Peroxidase compositions containing improved enhancers

Abstract

The present invention provides peroxidase-based pharmaceutical, prophylactic, and cosmetic compositions. The compositions are preferably formulated for topical use. More specifically, the present invention relates to peroxidase-based compositions that further comprise a compound that enhances and / or stabilizes the activity of antimicrobial peroxidase. The present invention also provides methods and uses that include the compositions of the present invention.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention belongs to the fields of medicine and biomedical science. The present invention broadly relates to both pharmaceutical and cosmetic compositions, and their related methods and uses. More specifically, the present invention relates to peroxidase-based compositions further comprising a compound that enhances and / or stabilizes the activity of antimicrobial peroxidase. [Background technology]

[0002] Background of the Invention Peroxidases are ubiquitous enzymes that are part of a larger group known as oxidoreductases, constituting the second largest class of enzymes applied in biotechnology processes. Peroxidases are used to catalyze a variety of oxidation reactions that use hydrogen peroxide and other substrates as electron donors; this reduction of peroxide to consume the electron-donating substrate makes peroxidases particularly useful in many biotechnology applications. Different peroxidases can be isolated from a variety of sources, including plants, animals, and microorganisms. Different peroxidase enzymes have versatile applications in bioenergy, bioremediation, dye decolorization, humic acid degradation, paper and pulp, and textile industries (Twala et al., AIMS Microbiol, 2020).

[0003] The antibacterial activity of peroxidase-based systems depends on the type of electron donor used. EP 514417 reports an antibacterial composition containing glucose oxidase, iodide, and thiocyanate ions. EP 514417 further demonstrates the enhancing effect of antioxidants on peroxidase activity.

[0004] US 4,476,108 describes a method for generating bactericidal free radicals in the oral cavity over a controlled period of time by applying a combination of peroxidase, peroxide, and a donor molecule source. The formulation is described as preferably used in a carrier liquid or paste. The carrier can be water, toothpaste, mouthwash, chewing gum, prophylactic paste, denture cleaner, and mouthwash gel. More specifically, US 4,476,108 discloses a short-acting (less than 2 minutes) antibacterial composition for oral application that uses peroxide, peroxidase, and donor molecules such as phenylethylamine, tyrosine, tryptophan, benzoic acid, salicylic acid, hydroquinone, dehydrophenylalanine, vanillin, and paraaminobenzoic acid.

[0005] Antibacterial activity against fungi and bacteria has been attributed to aromatic flavor compounds such as syringaldehyde and vanillin (Fitzgerald et al., J Agric Food Chem, 2005; and Fitzgerald et al., J Appl Microbial, 2004). However, antibacterial activity is only observed at high concentrations and after prolonged incubation.

[0006] US 20020119136 describes a class of dialkoxyphenolic compounds (e.g., acetosyringone and alkylsyringates) that have peroxidase-enhancing effects.

[0007] WO 2006 / 133523 describes a -OH group or (CH2) nImproved peroxidase-based antimicrobial compositions are described that contain benzene compounds (including guaiacol) substituted with OH groups (n=1, 2, 3, or 4) and one alkoxy group (—OR) with a chain length of 1, 2, 3, or 4 carbon atoms. These enhancers are currently used in Flaminal, manufactured by Flen Health for the treatment of acute and chronic wound types (e.g., traumatic wounds, burns, surgical wounds, venous and arterial ulcers, diabetic ulcers, pressure ulcers, etc.). (登録商標) Forte and Flaminal (登録商標) These enhancing agents are formulated into well-defined commercial compositions such as Hydro. However, given the highly specific nature of these enhancing agents, alternative and / or improved enhancing agents would be valuable for further improving peroxidase-based compositions.

[0008] Thus, there is an unmet need for improved, potent and / or stable peroxidase-based antimicrobial compositions. Summary of the Invention

[0009] Summary of the Invention As demonstrated in detail in the enclosed examples, the inventors have discovered a group of enhancers, including certain amino acids, amino acid mimics, and amino acid derivatives, that are particularly useful for improving peroxide-generating systems in antimicrobial compositions. In addition to the enhancers, the compositions contain a peroxide-generating system, a peroxidase, and a halide or pseudohalide. The inventors provide experimental evidence of the efficacy of the enhancers through both enzyme assays and antimicrobial activity assays (Minimal Inhibitory Concentration (MIC) assays, Minimal Bactericidal Concentration (MBC) assays, absolute bacterial counting experiments, and challenge tests). This finding is directly applicable to all types of compositions, including, but not limited to, therapeutic, prophylactic, and cosmetic compositions, and more particularly, topical compositions. Thus, compositions containing the above-mentioned ingredients can increase the rate of healing by modulating microbial load, if present, and by modulating the immune response through multiple mechanisms (both indirect and direct), as outlined below.

[0010] First, the compositions described herein modulate the microbial load, and therefore indirectly modulate the inflammatory condition, even when compared to known compositions that rely on other potentiators, such as, but not limited to, guaiacol. Indeed, by reducing the microbial load (i.e., reducing the number of microorganisms present in an infected wound), the compositions indirectly effectively alleviate the inflammatory condition.

[0011] In addition, the compositions described herein also modulate inflammation in a direct manner, i.e., by both reducing the secretion of pro-inflammatory molecules by immune cells and by exerting a neutralizing effect on already secreted pro-inflammatory molecules. Thus, even in the absence of infection, the compositions described herein exert a direct effect on inflammation by either reducing the secretion of pro-inflammatory markers (i.e., molecules) by immune cells, neutralizing already secreted pro-inflammatory markers, or a combination thereof.

[0012] Taking the above findings made by the inventors together, the compositions described herein are a powerful means of increasing the rate of wound healing through each of the above properties, particularly the combined effect of each of the above properties, ultimately reducing discomfort, mortality, and morbidity in subjects. As a further result of the antimicrobial effect, when the compositions are applied to a subject as a component of an ointment, gel, dressing, etc., they need to be changed less frequently.

[0013] Furthermore, the inventors have unexpectedly found that certain halides, such as potassium iodide, provide even greater improvements in the composition when compared to other halides, including those commonly used in the art.Finally, lactoperoxidase has been found to be a particularly potent peroxidase when used in the compositions described herein.

[0014] Accordingly, a first aspect of the present invention is directed to a composition comprising a peroxide-generating system, a peroxidase, a halide or pseudohalide, and an enhancer, wherein the enhancer is an organic compound characterized by (a) amino and carboxylate functional groups and by further comprising one or more side chains containing a functional group selected from the group consisting of a polar uncharged methanol, ethane-1-ol, acetamide, propenamide, or secondary butyl group; or (b) pyrrolidine and carboxylate functional groups.

[0015] In certain embodiments, the connection between the amino and / or carboxylate functional groups and the different side chains or pyrrolidines (in a and b) is (CH) n (wherein n=1, 2, or 3).

[0016] In certain embodiments, the enhancer has a free amino functional group (-NH + ), a free carboxylic acid functional group (—COH), and (i) -OH group; (ii)-(CH2) n an OH group (where n is 1, 2, or 3); (iii)-(CH2) n CONH2 groups (where n is 1, 2, or 3); (iv) -cyclic C4H9N group; or (v) Branched alkyl group and a side chain comprising:

[0017] In certain embodiments, the enhancer is a hydrophilic amino acid, amino acid mimic, or amino acid derivative having an octanol / water partition coefficient of about -1 to about -5.

[0018] In some embodiments, the enhancer is a hydrophilic natural or unnatural amino acid. In some embodiments, the enhancer is a naturally occurring amino acid having an octanol / water partition coefficient of about -1.72 to about -3.82.

[0019] In certain embodiments, the halide or pseudohalide is a water-soluble iodide salt. Preferably, the halide is potassium iodide, which has been found to further improve the antimicrobial activity of the composition.

[0020] In some embodiments, the enhancer is a naturally occurring amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof.

[0021] In one embodiment, the potentiator is present in the composition at a concentration of from 0.0005 mg / ml to 200 mg / ml, preferably from 0.005 mg / ml to 10 mg / ml, and most preferably from 0.05 to 1 mg / ml.

[0022] In certain embodiments, the peroxide generating system comprises an oxidase, preferably the peroxide generating system is glucose oxidase.

[0023] In some embodiments, peroxidase is lactoperoxidase, which has been found to work particularly well with the enhancer described herein.In some embodiments, lactoperoxidase is human lactoperoxidase or bovine peroxidase.Preferably, lactoperoxidase comprises the amino acid sequence with SEQ ID NO:1 that has at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably 100% sequence identity.

[0024] In some embodiments, the composition further comprises one or more solvents, diluents, buffers, solubilizers, colloids, fillers, dispersion media, amino acids, proteins, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, sweeteners, colorants, flavorings, coatings, antifungal agents, preservatives, antioxidants, adjuvants, viscosity modifiers, penetration enhancers, chelating agents, or any combination thereof. In further embodiments, the composition further comprises guaiacol.

[0025] In some embodiments, the enhancer increases the activity of the peroxide generating system and / or peroxidase by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% when compared to a reference (control) composition that does not contain the enhancer.

[0026] In some embodiments, the composition is characterized by a Minimum Inhibitory Concentration (MIC) and / or Minimum Bactericidal Concentration (MBC) and / or absolute count and / or enzyme activity against an inoculum of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans), and Aspergillus niger (A. niger), or any combination thereof, that is significantly reduced when compared to a reference (control) composition that does not contain a potentiator.

[0027] In some embodiments, the MIC and / or MBC is greater than or equal to an OD of 0.1. 600 The composition is measured by incubating the composition in a microbial inoculum characterized by the following: 37°C for 24 hours (Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans) or 25°C for 48 hours (Aspergillus niger).

[0028] In certain embodiments, the composition is a topical composition. In preferred embodiments, the topical composition is for application to the skin.

[0029] In some embodiments, the composition is a cream, lotion, foundation, ointment, suspension (oil-in-water and water-in-oil), patch, dressing, gel, or emulsion. In some embodiments, the composition is a deodorant, preferably a roll-on or stick deodorant. In some embodiments, the composition is an anti-acne preparation, preferably in the form of a lotion or cream. In some embodiments, the composition is contained in or applied to an impregnated material, such as a wound dressing. In further embodiments, the wound dressing is a dressing configured for wound cleansing and / or wound treatment. In some embodiments, the composition is an ear drop, nose drop, inhalant, vaginal solution, or rectal solution.

[0030] In some embodiments, the composition comprises: about 150 to about 4000 U / kg, preferably about 200 to about 3000 U / kg, more preferably about 300 to about 2500 U / kg of a peroxide generating system; about 10-100,000 U / kg, preferably about 10-4,000 U / kg, more preferably about 10-100 U / kg of a peroxidase, such as lactoperoxidase; about 0.01 to about 500 mg / kg, preferably about 0.1 to about 200 mg / kg, more preferably about 1 mg to about 100 mg / kg, even more preferably about 2 mg to about 75 mg / kg, and most preferably 5 to 50 mg / kg of a halide or pseudohalide; and an enhancer of 0.0005 mg / ml to 200 mg / ml, preferably 0.005 mg / ml to 10 mg / ml, more preferably 0.05 to 1 mg / ml Includes.

[0031] In certain embodiments, the composition is a pharmaceutical or cosmetic composition. In some embodiments, the composition is a sustained or controlled release composition.

[0032] In further aspects, the compositions described herein are contemplated for use as a medicament. In certain embodiments, the compositions described herein are contemplated for use in the treatment or prevention of skin disorders. In certain embodiments, the compositions described herein are contemplated for use in wound healing. In certain embodiments, the compositions described herein are contemplated for use as an antibacterial composition or for the treatment or prevention of microbial infections. In further aspects, the compositions described herein are contemplated for use as an anti-inflammatory, i.e., for reducing inflammation in a subject.

[0033] In further aspects, the use of the compositions described herein is contemplated for the manufacture of a medicament. In some embodiments, the use of the compositions described herein is contemplated for the manufacture of a medicament for the treatment or prevention of skin disorders. In some embodiments, the use of the compositions described herein is contemplated for the manufacture of a medicament for wound treatment. In some embodiments, the use of the compositions described herein is contemplated for the manufacture of a medicament for the treatment or prevention of microbial infections.

[0034] In a further aspect, the present invention contemplates a method of treating or preventing a skin disorder in a subject, the method comprising administering to the subject a composition described herein. In some embodiments, the present invention relates to a method of treating a wound, comprising administering to the subject a composition described herein. In some embodiments, the present invention relates to a method of treating or preventing a microbial infection, the method comprising administering to the subject a composition described herein. In some embodiments, the present invention relates to an indirect method of treating or preventing inflammation associated with the presence of a microbial infection, the method comprising administering to the patient a composition described herein.

