% hypochlorous acid was prepared.

[0355] 5 mL of Avenova (0.01% hypochlorous acid) was added to the flask.

[0356] 20 mg of calcium chloride was dissolved in the solution.

[0357] 50 mg of α-amylase from Aspergillus oryzae (Sigma-Aldrich catalog number 10065) was added and mixed until the α-amylase was dissolved and evenly distributed.

[0358] Test composition All liquid was pipetted from the wells by carefully tilting the plate and inserting the pipette tip into the corner of the bottom of the well to remove non-adherent cells without disturbing the biofilm growth on the bottom of the well.

[0359] A 10-minute timer was started when the first row was treated using an 8-channel pipette. Each row should be treated every 30 seconds, ensuring that the composition at the corners of the wells is released gradually to avoid mechanically disturbing the biofilm.

[0360] For column 1: 50 μl of DI water was added to column 1.

[0361] For column 2: 50 μl of 10 mg / ml α-amylase with cofactors was added to column 2.

[0362] For column 3: 50 μl of 10 mg / ml α-amylase with cofactors and hypochlorous acid that was left for 30 minutes was added to column 3.

[0363] For column 4: 50 μl of 10 mg / ml α-amylase with cofactors and hypochlorous acid that had been left for 1 hour was added to column 4.

[0364] For column 5: Combine 5 ml of 10 mg / ml α-amylase solution with hypochlorous acid. Add 50 μl of 10 mg / ml α-amylase with cofactors and hypochlorous acid to column 5.

[0365] Regarding column 6: 50 μl of 0.018% hypochlorous acid solution was added to column 7.

[0366] For column 7: 50 μl of 7.5% bleach (sodium hypochlorite) solution was added to column 7.

[0367] The plate was incubated at 37°C for the remaining 10 minutes.

[0368] Biofilm reduction test After 10 minutes, all fluid should be removed column by column every 30 seconds, starting with column 1. Fluid should be removed by tilting the plate, placing the pipette in the corner, and gently pipetting to remove non-adherent cells without disturbing biofilm growth on the well bottom. Fifty microliters of 0.1% crystal violet (CV) was added to all wells to stain adherent cells. All wells were carefully rinsed by immersing the plate in a DI water bath to remove any remaining crystal violet. The well plate was then dried, and the optical density of the biofilm was measured at 600 nm using a plate reader. After all measurements were completed, the well plate should be transferred to a bleach solution for disinfection and disposed of in a biological waste bin.

[0369] result The results showed that neither α-amylase with cofactors nor α-amylase with cofactors combined with hypochlorous acid spray demonstrated statistically significant reduction in biofilm across all time points, indicating that 0.01% hypochlorous acid did not inhibit enzymatic biofilm removal. While 0.01% hypochlorous acid slightly reduced biofilm, there was no additive benefit of increased biofilm removal when α-amylase was combined with cofactors. See Figure 13. [Example]

[0370] Enzymatic face and eyelid cleanser The various compositions described herein have been developed as enzyme-based facial and / or eyelid treatments.

[0371] This enzymatic facial and eyelid cleanser device utilizes alpha-amylase enzymes to disrupt the extracellular matrix of biofilms that form on the eyelids. Biofilms are structured communities of microorganisms attached to surfaces and surrounded by their own extracellular matrix (ECM). The ECM is primarily composed of polysaccharides, proteins, nucleic acids, and lipids. This matrix provides structural stability for the biofilm, protects the internal bacteria from external factors, and helps the biofilm adhere to surfaces.

[0372] Biofilms are aggregates of microorganisms, such as bacteria, embedded in a self-produced matrix of proteins and polysaccharides on the eyelids. These biofilms can lead to inflammation and infection of the eyelid margin, known as blepharitis, and contribute to dry eye disease by disrupting the tear film and causing tear evaporation.

[0373] The α-amylase isoform in the composition acts as an enzyme that breaks down polysaccharides in the biofilm matrix, thereby destabilizing the biofilm structure and facilitating its removal from the eyelids. As the ECM destabilizes, the biofilm becomes more susceptible to mechanical removal (e.g., by wiping). Furthermore, the bacteria within the biofilm lose their protective shield, making them more vulnerable to antimicrobial agents or the body's immune response.