[0035] In a further aspect, the present invention is directed to the use of an enhancer to improve the minimum inhibitory concentration (MIC) and / or the minimum bactericidal concentration (MBC) and / or the absolute number and / or enzymatic activity of a pharmaceutical composition comprising a peroxide generating system, a peroxidase, and a halide or pseudohalide, wherein the enhancer is a compound having a free amino functional group (-NH + ), a free carboxylic acid functional group (—COH), and (i) -OH group; (ii)-(CH2) n an OH group (where n is 1, 2, or 3); (iii)-(CH2) n CONH2 groups (where n is 1, 2, or 3); (iv) -cyclic C4H9N group; or (v) Branched alkyl group and a side chain comprising:

[0036] In certain embodiments, the enhancer is a natural or unnatural amino acid, preferably the enhancer is a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof.

[0037] In some embodiments, the use is an in vitro use. In some embodiments, the use is an in vitro use for preservation of skin grafts, cell lines, biomarkers, and / or biological sample material.

[0038] In a further aspect, the present invention is directed to the use of an enhancer for preserving the sterility of a pharmaceutical formulation, wherein the enhancer is as described herein. In certain embodiments, the enhancer is a compound having a free amino functional group (-NH + ), a free carboxylic acid functional group (—COH), and (i) -OH group; (ii)-(CH2) n an OH group (where n is 1, 2, or 3); (iii)-(CH2) n CONH2 groups (where n is 1, 2, or 3); (iv) -cyclic C4H9N group; or (v) Branched alkyl group and a side chain comprising:

[0039] These and further aspects and preferred embodiments of the present invention are set out in the following sections and in the appended claims, the subject matter of which is specifically incorporated into this specification. [Brief explanation of the drawings]

[0040] [Figure 1]Figure 1. Overview of GOX activity in the presence of proline (100–0.1 mg / ml) and cysteine ​​(1–0.1 mg / ml). Each result represents the mean enzyme activity (expressed in U / g) ± SD of at least two biological replicates. Statistical significance compared to the reference (i.e., GOX activity without the potentiator molecule candidate) was calculated using students' t-tests. Significance levels are defined as p<0.05 = *; p<0.01 = **, and p<0.001 = ***. [Figure 2] Figure 2. Summary of LPO activity in the presence of different molecules. Each result represents the mean enzyme activity (expressed in U / g) ± SD of at least two biological replicates. Statistical significance compared to the reference (LPO activity without the candidate enhancer molecule) was calculated using students' t-tests. Significance levels are defined as p<0.05 = *; p<0.01 = ** and p<0.001 = ***. [Figure 3] Figure 3. Summary of absolute counts of S. aureus in the presence of serine and isoleucine as potentiator candidates. Each result represents the average obtained from 3–7 biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 4] Figure 4. Summary of absolute counts of Staphylococcus aureus in the presence of serine, glutamine, and isoleucine as potential enhancers. Each result represents the average obtained from 3–7 biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 5] Figure 5. Summary of absolute numbers of P. aeruginosa in the presence of serine and proline as potential enhancers. Each result represents the average obtained from 3–7 biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 6] Figure 6. Summary of absolute numbers of P. aeruginosa in the presence of serine and proline as potential enhancers. Each result represents the average obtained from 3–7 biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 7]Figure 7. Summary of absolute numbers of P. aeruginosa in the presence of threonine as a potential enhancer. Each result represents the average obtained from two to three biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 8] Figure 8. Summary of absolute counts of P. aeruginosa in the presence of isoleucine as a potential enhancer. Each result represents the average obtained from two to three biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 9] Figure 9. Summary of absolute P. aeruginosa counts in the presence of glutamine as a potential enhancer. Each result represents the average obtained from two to three biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 10] Figure 10. Summary of absolute numbers of P. aeruginosa in the presence of asparagine as a potential enhancer. Each result represents the average obtained from two to three biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 11] Figure 11. Summary of absolute numbers of Candida albicans in the presence of serine and isoleucine as potential enhancers. Each result represents the average obtained from 3–7 biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 12] Figure 12. Summary of absolute counts of Aspergillus niger in the presence of proline as a potential enhancer. Each result represents the average obtained from two to four biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 13] Figure 13. Summary of absolute counts of Aspergillus niger in the presence of serine as a potential enhancer. Each result represents the average obtained from two to four biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 14] Figure 14. Summary of absolute counts of Aspergillus niger in the presence of threonine as a potential enhancer. Each result represents the average obtained from two to four biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 15] Figure 15. Summary of absolute counts of Aspergillus niger in the presence of glutamine as a potential enhancer. Each result represents the average obtained from two to four biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 16] Figure 16. Summary of absolute counts of Aspergillus niger in the presence of asparagine as a potential enhancer. Each result represents the average obtained from two to four biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 17] Figure 17. Summary of absolute counts of Aspergillus niger in the presence of isoleucine as a potential enhancer. Each result represents the average obtained from two to four biological replicates. X-axis: enzyme concentration (%), Y-axis: microbial concentration (log CFU / ml). [Figure 18] Figure 18. Summary of absolute numbers of S. aureus in the presence of proline as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 19] Figure 19. Summary of absolute numbers of P. aeruginosa in the presence of proline as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 20] Figure 20. Summary of absolute numbers of Candida albicans in the presence of proline as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 21] Figure 21. Summary of absolute counts of Aspergillus niger in the presence of proline as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 22] Figure 22. Summary of absolute numbers of Staphylococcus aureus in the presence of serine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 23] Figure 23. Summary of absolute numbers of P. aeruginosa in the presence of serine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 24]Figure 24. Summary of absolute numbers of Candida albicans in the presence of serine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 25] Figure 25. Summary of absolute numbers of Aspergillus niger in the presence of serine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 26] Figure 26. Summary of absolute numbers of S. aureus in the presence of threonine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 27] Figure 27. Summary of absolute numbers of P. aeruginosa in the presence of threonine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 28] Figure 28. Summary of absolute numbers of Candida albicans in the presence of threonine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 29] Figure 29. Summary of absolute numbers of Aspergillus niger in the presence of threonine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 30] Figure 30. Summary of absolute numbers of Staphylococcus aureus in the presence of glutamine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 31] Figure 31. Summary of absolute numbers of P. aeruginosa in the presence of glutamine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 32] Figure 32. Summary of absolute numbers of Candida albicans in the presence of glutamine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 33] Figure 33. Summary of absolute counts of Aspergillus niger in the presence of glutamine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 34]Figure 34. Summary of absolute counts of Staphylococcus aureus in the presence of asparagine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 35] Figure 35. Summary of absolute numbers of P. aeruginosa in the presence of asparagine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 36] Figure 36. Summary of absolute numbers of Candida albicans in the presence of asparagine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 37] Figure 37. Summary of absolute numbers of Aspergillus niger in the presence of asparagine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 38] Figure 38. Summary of absolute counts of Staphylococcus aureus in the presence of isoleucine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 39] Figure 39. Summary of absolute numbers of P. aeruginosa in the presence of isoleucine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 40] Figure 40. Summary of absolute numbers of Candida albicans in the presence of isoleucine as a potential enhancer. Each result represents the average obtained from 1 to 3 biological replicates. [Figure 41] Figure 41. Summary of absolute counts of Aspergillus niger in the presence of isoleucine as a potential enhancer. Each result represents the average obtained from one to three biological replicates. [Figure 42] Figure 42. Summary of absolute counts of Staphylococcus aureus in the presence of 0.0005% ethanolamine or proline as potential enhancers. Microbial load (expressed as log CFU / g alginogel) was plotted as a function of time (expressed in minutes). Each result represents the average obtained from three biological replicates. [Figure 43]Figure 43. Overview of absolute counts of Staphylococcus aureus in the presence of KI, KBr, and KCl at final concentrations of 0.03% and 0.003%. Microbial load (expressed in log CFU / g alginate gel) was plotted as a function of time (expressed in minutes). Each result represents one biological replicate. [Figure 44] Figure 44. Summary of IL-6 regulation. (A) Inhibition of IL-6 secretion by macrophages after 24 hours of no treatment, treatment with GOX + LPO (1:1) alone, or treatment with GOX + LPO (1:1) and 0.0025, 0.05, and 0.5% isoleucine. Results are expressed as absolute IL-6 concentration (pg / ml) and relative IL-6 secretion (%) compared to the no-treatment group. (B) Neutralization of secreted IL-6 after 3 hours of no treatment, treatment with GOX + LPO (1:1) alone, or treatment with GOX + LPO (1:1) and 0.05% isoleucine. Results are expressed as absolute IL-6 concentration (pg / ml) and relative IL-6 neutralization (%) compared to the no-treatment group. [Figure 45] Figure 45. Summary of TNFα regulation. Neutralization of secreted TNFα 3 hours after untreated, treatment with GOX + LPO (1:1) alone, or treatment with GOX + LPO (1:1) and 0.05% isoleucine. Results are expressed as absolute TNFα concentration (pg / ml) and relative TNFα neutralization (%) compared to the untreated group. [Figure 46] Figure 46. Summary of MMP9 regulation. (A) Inhibition of MMP9 secretion by macrophages 24 hours after treatment with no treatment, treatment with GOX + LPO (1:1) alone, or treatment with GOX + LPO (1:1) and 0.0025 and 0.05% isoleucine. Results are expressed as absolute MMP9 concentration (pg / ml) and relative MMP9 secretion (%) compared to the no-treatment group and represent the average of two biological replicates. (B) Neutralization of secreted MMP9 3 hours after treatment with no treatment, treatment with GOX + LPO (1:1) alone, or treatment with GOX + LPO (1:1) and 0.05% isoleucine. Results are expressed as absolute MMP9 concentration (pg / ml) and relative MMP9 neutralization (%) compared to the no-treatment group. [Figure 47]Figure 47. Summary of absolute Staphylococcus aureus counts after treatment of arginine gel with the enzyme combination GOX+EPO in the presence and absence of 0.0025% isoleucine as a potential enhancer. Arginogel without the enzyme combination GOX+EPO and without isoleucine served as negative controls. Microbial load (expressed as log(CFU / g arginine gel)) was plotted as a function of time (expressed in minutes). Each result is from one biological replicate. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0042] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms also encompass "consisting of" and "consisting essentially of," which enjoy the meanings established in patent language.

[0043] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges whether they are introduced by the phrase "from... to...", the phrase "between... and...", or other expressions.

[0044] As used herein, the term "about" or "approximately" when referring to a measurable value such as a parameter, amount, time duration, etc., is meant to encompass, to the extent appropriate for practice in the disclosed invention, a variation of the stated value and a variation from the stated value of no more than ±10%, preferably no more than ±5%, more preferably no more than ±1%, and even more preferably no more than ±0.1%. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself specifically and preferably disclosed.

[0045] The terms "one or more" or "at least one," such as one or more members or at least one member of a group of members, are themselves explicit, and by way of further illustration, the terms specifically encompass reference to any one of said members, or any two or more of said members, for example, >3, >4, >5, >6, or >7, etc., of any of said members, up to and including all said members. In another example, "one or more" or "at least one" can refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0046] The background to the invention discussion herein is included to explain the context of the invention and is not to be taken as an admission that any of the material referred to was published, known, or part of the general knowledge in any country as of the priority date of any claim.

[0047] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifiable citation. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of such documents specifically mentioned herein are incorporated by reference.

[0048] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which this invention belongs. As further guidance, term definitions are included to better understand the teachings of the present invention. When a particular term is defined in connection with a particular aspect of the present invention or a particular embodiment of the present invention, such connotation or meaning is meant to apply throughout this specification, i.e., in the context of other aspects or embodiments of the present invention, unless otherwise defined. For example, an embodiment directed to a product is also applicable to the corresponding features of the method and use.

[0049] In the following text, different aspects or embodiments of the present invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect or embodiment, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.

[0050] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, although some embodiments described herein include some features but not other features included in other embodiments, combinations of features from different embodiments are meant to form different embodiments within the scope of the present invention, as would be understood by one of ordinary skill in the art. For example, the appended claims encompass alternative combinations of the claimed embodiments, as would be understood by one of ordinary skill in the art.

[0051] Unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can be and have been carried out in a manner known per se, as would be apparent to one skilled in the art, for example by reference again to the standard handbooks, the general background referred to herein and the further references cited therein.

[0052] In the applicant's earlier application (WO 2006 / 133523), an enhancer that enhances and / or stabilizes the activity of antibacterial peroxidase is described. nA class of benzene compounds (including guaiacol) substituted with OH groups (n = 1, 2, 3, or 4) and one alkoxy group (-OR) with a chain length of 1, 2, 3, or 4 carbon atoms is taught to have an enhancing effect on peroxidase-based antibacterial compositions. Formulations of these compositions contain antibacterial enzyme systems based on an oxidase enzyme (i.e., glucose oxidase or GOX) and a peroxidase enzyme (i.e., lactoperoxidase or "LPO"), which, in the presence of air, water, and iodide ions, generate reactive oxygen species (interchangeably abbreviated "ROS"), i.e., hypoiodite, which kills pathogenic microorganisms in a nonspecific manner. The enhancing molecule guaiacol is described as stabilizing the ROS formed by LPO, prolonging their antibacterial reactivity. Similarly, other potentiators, such as ethanolamine, have been described in the art for use in antimicrobial compositions that rely on oxidase and peroxidase enzymes (e.g., US 2015 / 196025 A1).

[0053] The present disclosure provides alternative and improved enhancers that provide the beneficial effect of further improving the efficacy of peroxidase-generating systems, and ultimately peroxidase activity, in the compositions and uses described herein. The enhancers described herein generally outperform the enhancers described in WO 2006 / 133523 (guaiacol) and US 2015 / 196025 A1 (ethanolamine).

[0054] Thus, in a first aspect, the present invention relates to a composition comprising a peroxide-generating system, a peroxidase, a halide or pseudohalide, and an enhancer, wherein the enhancer is an organic compound characterized by (a) amino and carboxylate functional groups and by further comprising one or more side chains containing a functional group selected from the group consisting of polar uncharged methanol, ethane-1-ol, acetamide, propenamide, or secondary butyl groups; or (b) pyrrolidine and carboxylate functional groups.

[0055] In certain embodiments, the connection between the amino and / or carboxylate functional groups and the different side chains or pyrrolidines (in a and b) is (CH) n (wherein n=1, 2, or 3).

[0056] In certain embodiments, the compound is hydrophilic, ie, the partition coefficient of the compound is between −1.72 and −3.82.

[0057] In certain embodiments, the enhancer has a free amino functional group (-NH + ), a free carboxylic acid functional group (—COH), and (i) -OH group; (ii)-(CH2) n an OH group (where n is 1, 2, or 3); (iii)-(CH2) n CONH2 groups (where n is 1, 2, or 3); (iv) -cyclic C4H9N group; or (v) Branched alkyl group and a side chain comprising:

[0058] The term "peroxidase," as used herein and interchangeably referred to throughout the art by the term "peroxide reductase," refers to an enzyme in Enzyme Commission (EC) Class 1.11.1.x. The term is understood by those skilled in the art to refer to a group of enzymes that catalyze the oxidation of substrates by hydrogen peroxide or organic peroxides. Examples of suitable peroxidase enzymes in the context of the present invention are peroxidases capable of oxidizing one or more of chloride, iodide, bromide, or thiocyanate ions to the antimicrobial hypochlorite, hypoiodite, hypobromite, or hypothiocyanite ions, respectively. By way of example and not limitation, suitable peroxidase enzymes include lactoperoxidase, guaiacol peroxidase, plant peroxidase, Japanese radish peroxidase, horseradish peroxidase (HRP), soybean peroxidase (SBP), extensin peroxidase, heme peroxidase, oxyperoxidase, protoheme peroxidase, pyrocatechol peroxidase, scopoletin peroxidase, Coprinus cinereus peroxidase, and the like. peroxidase, and Arthromyces ramosus peroxidase.

[0059] In connection with the above, the term "peroxidase activity" is defined herein as the enzyme activity of converting peroxides, such as hydrogen peroxide, into less oxidizing species, such as water. As used herein, molecules with peroxidase activity are understood to include peroxide decomposition enzymes. The term "peroxide" is well known in the art and generally refers to a group of compounds containing the structure ROOR (wherein R can be any element). A characteristic example of a peroxide is hydrogen peroxide (HO), which is generally referred to as the term "peroxide."

[0060] Optionally, the peroxidase is a heme peroxidase. Alternatively, and also optionally, the peroxidase is a non-heme peroxidase. In further optional embodiments, the compositions described herein comprise at least one heme peroxidase and at least one non-heme peroxidase.

[0061] A particularly preferred peroxidase in the context of the present invention is lactoperoxidase. Lactoperoxidase (abbreviated as LPO) is a glycoprotein that is classified as an oxidoreductase naturally occurring in milk. Lactoperoxidase catalyzes the production of hypothiocyanate and water from hydrogen peroxide (H2O2) and thiocyanate. The origin of lactoperoxidase is not particularly limited, and therefore can be derived from any suitable source, such as plants, animals, microorganisms, or any products derived therefrom, such as human milk, cow's milk, mare's milk, sheep's milk, and goat's milk. In such an embodiment, lactoperoxidase can be derived from skim milk using any suitable method known to those skilled in the art, for example, ion chromatography (for example, as described by Borzouee et al., Adv Biomed Res, 2016). Alternatively, lactoperoxidase can be recombinantly produced (for example, as described by Watanabe et al., FEBS Lett, 1998). Particularly preferred lactoperoxidases in the context of the present invention are human (UniProt ID P22079) and bovine lactoperoxidase (UniProt ID P80025), which share the same amino acid sequence. Those skilled in the art will understand that when referring to human and / or bovine lactoperoxidase, all human and / or bovine isoforms are equally contemplated. By way of example and not limitation, a suitable bovine lactoperoxidase is Bos taurus lactoperoxidase. Optionally, the lactoperoxidase is a monomeric glycoprotein with a molecular weight of 77,500 Da, an IEP of 9.6, and an optimum pH of pH 6. Alternatively, the lactoperoxidase may be a modified glycoprotein with a molecular weight less than 77.5 Da, an IEP other than 9.6, and / or an optimum pH other than pH 6.

[0062] Lactoperoxidases having substantial sequence identity to SEQ ID NO: 1 are particularly contemplated in this context. However, because lactoperoxidase is initially translated as a protein containing additional propeptides and signal peptides, lactoperoxidases having substantial sequence identity to SEQ ID NO: 2 and / or SEQ ID NO: 3 are also contemplated. Thus, in a preferred embodiment, the lactoperoxidase comprises an amino acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97.5%, or even 100% sequence identity to SEQ ID NO: 1.

[0063] The amino acid sequence of Bos taurus lactoperoxidase is reproduced here: (SEQ ID NO. 1).

[0064] In an alternative preferred embodiment, the lactoperoxidase comprises an amino acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97.5%, or even 100% sequence identity to SEQ ID NO:2.

[0065] The amino acid sequence of Bos taurus lactoperoxidase (+ propeptide) is reproduced here: (SEQ ID NO: 2)

[0066] In yet another preferred embodiment, the lactoperoxidase comprises an amino acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97.5%, or even 100% sequence identity to SEQ ID NO:3.

[0067] The amino acid sequence of Bos taurus lactoperoxidase (+ propeptide + signal peptide) is reproduced here: (SEQ ID NO. 3).

[0068] In yet another preferred embodiment, the lactoperoxidase comprises three amino acid sequences each independently having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97.5%, or even 100% sequence identity to SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:5.

[0069] The signal peptide of isolated Bos taurus lactoperoxidase is reproduced here: MWVCLQLPVFLASVTLFEVAAS(SEQ ID NO:4).

[0070] The propeptide of isolated Bos taurus lactoperoxidase is reproduced here: DTIAQAASTTTISDAVSKVKIQVNKAFLDSRTRLKTTLSSEAPTTQQLSEYFKHAKGRTRTAIRNGQVWEESLKRLRR(SEQ ID NO:5).

[0071] Those skilled in the art will appreciate that, depending on the details of the synthesis and / or purification method, a methionine residue will be present at the N-terminus of the proposed peroxidase.

[0072] As used herein, a "peroxide-generating system" refers to any molecule or substance capable of generating peroxide. A peroxide-generating system may be a single molecule or a group of distinct molecules. Typically, a peroxide-generating system includes a peroxide-generating enzyme and a substrate. In certain embodiments, the peroxide-generating enzyme is an oxidoreductase enzyme. Suitable oxidoreductases include, but are not limited to, glucose oxidase, galactose oxidase, glycolate oxidase, lactate oxidase, L-gluconolactone oxidase, L-2-hydroxyacid oxidase, aldehyde oxidase, xanthine oxidase, D-aspartate oxidase, L-amino acid oxidase, D-amino acid oxidase, monoamine oxidase, pyridoxamine phosphate oxidase, diamine oxidase, and sulfite oxidase.

[0073] Substrates suitable for use in the peroxide-generating system of the present invention include the natural substrates of the enzymes and other substrates that allow for the production of peroxide. Beta-D-glucose is a specific substrate for glucose oxidase. Other suitable substrates include, but are not limited to, D-glucose, O-galactose, L-sorbose, ethanol, tyramine, 1,4-diaminobutane, 2-aminophenol, glycolate, L-lactate, 2-deoxy-D-glucose, L-gluconolactone, L-galaconolactone, D-mannonolactone, L-2-hydroxyisocaproate, acetaldehyde, butyraldehyde, xanthine, D-aspartate, D-glutamate, L-amino acids, and D-amino acids.

[0074] Alternatively, the peroxide generating system may be non-enzymatic, with hydrogen peroxide being generated instead by a molecule that spontaneously decomposes to produce peroxide, such as a perborate or percarbonate, more particularly sodium percarbonate or sodium perborate.

[0075] A preferred peroxidase-producing system in the context of the present invention is glucose oxidase (GOX), optionally in combination with glucose as a substrate. Glucose oxidase is an enzyme that oxidizes β-D-glucose to produce D-glucono-δ-lactone and hydrogen peroxide. The glucose oxidase used in the context of the present invention is not particularly limited. Optionally, glucose oxidase is obtained from microorganisms, such as, but not limited to, Aspergillus niger and Penicillium chrysogenum. Microbial glucose oxidase can be obtained by any suitable method described in the field for producing microbial enzymes. Furthermore, commercially available glucose oxidase or glucose oxidase derived from a commercially available microorganism may be used.

[0076] As used herein, "halide" refers to an ion of a halogen. Halides are binary compounds, one of which is a halogen atom and the other of which is an element or radical less electronegative (or more electropositive) than the halogen, forming compounds such as fluoride, chloride, bromide, iodide, astatide, or, theoretically, tennesside. Alkali metals combine directly with halogens under appropriate conditions to form halides of the general formula MX (X = F, Cl, Br, or I). By way of illustration and not limitation, suitable halides include ionic iodides, optionally in the form of water-soluble iodide salts such as alkali metal iodide salts, e.g., potassium iodide (Kl), sodium iodide (NaI), lithium iodide, ammonium iodide, or calcium iodide. Typical examples are sodium iodide and potassium iodide. In particular, potassium iodide has unexpectedly been found to further improve the antimicrobial properties of the compositions described throughout this specification when compared to other halides such as potassium chloride and / or potassium bromide.

[0077] The term "pseudohalide" refers to a polyatomic anion that resembles a halide in acid-base and redox chemistry. These include cyanide, thiocyanate, thiosulfate, and azide ions. Suitable sources of thiocyanate ions (SCN-) include sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, and other thiocyanate salts. Typical examples are sodium thiocyanate and potassium thiocyanate. According to certain embodiments, the composition of the present invention comprises lactoperoxidase and both thiocyanate and a halide as electron donors.

[0078] The term "potentiator" is broadly construed to refer to any functionality that enhances peroxidase activity and / or peroxidase-based antimicrobial activity. Potentiators contemplated in the context of the present invention are non-toxic compounds and / or compounds recognized as GRAS (Generally Accepted As Safe) compounds.

[0079] In the context of the present invention, the enhancer is preferably a hydrophilic compound (i.e., water-soluble), such as a hydrophilic amino acid, a hydrophilic amino acid mimic, or a hydrophilic amino acid derivative.Those skilled in the art will understand that the term "hydrophilic compound" or "hydrophilic molecule" refers to a molecule whose interaction with water and other polar substances is thermodynamically more favorable than its interaction with oil or other hydrophobic solvents.Hydrophilic molecules are typically charged and capable of hydrogen bonding.

[0080] The term "amino acid" refers to natural amino acids, naturally encoded amino acids, non-naturally encoded amino acids, unnatural amino acids, amino acid analogs, and amino acid mimics that function in a manner similar to natural amino acids, all D- and L-stereoisomers, where their structure allows for such stereoisomeric forms. Amino acids are referred to herein by their full name, their three-letter abbreviation, or their one-letter abbreviation. Amino acids are referred to herein by either their name, their commonly known three-letter symbol, or the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. A "naturally encoded amino acid" refers to an amino acid that is one of the 20 common amino acids, or pyrrolysine, pyrroline-carboxy-lysine, or selenocysteine. The 20 common amino acids are: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr). The term also contemplates amino acid analogs in which at least one individual atom is replaced with either a different atom, an isotope of the same atom, or a different functional group.