[0374] In summary, α-amylase isoforms from Aspergillus oryzae targeted and disrupted the polysaccharide component of the biofilm extracellular matrix, facilitating biofilm removal from the eyelid, which may be particularly useful in the management of conditions such as blepharitis and dry eye disease, where biofilms contribute to the pathology.

[0375] The enzyme compositions can be delivered via different delivery mechanisms, such as sterile wipes, sprays, or single- or multi-dose sterile applicators. In some cases, the compositions are cleansers. The compositions can be packaged as wipes, sprays, pads, or another suitable applicator infused with α-amylase powder or solution sourced from Aspergillus oryzae.

[0376] 16.1 Sterile wipes The compositions of the present disclosure are packaged as sterile wipes pre-filled with the composition. These wipes are packaged individually or in sets. Such wipes can be further packaged in containers or as part of kits containing other reagents.

[0377] How to use the composition packaged as a sterile wipe: The subject gently closed their eye and used the sterile wipe to clean the eyelid, for example, in a horizontal motion from the inner corner to the outer corner of the eye. The subject ensured that the composition was applied preferentially to the base of the eyelashes, where biofilm is more likely to accumulate.

[0378] The wipes are individually wrapped for single use, reducing the risk of contamination. The wipes are portable, making them convenient for travel or on-the-go use.

[0379] 16.2 Spray The enzyme composition could be formulated as a solution (eg, a spray or drops) in a bottle.

[0380] To use the composition packaged as a spray: The spray bottle containing the enzyme composition was applied directly to the eyelid or sprayed onto a clean cloth or cotton pad. The subject could close their eyes and gently spray the solution directly onto their eyelid. Alternatively, the subject could spray the solution onto a clean cloth or cotton pad. If using a cloth or pad, the eyelid was gently wiped from the inner corner to the outer corner.

[0381] The spray bottle is easy to use and allows for quick application, is suitable for multiple uses, and does not come into direct contact with the eyelids, reducing the risk of contamination.

[0382] 16.3 Single- or multi-dose sterile containers The single or multi-dose sterile container was used for controlled delivery of the enzyme solution to a sterile applicator, e.g., a wipe or sponge, which was then applied to the eyelid. The applicator could be attached to the sterile container or could be separate.

[0383] Directions for use of the composition packaged in a single or multi-dose sterile container: The subject closes the eye and uses the applicator to apply the solution along the eyelid margin and eyelashes. The subject carefully cleanses the eyelid in a horizontal motion, moving from the inner corner to the outer corner. The applicator supports targeted application of the solution / gel, which may be beneficial for those requiring more controlled application or with sensitivity issues.

[0384] While the present invention is fulfilled by embodiments in many different forms, as detailed in connection with the preferred embodiments of the present invention, it should be understood that the present disclosure should not be considered as an exemplification of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations can be made by those skilled in the art without departing from the spirit of the invention. The scope of the invention will be determined by the appended claims and their equivalents. The abstract and title of the invention should not be construed as limiting the scope of the invention, as their purpose is to enable the appropriate authorities and the general public to quickly determine the general nature of the invention. In the following claims, unless the term "means" is used, no feature or element recited therein should be construed as a means-function limitation pursuant to 35 U.S.C. § 112, paragraph 6.

Claims

1. A method for treating one or more of blepharitis and dry eye, comprising administering a concentration of CaCl ２ 20. A method for treating a subject comprising administering to the subject a therapeutically effective amount of a composition comprising an active α-amylase enzyme or functional fragment thereof stabilized with α-amylase and one or more pharmaceutically acceptable excipients.

2. The method of claim 1 , wherein the composition is present in a topical applicator.

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

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

5. The method of claim 3 , wherein the wipe comprises a dry component.

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

7. The method of claim 1 , wherein the composition is applied to the eyelid.

8. 10. The method of claim 1, wherein the administration promotes removal of biofilm in the subject's eye or in the area surrounding the subject's eye.