[0081] As used herein, "amino acid mimic" refers to any molecule that has the same functional properties as the "mimicking" amino acid, where the general chemical formula of the amino acid mimic may deviate from the typical R-CH(NH)-COOH formula and is intended to be illustrative and not limiting, as R-CH(NH)(CH). nThe amino acid mimic may have the formula -COOH (where n is any integer). Those skilled in the art will understand that the term "amino acid mimic" does not include amino alcohols. In particular, those skilled in the art will understand that the term "amino acid mimic" does not include 1,2 amino alcohols, such as ethanolamine, which contain both a primary alcohol and a primary amine.

[0082] As used herein, "amino acid derivative" refers to a molecule that contains or substantially contains the same chemical formula as an amino acid, in which one or more functional groups have been modified, added, or deleted. Thus, the term encompasses any derivative of an amino acid resulting from reaction at the amino group, carboxy group, side chain functional group, or from replacement of any hydrogen with a heteroatom.

[0083] In some embodiments, the enhancer has a free amino functional group (-NH + ), a free carboxylic acid functional group (—COH), and a side chain containing an OH group. In an alternative embodiment, the enhancer is a hydrophilic amino acid, amino acid mimic, or amino acid derivative characterized by a free amino functional group (—NH + ), a free carboxylic acid functional group (-CO2H) and -(CH2) n In yet another alternative embodiment, the enhancer is a hydrophilic amino acid, amino acid mimic, or amino acid derivative characterized by a side chain containing a free amino functional group (-NH + ), a free carboxylic acid functional group (-CO2H) and -(CH2) n In yet another alternative embodiment, the enhancer is a hydrophilic amino acid, amino acid mimic, or amino acid derivative characterized by a side chain containing a free amino functional group (-NH + ), a free carboxylic acid functional group (—COH), and a side chain containing a cyclic C4H9N group. In yet another alternative embodiment, the enhancer is a hydrophilic amino acid, amino acid mimic, or amino acid derivative characterized by a free amino functional group (—NH +), a free carboxylic acid functional group (—COH), and a side chain containing a branched alkyl group.

[0084] In certain embodiments, the enhancer is characterized by the presence of amino and carboxylate functional groups and by further comprising a functional group selected from the group consisting of a polar uncharged methanol, ethane-1-ol, acetamide, propenamide, or secondary butyl group; and the amino and carboxylate functional groups and (CH) n where n=1, 2, or 3. In certain embodiments, the enhancer is an organic compound characterized by the presence of a pyrrolidine and a carboxylate functional group; and by a connection between the amino and carboxylate functional groups and a side chain consisting of (CH2)n, where n=1, 2, or 3.

[0085] The term "pyrrolidine," referred to interchangeably throughout the art as "tetrahydropyrrole," is known to those skilled in the art and refers to an organic compound characterized by the molecular formula (CH)NH. Pyrrolidine is a cyclic secondary amine that is alternatively classified as a saturated heterocycle.

[0086] In certain embodiments, the enhancer is a hydrophilic amino acid, amino acid mimic, or amino acid derivative having an octanol / water partition coefficient (LogP) of about -1 to about -5. In further embodiments, the enhancer is a natural amino acid having an octanol / water partition coefficient (LogP) of about -1.25 to about -4.5. In further embodiments, the enhancer is a natural amino acid having an octanol / water partition coefficient (LogP) of about -1.5 to about -4. In further embodiments, the enhancer is a natural amino acid having an octanol / water partition coefficient (LogP) of about -1.72 to about -3.82.

[0087] (n-) Octanol / water partition coefficient (alternatively referred to in the art as "partition coefficient" and "K ow") is the partition coefficient for a two-phase system consisting of n-octanol and water. K ow is often referred to by the symbol P and is commonly expressed as a base 10 logarithm (i.e., LogP). It is also called the n-octanol-water partition ratio. The partition coefficient of a molecule indicates the lipophilicity (fat solubility) and hydrophilicity (water solubility) of the molecule. A higher partition coefficient value indicates a more hydrophobic nature of a given molecule, while a lower partition coefficient value indicates a more hydrophilic nature of a given molecule. Exemplary partition coefficient values ​​for a non-exhaustive list of possible amino acids suitable to act as enhancers in the context of the present invention are provided below:

[0088] JPEG2025539810000001.jpg4865

[0089] In some embodiments, the enhancer is a hydrophilic natural or unnatural amino acid. In further embodiments, the enhancer is a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof.

[0090] In some embodiments, the enhancer is present in the composition at a concentration of about 0.0005 mg / ml to about 200 mg / ml, preferably about 0.005 mg / ml to about 10 mg / ml, and most preferably about 0.05 to about 1 mg / ml. In some embodiments, the enhancer is present in the composition at a concentration of at least about 0.0001 mg / ml, preferably at least about 0.005 mg / ml, and most preferably at least about 0.05 mg / ml.

[0091] In a preferred embodiment, the enhancer is a naturally occurring amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof, and the enhancer is present in the composition at a concentration of about 0.0005 mg / ml to about 200 mg / ml, preferably about 0.005 mg / ml to about 10 mg / ml, and most preferably about 0.05 to about 1 mg / ml.

[0092] Optionally, the composition comprises an enhancer that is a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof, and a water-soluble iodide salt. In a preferred embodiment, the composition comprises an enhancer that is a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof, lactoperoxidase, a water-soluble iodide salt, and glucose oxidase.

[0093] Optionally, the composition comprises an enhancer which is a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof; a peroxidase such as lactoperoxidase; a halide or pseudohalide such as a water-soluble iodide salt (e.g., potassium iodide, KI); a peroxide generating system such as glucose oxidase; and a further enhancer which is a benzene molecule substituted with -OH or (CH2)nOH groups (n=1, 2, 3, or 4) and one alkoxy group (-OR) having a chain length of 1, 2, 3, or 4 carbon atoms.

[0094] Optionally, the composition comprises an enhancer that is a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof, and a further enhancer that is lactoperoxidase, a water-soluble iodide salt, glucose oxidase, guaiacol, vanillin, or a combination thereof.

[0095] "Guaiacol" is described in the art and refers to an organic compound characterized by the chemical formula CH(OH)(OCH). The source of guaiacol optionally used in the context of the present invention is not particularly limited and can be obtained, for example, from laurel tree, wood creosote, or the essential oils of celery seeds, tobacco leaves, orange leaves, and lemon peel. Suitable means for chemically synthesizing guaiacol include, but are not limited to, hydrolysis of o-anisidine with diazonium derivatives or by demethylation of catechol followed by selective monodemethylation. As described herein, the phenolic aldehyde "vanillin" may be derived from guaiacol or lignin and is characterized by the chemical formula CHO. The source of vanillin in the context of the present disclosure is not particularly limited and therefore vanillin can equally be obtained by biosynthesis by vanilla (V. planifolia), chemical synthesis from eugenol, or genetically modified microorganisms.

[0096] The terms "formulation" and "composition" may be used interchangeably herein. In any of the embodiments relating to one or more compositions described herein, it is clear that the composition may also include one or more pharmaceutically or cosmetically acceptable carriers (i.e., excipients). As used herein, the terms "pharmaceutically acceptable" or "cosmetically acceptable" mean, consistent with the art, compatible with other ingredients of a pharmaceutical or cosmetic composition and not deleterious to the recipient thereof.

[0097] In any of the embodiments described herein, the composition may include one or more excipients. The term "excipient", which is used interchangeably with "carrier" herein and in the art, includes any solvent, diluent, buffer (including but not limited to neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate, or phosphate buffer), solubilizer (including but not limited to Tween 80 or Polysorbate 80), colloid, dispersion medium, vehicle, filler, chelating agent (including but not limited to EDTA or glutathione), amino acid, protein, disintegrant, binder, lubricant, wetting agent, stabilizer, emulsifier, sweetener, colorant, flavor, fragrance, thickener, any agent suitable for achieving a depot effect, coating, antifungal agent, any preservative (including but not limited to benzalkonium chloride or benzyl alcohol), antioxidant (including but not limited to ascorbic acid, sodium metabisulfite), tonicity controlling agent, etc. The term "excipient" may refer to an excipient, such as an absorption retardant, an adjuvant, a bulking agent (including, but not limited to, lactose, mannitol), and any other ingredient that may affect any parameter or property of the composition or of the polypeptide contained in the composition described herein. Those skilled in the art will understand that one or more excipients can be used in the composition, provided that the one or more excipients are compatible with one or more pharmaceutical ingredients (i.e., in the context of the present invention, at least the polypeptide comprising the variant amino acid sequence) and result in a pharmaceutically acceptable formulation.

[0098] In certain embodiments, the excipient is an active pharmaceutical ingredient excipient, a binding excipient, a carrier excipient, a co-processing excipient, a coating system excipient, a controlled release excipient, a diluent excipient, a disintegrating excipient, a dry powder inhalation excipient, an effervescent system excipient, an emulsifying excipient, a lipid excipient, a lubricating excipient, a modified release excipient, a penetration enhancer excipient, a permeation enhancer excipient, a pH adjusting excipient, a plasticizing excipient, a preservative excipient, a solubilizing excipient, a solvent excipient, a sustained release excipient, a sweetening excipient, a taste making excipient, a stimulant ... The excipient may be a thickening excipient, a viscosity-adjusting excipient, a filler excipient, a compression excipient, a dry granulation excipient, a hot-melt extrusion excipient, a wet granulation excipient, a rapid-release excipient, an increased bioavailability excipient, a dispersing excipient, a solubility-enhancing excipient, a stabilizing excipient, a capsule-filling excipient, or any combination thereof. Those skilled in the art will recognize that the use of such vehicles and agents for pharmaceutically active substances is common practice, and therefore the incorporation of these excipients is well known in the art. It is clear that all of the components used should be non-toxic at the concentrations contained in the final composition and should not negatively interfere with the activity of one or more pharmaceutically active ingredients (in this context, at least the peroxide-generating system and peroxidase). In some embodiments, multiple excipients that those skilled in the art would classify as belonging to the same group of excipients are added to the composition. In further embodiments, multiple excipients are added to the composition, with different excipients belonging to different groups. In some embodiments, an excipient may perform multiple functions and / or may be classified by one skilled in the art as belonging to different groups or classes of excipients.

[0099] The composition may further contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, preservatives, complexing agents, tonicity adjusting agents, wetting agents, etc., non-limiting examples of which include sodium acetate, sodium lactate, sodium phosphate, sodium hydroxide, hydrogen chloride, benzyl alcohol, parabens, EDTA, sodium oleate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. In some embodiments, at least one additional component is combined with the composition before administration. In further embodiments, the additional component is combined with the composition immediately before administration. In some embodiments, the amount of additional component added to the composition is calculated based on certain patient parameters, including, but not limited to, the patient's age, weight, sex, severity of the disease state, and other known or suspected disease states.

[0100] According to one embodiment of the present invention, the composition of the present invention comprises both peroxidase enzyme and electron donor for peroxidase.Usually, when lactoperoxidase is used, iodide ion and / or thiocyanate ion are added as donor for lactoperoxidase enzyme.According to another embodiment, the enhancer of the present invention partially or completely replaces donor molecule.This has the additional advantage that toxic compounds such as thiocyanate can be completely or partially replaced with less toxic or non-toxic compounds.

[0101] Thus, in certain embodiments, the antibacterial and anti-inflammatory active compounds in the compositions of the present invention consist of a peroxide or peroxide-generating system, lactoperoxidase, a halide, and an enhancer selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof. In further specific embodiments, the antibacterial and anti-inflammatory active compounds in the compositions of the present invention consist of a peroxide or peroxide-generating system, lactoperoxidase, iodide anion, and an enhancer selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof.

[0102] The enhancers described herein increase the activity of the peroxide generating system and / or peroxidase by at least about 5%, preferably at least about 10%, more preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 100%.

[0103] Optionally, the enhancer increases the activity of the peroxide generating system and / or peroxidase by at least 1.2-fold, preferably at least 1.5-fold, preferably at least 1.75-fold, preferably at least 2-fold, more preferably at least 2.5-fold, and most preferably at least 5-fold.

[0104] Those skilled in the art will understand that the relative increase in activity of a peroxide-generating system and / or peroxidase described herein should be interpreted relative to the reference activity in an appropriate control condition, i.e., a composition that does not contain an enhancer described herein.

[0105] A suitable control composition is therefore a composition containing a peroxide-generating system, peroxidase, halide, or pseudohalide that is identical in activity to the composition under investigation. An alternative suitable control composition is therefore a composition containing a peroxide-generating system, peroxidase, halide, or pseudohalide that is identical in activity to the composition under investigation, to which an appropriate amount of an enhancer that is the subject of WO 2006 / 133523 is added. Suitable control enhancers for use in the control composition include benzene molecules substituted with an -OH group or a (CH)OH (n = 1, 2, 3, or 4) group and one alkoxy group (-OR) with a chain length of 1, 2, 3, or 4 carbon atoms. Optionally, the control enhancer is guaiacol or vanillin.

[0106] The compositions of the present invention are characterized by strong antimicrobial activity, which is further enhanced by the enhancers described herein. "Antimicrobial" means killing or slowing the growth of microbes. Microbes include bacteria such as gram-negative bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa), gram-positive bacteria (e.g., Staphylococcus aureus, Pseudomonas aeruginosa), and spore-forming bacteria. Microorganisms include fungi such as molds (e.g., Aspergillus niger, Penicillium funiculosum), yeasts (e.g., Candida albicans, Saccharomyces cerevisiae, Pityrosporum ovale), and dermatophytes (e.g., Trichophyton rubrum). Thus, the term antimicrobial includes the terms "bactericidal," "bacteriostatic," "fungicidal," and "fungistatic." Microorganisms also include microalgae such as Chlorella spp. and Spyrogyra spp., and viruses such as herpesvirus, picornavirus, chickenpox, and warts.

[0107] Optionally, the composition is characterized by a minimum inhibitory concentration (MIC) and / or minimum bactericidal concentration (MBC) and / or absolute count and / or enzyme activity against an inoculum of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, or any combination thereof, that is significantly reduced when compared to a control composition described herein. The phrase "minimum inhibitory concentration" is known to those skilled in the art and is to be interpreted in accordance with generally accepted interpretations in the field. Thus, as used herein, minimum inhibitory concentration refers to the lowest concentration of an antimicrobial composition for inhibiting the growth of a particular bacterium or distinct group of bacteria. In some embodiments, the minimum inhibitory concentration and / or minimum bactericidal concentration and / or absolute count and / or enzyme activity against an inoculum of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, or any combination thereof, is reduced by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 25%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 60%, more preferably at least about 70%, and most preferably at least about 75% when compared to a control composition.

[0108] Optionally, the composition is characterized by a minimum bactericidal concentration (MBC) against an inoculum of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, or any combination thereof, that is significantly reduced when compared to a control composition described herein. The phrase "minimum bactericidal concentration" is known to those of skill in the art and should be interpreted according to generally accepted interpretations in the field. Thus, as used herein, minimum bactericidal concentration refers to the lowest concentration of an antimicrobial composition that will kill a particular bacterium or distinct group of bacteria. In some embodiments, the minimum bactericidal concentration against an inoculum of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, or any combination thereof, is reduced by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 25%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 60%, more preferably at least about 70%, and most preferably at least about 75% when compared to a control composition described herein.

[0109] Optionally, both the minimum inhibitory concentration and the minimum bactericidal concentration of the compositions described herein are reduced by about 10% to about 75%, preferably about 20% to about 65%, and more preferably about 30% to about 50%, when compared to a control composition. Methods for measuring the minimum inhibitory concentration and / or minimum bactericidal concentration of a composition are well-described in the art and are therefore known to those skilled in the art. Preferably, the minimum inhibitory concentration and / or minimum bactericidal concentration are measured by incubating the composition in a microbial inoculum characterized by an OD of 0.1 for 24 hours at 37°C for Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, and for 48 hours at 20-25°C for Aspergillus niger. Optionally, the minimum inhibitory concentration and / or minimum bactericidal concentration are reduced to the above-mentioned extents when compared to a control composition containing a peroxide-generating system, peroxidase, halide, or pseudohalide whose activity is identical to that of the composition under investigation. Therefore, an alternative suitable control composition would be one containing a peroxide generating system, peroxidase, halide or pseudohalide that is identical in activity to the composition under investigation, optionally with the addition of an appropriate amount of an enhancer that is the subject of WO 2006 / 133523. Thus, suitable control enhancers for use in the control composition include -OH, or (CH) n It includes a benzene molecule substituted with an OH (n=1, 2, 3, or 4) group and one alkoxy group (—OR) having a chain length of 1, 2, 3, or 4 carbon atoms. Optionally, the contrast enhancer is guaiacol or vanillin.

[0110] Preferably, the compositions described herein are preferably topical compositions. It is understood that topical compositions are formulated to be particularly useful for topical administration (i.e., application) of the compositions described herein to discrete tissue surfaces of a subject. In this context, "cutaneous application" refers to administration to the skin of a subject. Topical administration can also include the use of transdermal administration means, such as, but not limited to, transdermal patches, wound dressings, or any impregnated material, as further discussed below. Optionally, the composition is a topical therapeutic composition for cutaneous application. Alternatively, the composition is a topical prophylactic composition for cutaneous application. Still alternatively, the composition is a topical cosmetic composition for cutaneous application.

[0111] The terms "subject," "individual," or "patient" can be used interchangeably herein and typically refer to humans, preferably humans, but can also include reference to non-human animals, preferably warm-blooded animals, more preferably mammals, such as non-human primates, rodents, canines, felines, equines, ovine, porcines, and the like. The term "non-human animals" includes all vertebrates, e.g., mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows, and non-mammals such as chickens, amphibians, and reptiles. In some embodiments, the subject is a non-human mammal. Preferred subjects are human subjects, including all genders and all age categories. Both adult subjects, newborn subjects, and fetuses are intended to be covered by the term "subject." Thus, both adult and neonatal subjects are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice. Preferred subjects in the context of the present invention are human subjects.

[0112] Optionally, the composition is a cream, lotion, foundation, ointment, suspension (oil-in-water and water-in-oil), (hydro)gel, foam, emulsion, paste, paint, spray, oxymel, liniment, insufflation, patch, or dressing.

[0113] The term "emulsion" refers to any mixture of at least two immiscible (i.e., immiscible, unmixable) liquids, where the first liquid is distributed in droplets (dispersed phase) throughout the second liquid (dispersion medium). The composition may therefore be an oil-in-water or water-in-oil emulsion. Emulsions are widely used in skin care formulations. In this context, "suspension" broadly refers to a heterogeneous mixture containing solids dispersed in a liquid phase, undissolved, and large enough to allow settling.

[0114] "Cream" generally refers to a water-in-oil emulsion in which an aqueous phase is dispersed in an oil phase, but may equally well be an oil-in-water emulsion in which an oil is dispersed in an aqueous base. It is generally accepted that creams differ from emulsions in that an emulsion is a stable suspension of small, immiscible droplets of a fluid that is immiscible with the other fluid portions of the emulsion, whereas creams represent a specific subset of emulsions that are more viscous and usually contain more lipophilic and / or surfactant components.

[0115] A "lotion" is a liquid composition with low to medium viscosity. Generally, lotions are less viscous than creams, although the viscosities of the two may be comparable. Lotions optionally contain finely divided substances that are insoluble in the dispersion medium, using suspending and dispersing agents. Alternatively, lotions may have a dispersed phase that is immiscible with the vehicle and is usually dispersed by an emulsifier or other suitable stabilizer. In one embodiment, the lotion is in the form of an emulsion with a viscosity between 100 and 1000 centistokes. The fluidity of lotions allows them to be applied quickly and evenly to large surface areas. Lotions are typically intended to dry on the skin, leaving a thin film of their ingredients on the subject's skin surface.

[0116] "Ointment" typically refers to a more viscous oil-in-water cream, i.e., a semi-solid substance containing an ointment base and optionally one or more pharmaceutically active ingredients (in the context of the present invention, peroxidase and a peroxide-generating system). Examples of suitable ointment bases include hydrocarbon bases, absorption bases, water-removable bases, and water-soluble bases.

[0117] "Pastes" generally differ from ointments in that they contain a greater proportion of solids. Overall, pastes are more absorbent and less greasy than ointments of substantially similar composition. The base may be anhydrous (e.g., liquid or soft paraffin) or water-soluble (e.g., glycerol or mucilage).

[0118] A "(hydro)gel" is an aqueous colloidal suspension of colloids in which particles are in the external or dispersed phase and water is in the internal or dispersed phase. Different ingredients suitable for producing pharmaceutically acceptable hydrogels are known to those skilled in the art. Hydrogels are generally used to retain or absorb moisture or water. Suitable hydrogels in the context of the present invention are prepared using hydrocolloids such as alginates and polyacrylates (e.g., carbopol) and cellulose and its derivatives, such as carboxymethylcellulose (CMC). Other suitable hydrocolloids are aluminum hydroxide, silicon dioxide or silicic acid, starch, glycogen, gelatin, pectin, chitosan, chitin, gum arabic, locust bean gum, karaya gum, tragacanth gum, ghatti gum, agar, carrageenan, carob gum, guar gum, xanthan gum, and glyceryl polymethacrylate. The hydrogel can be used as is. Hydrogels for use as contemplated by the present disclosure can be formulated with different concentrations of colloid, depending on the consistency desired.

[0119] As used herein, "foam" refers to a dispersion of gas particles in a liquid medium. Examples of liquid media in foams include oil-in-water emulsions, water-in-oil emulsions, ethanol, water, solvents, liquid oils, propylene glycol, and glycerin. (a) It will be understood by those skilled in the art that foams can be generated by reducing the surface tension of a liquid mixed into a gaseous substance, causing bubble formation. The usefulness of foams derives from the fact that they are easily applied to a wide area of ​​skin, do not leave an oily or greasy film, and are rapidly absorbed into the skin.

[0120] "Mousse" refers to a substance that is similar to foam, but is usually less aqueous. "Sprays" are formulations of drugs in aqueous, alcoholic, or glycerin-containing vehicles. They are applied to mucous membranes or (broken) skin with an atomizer or nebulizer.

[0121] "Paints" are liquids for application to the skin or mucous membranes, usually with a soft brush. Skin paints often have volatile solvents that evaporate quickly, leaving behind a dry or resinous film of the drug. Paints can be made viscous by the addition of glycerin, which provides adhesive properties and helps adhere to the affected area, prolonging the drug's action.

[0122] Oxymel is a preparation that uses a mixture of acid and honey as the vehicle. "Liniments" are liquid, semi-liquid, or sometimes semi-solid preparations intended to be applied to the skin. They can be emulsions, alcoholic or oily solutions. Many are applied to the skin by massage, but some are applied with a dressing or brush.

[0123] An "insufflation" is a medicated powder that is sprayed by an insufflator (a device similar to an atomizer or pressurized atomizer).

[0124] Optionally, the composition is a deodorant, preferably a roll-on or stick deodorant. The term "deodorant," or simply "deo," refers to a cosmetic product applied to the skin that does not prevent perspiration itself, but masks the odor of sweat or prevents the occurrence of sweat odor by applying a stronger (more pleasant) fragrance. Thus, the compositions or formulations described herein can be considered products for promoting personal hygiene of a subject. The term "antiperspirant" refers to a means of suppressing perspiration (e.g., by reducing or stopping perspiration) so that sweat odor is also reduced. However, in today's cosmetics, the term "deodorant" refers to both "deodorant" and "antiperspirant," and most deodorants are a combination of both.

[0125] Optionally, the composition is an anti-acne agent, and is preferably in the form of lotion or cream.Acne vulgaris (referred to herein and in the field as the common name "acne") is a skin disease that typically, but not exclusively, affects adolescents.This condition has been described many times throughout the field, and therefore is known to those skilled in the art.Simply put, acne is caused by the blockage of hair follicles in the skin by dead skin cells or oil produced in the skin.Acne is typically characterized by the presence of blackheads (open comedones), milia (closed comedones), pimples, oily skin appearance, and optionally skin scars. Optionally, the composition is ear drops, nose drops, inhalant, vaginal, or rectal solution.