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

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

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

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

13. 10. The method of claim 1, wherein the composition comprises between 1 mg / mL (w / v) and 20 mg / mL (w / v) of active α-amylase enzyme.

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

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

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

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

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

19. 10. The method of claim 1, wherein the administration is topical administration to the outside of the eye.

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

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

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

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

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

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

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

27. 26. The method of claim 25, wherein the composition is formulated as a liquid.

28. 26. The method of claim 25, wherein the composition is formulated as a gel.

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

30. An ophthalmic composition comprising a therapeutically effective amount of a functionally active α-amylase enzyme for treating blepharitis or a dry eye condition, CaCl2 in a concentration to stabilize the α-amylase enzyme, and one or more pharmaceutically acceptable excipients.

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

32. 32. The ophthalmic composition of claim 31, wherein the alpha-amylase enzyme shares 95% sequence homology with the alpha-amylase peptide sequence from Aspergillus oryzae.

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

34. 31. The ophthalmic composition of claim 30, wherein the concentration of CaCl2 ranges from 0.2% to 0.8% (w / v).

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

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

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

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

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

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

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

42. 36. The ophthalmic composition of claim 35, wherein the composition further comprises between 0.01% (v / v) and 1.5% (v / v) glycerin.

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

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

45. 36. The ophthalmic composition of claim 35, wherein the composition further comprises between 0.2% (v / v) and 1.0% (v / v) of polysorbate.

46. 36. The ophthalmic composition of claim 35, wherein the composition further comprises between 0.2% (v / v) and 2.0% (v / v) propylene glycol.

47. 36. The ophthalmic composition of claim 35, wherein the composition further comprises between 0.2% (v / v) and 6.0% (v / v) polyvinyl alcohol.

48. 36. The ophthalmic composition of claim 35, wherein the composition further comprises between 0.1% (v / v) and 4.0% (v / v) povidone.

49. 36. The ophthalmic composition of claim 35, wherein the composition further comprises between 0.001% (v / v) and 0.1% (v / v) of benzalkonium chloride (BAK).

50. 36. The ophthalmic composition of claim 35, wherein the composition further comprises between 0.0001% (v / v) and 0.01% (v / v) of polyquad (polyquaternium-1).

51. 36. The ophthalmic composition of claim 35, wherein the composition further comprises a stabilized oxychloro complex.

52. 36. The ophthalmic composition of claim 35, wherein the composition further comprises sodium perborate.

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

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

55. 36. The ophthalmic composition of claim 35, wherein the composition further comprises a citrate buffer.

56. 36. The ophthalmic composition of claim 35, wherein the composition further comprises polyhexanide (polyhexamethylene biguanide).

57. 31. The ophthalmic composition of claim 30, wherein the composition is formulated as a solution.

58. 31. The ophthalmic composition of claim 30, wherein the composition is formulated as a gel.

59. A composition comprising any combination of components as recited in claims 30-58.

60. Functionally active α-amylase enzyme at a concentration ranging from 1 mg / mL to 20 mg / mL and CaCl at a concentration ranging from 0.4 to 0.8% (w / v) to stabilize the α-amylase enzyme. ２ and polyvinyl alcohol (PVA) at a concentration ranging from 0.1% to 5%, and at least one pharmaceutically acceptable excipient.

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

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

63. CaCl ２ 61. The composition of claim 60, wherein the concentration of is 0.4%.

64. Functionally active α-amylase enzyme at a concentration ranging from 1 mg / mL to 20 mg / mL and CaCl at a concentration ranging from 0.2% to 0.8% (w / v) to stabilize the α-amylase enzyme. ２ and polyvinyl alcohol (PVA) at a concentration ranging from 0.1% to 5%, and at least one pharmaceutically acceptable excipient.

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

66. 65. The topical applicator of claim 64, wherein the topical applicator is a wipe.

67. 65. The topical applicator of claim 64, wherein the wipes are individually packaged.

Images