[0126] Optionally, the composition is contained in and / or applied to an impregnated material such as a wound dressing. In a preferred embodiment, the wound dressing is a dressing configured for wound cleansing and / or wound treatment. Numerous types of dressings have been described in the art, including, but not limited to, gauze dressings, tulle dressings, alginate dressings, polyurethane dressings, film dressings, polysaccharide paste dressings, granular dressings, foam dressings, silicone dressings, synthetic polymer scaffold dressings, hydrocolloid dressings, occlusive dressings, or combinations thereof. The dressing may be adhesive or non-adhesive. As used herein, the term "occlusive dressing" refers to a dressing that prevents air and / or bacteria from contacting the wound and contains one or more of the following: moisture, heat, body fluids, and medication. Those skilled in the art will be able to select the appropriate wound healing dressing for use with a particular wound, and such selection may depend on parameters such as, but not limited to, the type of wound, the size of the wound, and the progress of healing of the wound. Optionally, the dressing is a hydrogel dressing. Hydrogel dressings are composed largely of water within a network of fibers that maintain the integrity of the polymer gel. Water is released from the dressing to maintain an appropriate moisture level in the wound. Examples of hydrogel dressings include Tegagel (登録商標) , Intrasite (登録商標) These include, but are not limited to:

[0127] The method and protocol for producing any of the above-mentioned dressings have been described in the art.The composition of the subject of the present disclosure can be incorporated into / onto said dressing when said dressing is produced, but can also be applied to pre-produced dressings.For illustration and not limitation, the dressing or a part thereof before production can be impregnated with the composition as described herein.Alternatively, the dressing or a part thereof before production can be coated with the composition as described herein.

[0128] In certain embodiments, the composition is included in a skin replacement (i.e., a skin substitute or dermal substitute). The skin substitute provides a three-dimensional biomatrix that performs the function of the dermal layer of skin, capable of temporarily or permanently covering open skin wounds. The material of the skin substitute is not particularly limited and may therefore include biological materials, synthetic materials, or combinations thereof. Non-limiting examples of biological materials include, but are not limited to, human or porcine skin and human or porcine intestinal submucosa. The biological skin substitute may include different components, including, but not limited to, collagen, glycosaminoglycans, fibronectin, hyaluronic acid, elastin, and any combination thereof.

[0129] Typically, a combination of a peroxide generating system, peroxidase, and a potentiator, optionally in combination with one or more halides and / or pseudohalides, is present in the pharmaceutical compositions of the present invention as the active antibacterial and anti-inflammatory component.

[0130] The composition may contain about 10 to 100,000 U / kg, preferably about 10 to 4,000 U / kg, more preferably about 10 to 100 U / kg of peroxidase. In a more preferred embodiment, the composition contains about 10 to about 100 U / kg of lactoperoxidase.

[0131] The composition may contain from about 0.0005 mg / ml to about 200 mg / ml, preferably from about 0.005 mg / ml to about 10 mg / ml, (more preferably from about 0.05 to about 1 mg / ml. In some embodiments, the composition contains a free amino functional group (—NH3+), a free carboxylic acid functional group (—CO2H), and (i) -OH group; (ii)-(CH2) n an OH group (where n is 1, 2, or 3); (iii)-(CH2) nCONH2 groups (where n is 1, 2, or 3); (iv) -cyclic C4H9N group; or (v) Branched alkyl group and a side chain comprising: is present in the composition at a concentration of at least about 0.0001 mg / ml, preferably at a concentration of at least about 0.005 mg / ml, more preferably at a concentration of at least about 0.05 mg / ml, more preferably at a concentration of at least about 0.01 mg / ml, and most preferably at a concentration of at least about 0.1 mg / ml.

[0132] In an even more preferred embodiment, the composition comprises from about 0.01 to about 100 mg / ml of an enhancer selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof.

[0133] The composition may contain from about 0.01 to about 500 mg / kg of one or more halides and / or pseudohalides. In a preferred embodiment, the composition contains from about 0.1 to about 200 mg / kg, more preferably from about 1 mg to about 100 mg / kg, even more preferably from about 2 mg to about 75 mg / kg, and most preferably from 5 to 50 mg / kg of one or more halides and / or pseudohalides. In a further preferred embodiment, the composition contains from about 0.1 to about 200 mg / kg of one or more water-soluble iodide salts, optionally wherein the one or more water-soluble iodide salts are potassium iodide.

[0134] The composition may contain about 50 to about 10,000 U / kg of peroxide-generating enzyme (i.e., the enzyme portion of the peroxide-generating system). In a preferred embodiment, the composition contains about 100 to about 5,000 U / kg of peroxide-generating enzyme, more preferably about 150 to about 4,000 U / kg, more preferably about 200 to about 3,000 U / kg, and more preferably about 300 to about 2,500 U / kg of peroxide-generating enzyme. In an even more preferred embodiment, the composition contains about 300 to about 2,500 U / kg of glucose oxidase.

[0135] In certain embodiments, the composition comprises: about 150 to about 4000 U / kg, preferably about 200 to about 3000 U / kg, more preferably about 300 to about 2500 U / kg of a peroxide generating system; about 10 to about 100,000 U / kg, preferably about 10 to 4,000 U / kg, more preferably about 10 to 100 U / kg of peroxidase; about 0.01 to about 500 mg / kg, preferably about 0.1 to about 200 mg / kg, more preferably about 1 mg to about 100 mg / kg, even more preferably about 2 mg to about 75 mg / kg, and most preferably 5 to 50 mg / kg of a halide and / or pseudohalide; and a free amino functional group (-NH3+) and a free carboxylic acid functional group (-CO2H), (i) -OH group; (ii)-(CH2) n an OH group (where n is 1, 2, or 3); (iii)-(CH2) n CONH2 groups (where n is 1, 2, or 3); (iv) -cyclic C4H9N group; or (v) Branched alkyl group and a side chain comprising:

[0136] In a further embodiment, the composition comprises: about 150 to about 4000 U / kg, preferably about 200 to about 3000 U / kg, more preferably about 300 to about 2500 U / kg of glucose oxidase; about 10 to about 100,000 U / kg, preferably about 10 to 4,000 U / kg, more preferably about 10 to 100 U / kg of lactoperoxidase; about 0.01 to about 500 mg / kg, preferably about 0.1 to about 200 mg / kg, of a water-soluble iodide salt, such as potassium iodide; and 0.0005 mg / ml to 200 mg / ml, preferably 0.005 mg / ml to 10 mg / ml, most preferably 0.05 to 1 mg / ml of a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof.

[0137] In certain embodiments, where the compositions described herein are topical (skin) compositions, the compositions may serve therapeutic and / or non-therapeutic purposes.

[0138] The composition may be a topical therapeutic composition. As used herein, the terms "therapeutic," "therapy," and the like refer to a treatment aimed at changing a subject's body or a part of a subject's body from an undesirable physiological condition, disease, or disorder caused by an infectious agent to a desired condition, such as a less severe condition (e.g., improvement or alleviation), or a normal, healthy condition (e.g., restoring the subject's health, physical integrity, and physical well-being), maintaining (i.e., not worsening) the undesirable physiological condition (e.g., stabilizing), or slowing progression to a more severe or worse condition compared to the undesirable physiological change or disorder. The subject to whom the topical therapeutic composition is applied may have been diagnosed with a disease or condition or may be suspected of having a disease or condition. Measurable relief includes a statistically significant decrease in a measurable marker or symptom. As used herein, statistical significance refers to a p-value of less than 0.05, which, as one skilled in the art will understand, is a commonly accepted cutoff score in statistical analysis. "Treatment" encompasses both curative treatment and treatment directed at alleviating symptoms and / or slowing progression and / or stabilizing disease. The compositions disclosed herein have the advantage of increased antimicrobial activity, and thus the compositions disclosed herein increase the rate of healing by modulating the microbial load and, indirectly, the inflammatory state resulting from the nature of the microorganisms, thereby reducing the need for more frequent changes of ointments, gels, dressings, etc., and reducing discomfort for the treated patient. Thus, the present invention provides pharmaceutical compositions suitable for reducing discomfort, mortality, and morbidity.

[0139] In addition to beneficially modulating any inflammation that may occur due to a reduction in microbial load, the compositions described herein also modulate inflammation in a more direct manner. Indeed, the inventors have found that the compositions can reduce the secretion of pro-inflammatory molecules by immune cells. Furthermore, a further advantage of the compositions described herein has been found to be their ability to directly neutralize already secreted pro-inflammatory molecules. In a preferred embodiment, the compositions described herein reduce the secretion of one or more pro-inflammatory molecules selected from the group consisting of IL-6 (interleukin 6), tumor necrosis factor alpha (TNFα), and matrix metallopeptidase 9 (MMP9) compared to the secretion of the one or more pro-inflammatory molecules in the absence of the enhancer that is the subject of the present disclosure. In complementary preferred embodiments, the compositions described herein neutralize the secretion of one or more pro-inflammatory molecules selected from the group consisting of IL-6, TNFα, and MMP9, preferably IL-6 and / or MMP9, relative to the secretion of said one or more pro-inflammatory molecules in the absence of the enhancer that is the subject of the present disclosure.

[0140] Those skilled in the art will recognize that to achieve effective therapeutic treatment, a therapeutically effective dose must be administered to the subject. Thus, in the context of the present disclosure, "effective amount" refers to the amount required to achieve a physiological effect. The physiological effect can be achieved by one dose or multiple doses. "Therapeutically effective amount" or "therapeutically effective dose" refers to the amount of the composition described herein that, when administered, results in a clinically positive response in treating a subject suffering from one or more wounds.

[0141] "Diagnosed with," "diagnosing," and "diagnosis" refer to the process of recognizing, determining, or concluding a disease, condition, or (adverse) effect in a subject based on symptoms and signs and / or from the results of various diagnostic procedures (e.g., knowing the presence, absence, and / or amount of one or more biomarkers or clinical symptoms characteristic of the diagnosed disease or condition). A "diagnosis" of a disease, condition, or (adverse) effect taught herein in a subject may specifically mean that the subject has such disease or condition. A subject may be diagnosed as not having such despite exhibiting one or more conventional symptoms or signs reminiscent of such. A "diagnosis" of a disease or condition taught herein in a subject may specifically mean that the subject has a skin disorder. Alternatively, a subject may be diagnosed as not having a particular disease despite exhibiting one or more conventional symptoms or signs reminiscent of such.

[0142] The composition may also be a topical prophylactic composition. A "prophylactic composition" in this context refers to a composition applied to a subject to inhibit a disabling disease or condition that is not yet characterized by clinical symptoms at the time of application. Those skilled in the art will understand that the purpose of treatment is to prevent, for example, the onset, development, and progression of a bacterial infection, for example, in a subject's wound, i.e., in the subject's skin. For example, a topical prophylactic composition in the context of the present invention may be applied before a burn wound becomes apparent, or as part of a preparatory treatment before surgery, or as a preventative treatment to prevent bedsores. The term "to prevent," or alternative forms thereof, such as "preventing" or "prevention," generally means to keep something from occurring, occurring, or not existing, or to delay the occurrence or onset of something, especially by one or more precautionary (preventative) measures.

[0143] The composition may be a topical cosmetic composition. Terms such as "cosmetics," "cosmetic composition," and "cosmetic adjuvant" refer to a means suitable for enhancing the appearance of cleanliness, (personal) hygiene, and / or physical cleanliness. The cosmetic uses or methods contemplated herein address normal, natural, or physiological processes and can be distinguished from therapies, including curative and preventative treatments, which aim to restore a subject to a pathological condition or at least alleviate the symptoms of pain and suffering caused by a pathological condition, or to prevent a pathological condition in the first place. Therefore, the cosmetic uses or methods contemplated herein can be described as "non-therapeutic." The cosmetic uses or methods contemplated herein generally employ cosmetic compositions configured for topical application to the skin. The cosmetic purpose of the composition is not particularly limited and may therefore include cleansing the skin, maintaining skin moisture balance, stimulating skin metabolism, protecting the skin from harmful environmental factors, such as, but not limited to, UV radiation, and any combination thereof.

[0144] The release profile of the contents (i.e., ingredients) of the topical composition is not particularly limited in the present invention and can be adjusted and optimized by those skilled in the art. Optionally, the topical composition is an immediate-release composition. In such embodiments, the ingredients of the composition are released onto the skin of a subject in an unlimited manner. Optionally, at least about 75% of the peroxidase, peroxide-generating system, and enhancer are released from the topical composition within about 6 hours of application, preferably within about 4 hours of application, more preferably within about 2 hours of application, more preferably within 1 hour of application, more preferably within 30 minutes of application, and most preferably within about 15 minutes of application. Alternatively, the composition is a sustained-release or controlled-release composition. In such embodiments, the ingredients of the composition are released onto the skin of a subject in a limited manner. Optionally, the composition releases less than 50% of the peroxidase, peroxide generating system, and enhancer within 2 hours, preferably less than 50% of the peroxidase, peroxide generating system, and enhancer within 4 hours, preferably less than 50% of the peroxidase, peroxide generating system, and enhancer within 6 hours, and preferably less than 50% of the peroxidase, peroxide generating system, and enhancer within 12 hours.

[0145] In a further aspect, the compositions are contemplated for use as medicines (both therapeutic and prophylactic). In other words, the use of the compositions described herein is contemplated for the manufacture of medicines. In yet another way, the use of the compositions described herein is contemplated as medicines.

[0146] Optionally, the compositions are contemplated for use in the treatment of skin disorders (both therapeutic and preventative). In other words, the use of the compositions described herein is contemplated for the manufacture of a medicament for treating skin disorders. In other words, the use of the compositions described herein is contemplated as a medicament for treating skin disorders. As used herein, the term "skin disorder" generally refers to any abnormal condition of the skin, including, but not limited to, inflammatory conditions caused by microbial infection. Those skilled in the art will understand that inflammation is a mechanism of disease caused by infection (i.e., "microbial infection"). Inflammatory skin disorders caused by microbial infection are derived from an innate immune response to infection by microorganisms, such as viruses, microorganisms, fungi, parasites, or any combination thereof.

[0147] By way of illustration and not limitation, viral infections of the skin include shingles (herpes zoster), chickenpox, molluscum contagiosum, warts, measles, and hand, foot and mouth disease.

[0148] Bacterial infections of the skin include, by way of illustration and not limitation, carbuncles, dermatitis, erythema, folliculitis, furuncles, impetigo, lymphadenitis, small skin abscesses (pockets of pus in the skin), cellulitis, erysipelas, large skin abscesses, lymphangitis, and necrotizing skin infections.

[0149] Fungal infections of the skin include, by way of illustration and not limitation, body ringworm infections (tinea corporis), tinea pedis (athlete's foot), pubic ulcers, scalp ringworm infections (tinea capitis), tinea versicolor (pityriasis versicolor), cutaneous candidiasis, and nail fungus (tinea unguium).

[0150] Optionally, the composition is intended for use in treating skin wounds. In other words, the use of the composition described herein is intended for the manufacture of a medicament for treating skin wounds. In other words, the use of the composition described herein is intended as a medicament for treating skin wounds. The nature and / or cause of the skin wound is not particularly limited in the present invention, but includes, but is not limited to, acute wounds caused by injury and surgically induced wounds. The cause of the injury is not limited in the present invention, and therefore includes both accidental injury and maliciously caused injury (i.e., combat wounds).

[0151] Optionally, the compositions described herein are intended for use in the treatment of burn wounds. As used herein, "burn" refers to a specific type of tissue injury caused by contact with heat, flame, chemicals, electricity, or radiation. Generally, first-degree burns are characterized by redness; second-degree burns are characterized by the presence of one or more blistered spots (i.e., rash); and third-degree burns are characterized by the presence of necrosis. Throughout the field, first- and second-degree burns are generally referred to as partial-thickness burns (i.e., tissue destruction extending from the epidermis to the dermis but not penetrating the dermis). Third-degree burns are generally referred to as full-thickness burns (i.e., destruction completely penetrating the dermis).

[0152] The pharmaceutical compositions and hydrogels optionally described herein are intended for use in the treatment of chronic wounds. The term "chronic wound" refers to any wound that does not show sufficient signs of healing even after applying the standard wound healing process (e.g., but not limited to, diabetic foot ulcers, venous leg ulcers, and pressure ulcers). Thus, wounds can be clinically classified as acute or chronic based on their healing time. The term "chronic wound" can be used interchangeably with synonyms such as, but not limited to, "recalcitrant wound," "difficult to heal wound," "non-healing wound," and "complex wound." Chronic wounds are characterized by a dysregulated healing process due to many factors that prolong one or more of the wound healing processes.

[0153] Thus, the present invention also contemplates the use of the compositions described herein in methods of treatment, such as methods of treating skin disorders, methods of treating wounds, such as skin wounds, and methods of treating microbial infections. The methods of treatment comprise administering a composition described herein to a subject. In embodiments in which the composition is a topical skin composition, the method comprises administering the composition to (a portion of) the skin of a subject. In further embodiments, the method of treatment comprises a single application of the composition to (a portion of) the skin of a subject. In alternative embodiments, the method of treatment comprises monthly, preferably biweekly, more preferably weekly, and most preferably daily administration of the composition to (a portion of) the skin of a subject.

[0154] The compositions that are the subject of the present invention may be used in combination with additional pharmaceutically active ingredients and / or drugs. The term "pharmaceutically active ingredient", which is used interchangeably with "pharmaceutically active agent" throughout this disclosure, should be interpreted according to the World Health Organization's definition of the term: "a substance used in a finished pharmaceutical product (FPP) that exhibits pharmacological activity or otherwise has a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or that is intended to have a direct effect in the restoration, correction or modification of human physiological function."

[0155] For example, the composition can be used in combination with an analgesic and / or an anti-inflammatory agent. Additional pharmaceutically active ingredients may be included in the composition of the present invention. Alternatively, the additional pharmaceutically active ingredients may be included in a separate composition, i.e., an additional and physically distinct composition. As used herein, the term "analgesic" should be interpreted in its broadest sense and can refer to any compound, substance, or composition that can reduce or suppress pain in a subject, and therefore any product that can achieve analgesia in a subject. Throughout the field, any analgesic may be interchangeably referred to as a "painkiller" or a "pain reliever." Analgesics can be used in the treatment and / or prevention of pain (i.e., pain prophylaxis). Analgesics can act on the peripheral and / or central nervous system. In the field, analgesics are generally classified according to their mechanism of action. Different classes of analgesics include, but are not limited to, acetaminophen (i.e., paracetamol), nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, muscle relaxants, anxiolytics, antidepressants, anticonvulsants, and corticosteroids.

[0156] It is clear that additional pharmaceutically active agents having antibacterial properties may further be incorporated into the composition. Optionally, the subject composition of the present invention may be combined with at least one additional antiviral agent, at least one additional antibacterial agent, at least one additional antifungal agent, or at least one antiparasitic agent.

[0157] In a further aspect, the present invention is directed to the use of a potentiator as described herein to improve the minimum inhibitory concentration (MIC) and / or minimum bactericidal concentration (MBC) and / or absolute number and / or enzymatic activity of a pharmaceutical composition comprising a peroxide generating system, a peroxidase, and a halide or pseudohalide, wherein the potentiator is as described herein above. In certain embodiments, the potentiator is a compound having a free amino functional group (-NH3 + ), a free carboxylic acid functional group (—COH), and (i) -OH group; (ii)-(CH2) n an OH group (where n is 1, 2, or 3); (iii)-(CH2) n CONH2 groups (where n is 1, 2, or 3); (iv) -cyclic C4H9N group; or (v) Branched alkyl group and a side chain comprising:

[0158] The use of the enhancers described herein to improve the minimum inhibitory concentration (MIC) and / or minimum bactericidal concentration (MBC) and / or absolute number and / or enzyme of a pharmaceutical composition comprising glucose oxidase, lactoperoxidase, and a water-soluble iodide salt is also contemplated. In certain embodiments, the enhancer is a natural or unnatural amino acid, preferably a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof.

[0159] The use to improve the minimum inhibitory concentration (MIC) and / or minimum bactericidal concentration (MBC) and / or absolute number and / or enzyme activity of a pharmaceutical composition is an in vitro use. In some embodiments, the use is an in vitro use for preservation of skin grafts, cell lines, biomarkers, and / or any other biological sample. The term "in vitro" generally refers to outside or exterior to the animal or human body.

[0160] The term "ex vivo" typically refers to tissues or cells removed from an animal or human body and maintained or grown outside the body (e.g., in a culture vessel). As used herein, the term "in vitro" should be understood to include "ex vivo." The term "in vivo" generally refers to inside, on, or within an animal or human body.

[0161] As used herein, the term "biological sample" or "sample" refers to a biological material separated from its natural environment. The sample may correspond to or include a tissue sample, a biological fluid sample (e.g., blood, plasma), or a cell sample, e.g., a hematopoietic cell sample. The term "biomarker," often referred to in the art as "marker," is widespread in the art and generally refers broadly to a biological component or molecule, more particularly an endogenous biological component or molecule, or a detectable portion thereof, whose qualitative and / or quantitative assessment in a subject, such as by evaluating a biological sample from the subject, is predictive (e.g., predictive, diagnostic, and / or prognostic) or informative regarding one or more aspects of the subject's phenotype and / or genotype, e.g., regarding the subject's status for a given disease or condition. In certain embodiments, where the use is an in vitro use for the preservation of a biological sample (such as, but not limited to, a skin graft sample) intended for transplantation onto or into a living subject in an otherwise unrelated manner, the composition may be applied directly to the sample, or the sample may be placed in a liquid or semi-liquid environment, wherein the environment comprises the composition that is the subject of the present invention.

[0162] In a further aspect, the present invention is directed to the use of an enhancer described herein to preserve the sterility of a pharmaceutical, prophylactic, or cosmetic composition. In certain embodiments, the enhancer comprises a free amino functional group (-NH3+), a free carboxylic acid functional group (-CO2H), and (i) -OH group; (ii)-(CH2) n an OH group (where n is 1, 2, or 3); (iii)-(CH2) n CONH2 groups (where n is 1, 2, or 3); (iv) -cyclic C4H9N group; or (v) Branched alkyl group and a side chain comprising:

[0163] Optionally, the use of the enhancer described herein is envisioned for maintaining the sterility of pharmaceutical, prophylactic, or cosmetic compositions. In certain embodiments, the enhancer is a natural or unnatural amino acid, preferably a natural amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof. As used herein, "sterility" refers to the complete or substantially complete absence of any living organisms, such as microorganisms, in a liquid or on a surface.

[0164] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims. Aspects and embodiments of the present invention disclosed herein are further supported by the following non-limiting examples. The following specific experimental examples are provided to support the claimed invention, but should not be construed as limiting the scope of the invention. [Example]

[0165] Example Example 1. Enzyme activity assay 1.1. Glucose oxidase (GOX) activity The native enzyme GOX is an oxidoreductase that catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide (HO) in the presence of water and air. A standard spectrophotometric assay was performed to measure GOX enzymatic activity. Briefly, a second oxidoreductase, peroxidase, and a chromogenic oxygen acceptor, o-dianisidine, were added to the reaction mixture (GOX + glucose + water + air) to form a colored compound that could be measured spectrophotometrically. GOX was tested at four different concentrations: 100, 50, 10, and 1 mg / ml. Each reaction mixture was tested in duplo. When GOX activity was assessed in the presence of a potential enhancer (a molecule that could potentially enhance GOX enzymatic activity), the latter was added at low and high concentrations ranging from 100 to 0.1 mg / ml. A blank consisted of the reaction mixture omitting the enzyme. After mixing all reaction components, the absorbance was immediately measured every minute for 5 minutes at 35°C using a plate reader (Spectramax ID3, Molecular Devices). This test was repeated at least twice for each combination evaluated. The results were further analyzed in MS Excel and expressed as U / g. Mean blank-corrected data ± SD were plotted. Statistical significance compared to the combination without the potentiator candidate (reference) was calculated using students' t-tests. Significance was achieved when the obtained p-value was lower than 0.05. The significance levels were defined as p<0.05 = *; p<0.01 = ** and p<0.001 = ***.

[0166] The results showed that adding proline (0.1–100 mg / ml) to the GOX enzyme reaction solution significantly enhanced the enzyme activity of GOX when compared with that of the reference (without the potentiator candidate) ( Figure 1 ):

[0167] Proline (100 mg / ml): As a result, GOX activity was significantly improved compared to the reference. The significance level was p<0.001.

[0168] Proline (0.1 mg / ml): As a result, GOX activity was significantly improved compared to the control. The significance level was p<0.05.

[0169] In contrast, the addition of other amino acids with structures similar to proline, such as cysteine, significantly reduced GOX activity compared with the reference (Figure 1). Therefore, the above-mentioned improvement in GOX activity is unlikely to be group-specific (general amino acids / proline-like amino acids).

[0170] 1.2. Lactoperoxidase (LPO) activity Lactoperoxidase is a member of the heme peroxidase family of enzymes, which reacts with the catalytic anion (I) derived from KI present in the substrate. - ) and catalyzes the oxidation of H2O2 to hypojodite (IO -This hypogeeitol is a reactive oxygen species (ROS). To measure the enzymatic activity of LPO, a standard spectrophotometric assay is performed. Briefly, HO in combination with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonzuur (ABTS)) is catalyzed by LPO, resulting in the formation of a colored compound that can be measured spectrophotometrically. LPO was tested at three different concentrations: 50, 10, and 1 mg / ml. Each reaction mixture was tested in duplicate. When LPO activity was evaluated in the presence of potential enhancers (molecules that may be able to enhance the enzymatic activity of LPO), the latter were added at low and high concentrations ranging from 100 to 0.1 mg / ml. A blank consisted of the reaction mixture without HO. The absorbance was immediately measured every 12 seconds for 5 minutes at 20 °C using a plate reader (Spectramax ID3, Molecular Devices). This test was repeated at least twice for each combination evaluated. The results were analyzed by MS. Further analysis was performed in Excel and expressed as U / g. Mean blank-corrected data ± SD were plotted. Statistical significance compared to the combination without the potentiator candidate (reference) was calculated using students' t-tests. Significance was achieved when the obtained p-value was lower than 0.05. Significance levels were defined as p<0.05 = *; p<0.01 = ** and p<0.001 = ***.

[0171] The results showed that adding serine (100 mg / ml), asparagine (1–0.1 mg / ml), glutamine (0.1 mg / ml), and threonine (1–0.1 mg / ml) to the LPO enzyme reaction solution significantly enhanced the enzyme activity of LPO when compared with that of the reference (without any potential enhancer) (Figure 2):

[0172] Serine (100 mg / ml) and asparagine (1 mg / ml): As a result, LPO activity was significantly increased compared to the control. The significance level was p<0.01.

[0173] Asparagine (0.1 mg / ml), glutamine (0.1 and 1 mg / ml), and threonine (0.1 and 1 mg / ml): LPO activity was significantly increased compared to the control. The significance level was p<0.001.

[0174] In contrast, the addition of other amino acids, such as leucine, did not significantly affect LPO activity compared with the reference (Figure 2), and therefore the above-mentioned enhancement of LPO activity is unlikely to be group-specific.

[0175] Example 2. Antibacterial activity assay 2.1.MIC measurement The minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial agent at which microbial growth is completely inhibited after overnight incubation (measured by visual assessment or absorbance measurement). This endpoint measurement is used to define the susceptibility of a microorganism to an antimicrobial agent. Here, the antimicrobial agent is the natural enzyme combination GOX + LPO. The MIC of this enzyme combination GOX + LPO was investigated by itself and in combination with a potential potentiator (a molecule that has no antimicrobial effect by itself but can potentiate the antimicrobial effect of an antimicrobial agent).

[0176] To determine the MICs of the described combinations, a classical broth microdilution assay was used to measure: (a) the gram-positive bacterium Staphylococcus aureus (ATCC 62141); (登録商標) 6538 TM (The results are shown below.) (b) Pseudomonas aeruginosa ATCC, a gram-negative bacterium (登録商標) 9027 TM (The results are shown below.) (c) Candida albicans ATCC (登録商標) 10231 TM (Results shown below), and (d) the fungus Aspergillus niger ATCC (登録商標) 16404 TMThe susceptibility of a series of microorganisms commonly found in infected wounds, including 1,2-dimethyl-2-propanol (DMSO) and 1,2-dimethyl-2-propanol (DMSO) (results shown below), was measured. Briefly, serial dilutions of the enzyme GOX+LPO solution were prepared in a clear, flat-bottom 96-well test plate to final concentrations ranging from 0.005 to 0.00000977% (v / v). Each well was filled with a fixed amount of enzyme substrate containing 3.3% (w / v) glucose and 0.084% (w / v) potassium iodide, and the OD was measured. 600 A 0.1% microbial inoculum was added. When enzyme combinations were evaluated in the presence of a potential potentiator, the latter was added along with the enzyme substrate at final concentrations ranging from 100% to 125% (v / v). A blank (no microbial inoculum added) was included for each combination evaluated. To evaluate the performance of the test and select the enzyme GOX + LPO concentration at which no microbial growth was detected (i.e., the absorbance was equal to that of the background solution alone), a positive (inoculum only) and negative (background solution only) were included. The 96-well test plate was then incubated for 24 hours at 37°C (Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans) or 25°C (Aspergillus niger). After incubation, the absorbance of each well was measured using a plate spectrophotometer (Spectramax ID3, Molecular Devices). This test was repeated at least five times for each evaluated combination. The collected data were further analyzed using MS Excel. The mean absorbance data was visualized in a tabular format with the MICs highlighted.

[0177] Staphylococcus aureus The results showed that the addition of serine and isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.00125% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of S. aureus (Table 1):

[0178] Serine (0.0025%-0.00125%): As a result, the MIC improved by 2× compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when serine (0.0025%-0.00125%) was added to the reaction mixture containing 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0179] Isoleucine (0.0025%-0.00125%): As a result, the MIC improved by 2× compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when isoleucine (0.0025%-0.00125%) was added to the reaction mixture containing 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0180] Increasing the concentration of the enhancer molecule gave the following results (Table 2): Proline (0.01%): As a result, the MIC improved by 4x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000195% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when proline (0.01%) was added to the reaction mixture containing 0.005% to 0.0000195% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0181] Proline (10%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when proline (10%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0182] Serine (10%): Growth inhibition occurred at all concentrations of the GOX+LPO enzyme solution tested, ranging from 0.005% to 0.0000977% (v / v). Thus, the MICs were equal to or less than 0.0000977% (v / v) (lower concentrations were not tested), indicating a difference of at least 6×. In other words, S. aureus was more susceptible to the addition of serine (10%) to reaction mixtures containing GOX+LPO enzyme solutions ranging from 0.005% to 0.00000977% (v / v).

[0183] Serine (0.01%-0.005%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when serine (0.01%-0.005%) was added to the reaction mixture containing 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0184] Threonine (2%): As a result, the MIC improved by 4x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000195% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when threonine (2%) was added to the reaction mixture containing 0.005% to 0.0000195% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0185] Threonine (0.01%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when threonine (0.01%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0186] Glutamine (1% to 0.01%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when glutamine (1% to 0.01%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0187] Asparagine (0.5%-0.01%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, S. aureus showed greater susceptibility when asparagine (0.5%-0.01%) was added to the reaction mixture containing 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution.

[0188] Isoleucine (0.5%-0.005%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when isoleucine (0.5%-0.005%) was added to the reaction mixture containing 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0189] Pseudomonas aeruginosa The results showed that the addition of proline and serine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.00125% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 3):

[0190] Serine (0.0025-0.00125%): As a result, the MIC improved by 2× compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when serine (0.0025-0.00125%) was added to the reaction mixture containing 0.005%-0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0191] Proline (0.0025-0.00125%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when proline (0.0025-0.00125%) was added to the reaction mixture containing 0.005%-0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0192] Increasing the concentration of the enhancing molecule gave the following results (Table 4): Serine (0.01-0.005%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when serine (0.01-0.005%) was added to the reaction mixture containing 0.005%-0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0193] Proline (0.01-0.005%): As a result, the MIC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when proline (0.01-0.005%) was added to the reaction mixture containing 0.005%-0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0194] The results showed that the addition of threonine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 5):

[0195] Threonine (1%): As a result, the MIC improved by 8x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when threonine (1%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0196] Threonine (0.1%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when threonine (0.1%) was added to the reaction mixture containing 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater sensitivity.

[0197] The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 6):

[0198] Isoleucine (1%): As a result, the MIC improved by 8x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when isoleucine (1%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0199] Isoleucine (0.1%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when isoleucine (0.1%) was added to the reaction mixture containing 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater sensitivity.

[0200] The results showed that the addition of glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 7):

[0201] Glutamine (1%): As a result, the MIC improved by 8x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when glutamine (1%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0202] Glutamine (0.1%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when glutamine (0.1%) was added to the reaction mixture containing 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0203] The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 8):

[0204] Asparagine (1%): As a result, the MIC improved by 8x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when asparagine (1%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0205] Asparagine (0.1%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when asparagine (0.1%) was added to the reaction mixture containing 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0206] 2.1.3. Candida albicans The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at a final concentration of 0.01% (v / v) resulted in a difference in the MIC when compared to that of the reference (without potentiator candidate), indicating a difference in the susceptibility of Candida albicans (Table 9):

[0207] Isoleucine (0.01%): As a result, the MIC improved by 4x compared to the reference and by 2x compared to the potentiating molecule guaiacol. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Candida albicans, instead of 0.000156% (v / v). In other words, when isoleucine (0.01%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, Candida albicans showed greater sensitivity.

[0208] 2.1.4. Aspergillus niger The results showed that the addition of proline to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 10):

[0209] Proline (1% to 0.01%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, when proline (1% to 0.01%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, Aspergillus niger showed greater sensitivity.

[0210] The results showed that the addition of serine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 11):

[0211] Serine (1%-0.01%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, when serine (1%-0.01%) was added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution, Aspergillus niger showed greater sensitivity.

[0212] The results showed that the addition of threonine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 12):

[0213] Threonine (1%-0.01%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, when threonine (1%-0.01%) was added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution, Aspergillus niger showed greater sensitivity.

[0214] The results showed that adding glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 13):

[0215] Glutamine (1%-0.01%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, when glutamine (1%-0.01%) was added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution, Aspergillus niger showed greater sensitivity.

[0216] The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.05% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 14):

[0217] Asparagine (1%-0.05%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, Aspergillus niger was more sensitive to asparagine (1%-0.05%) added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution.

[0218] The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.005% (v / v) resulted in differences in the MICs when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 15):

[0219] Isoleucine (1%-0.005%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, Aspergillus niger showed greater sensitivity when isoleucine (1%-0.005%) was added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution.

[0220] [Table 1]

[0221] [Table 2]

[0222] [Table 3]

[0223] Table 4

[0224] Table 5

[0225] Table 6

[0226] Table 7

[0227] Table 8

[0228] Table 9

[0229] Table 10

[0230] Table 11

[0231] Table 12

[0232] Table 13

[0233] Table 14

[0234] [Table 15]

[0235] 2.2.MBC measurement The minimum bactericidal concentration (MBC) is defined as the lowest concentration of an antimicrobial agent that completely kills a microbial population after overnight incubation (measured by standard CFU enumeration techniques). This endpoint measurement, together with the MIC (see above), is used to define the susceptibility of a microorganism to an antimicrobial agent. Here, the antimicrobial agent is the natural enzyme combination GOX+LPO. The MBC of this enzyme combination GOX+LPO was investigated both by itself and in combination with a potential potentiator (i.e., a molecule that has no antimicrobial effect by itself but can potentiate the antimicrobial effect of an antimicrobial agent).

[0236] To determine the MBC of the described combinations, a classical broth microdilution assay combined with standard CFU enumeration techniques was used to measure (a) the MBC of the Gram-positive bacterium Staphylococcus aureus ATCC 61444; (登録商標) 6538 TM (The results are shown below.) (b) Pseudomonas aeruginosa ATCC, a gram-negative bacterium (登録商標) 9027 TM (The results are shown below.) (c) Candida albicans ATCC (登録商標) 10231 TM (results shown below), and (d) the fungus Aspergillus niger ATCC (登録商標) 16404 TMThe susceptibility of a panel of microorganisms commonly found in infected wounds, including (results shown below), was determined. Briefly, a broth microdilution assay was set up as previously described (see 2.1). After incubation, the MBC was determined by examining the presence of viable microorganisms at the MIC and subsequent higher concentrations (MIC x 2, MIC x 4, etc.) using standard CFU enumeration techniques. This test was repeated at least five times for each combination evaluated. The collected data was further analyzed in MS Excel. The mean concentration data (expressed as log CFU / ml) were visualized in a tabular format with the MBC highlighted.

[0237] Staphylococcus aureus The results showed that the addition of serine and isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.00125% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of S. aureus (Table 16):

[0238] Serine (0.0025%-0.00125%): As a result, the MBC improved 4x compared to the reference and 2x compared to the potentiating molecule guaiacol. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when serine (0.0025%-0.00125%) was added to a reaction mixture containing 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0239] Isoleucine (0.00125%): As a result, the MBC improved 4x compared to the reference and 2x compared to the potentiating molecule guaiacol. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when isoleucine (0.00125%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater sensitivity.

[0240] Increasing the concentration of the enhancer molecule gave the following results (Table 17): Serine (10% to 0.005%): As a result, the MBC improved 4x compared to the reference and 2x compared to the potentiating molecule guaiacol. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when serine (10% to 0.005%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0241] Isoleucine (0.005%): As a result, the MBC improved 4x compared to the reference and 2x compared to the potentiating molecule guaiacol. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when isoleucine (0.005%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater sensitivity.

[0242] Glutamine (0.1%): As a result, the MBC improved 4x compared to the reference and 2x compared to the potentiating molecule guaiacol. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of S. aureus, instead of 0.0000781% (v / v). In other words, when glutamine (0.1%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, S. aureus showed greater susceptibility.

[0243] Pseudomonas aeruginosa The results showed that the addition of proline and serine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.00125% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 18):

[0244] Serine (0.0025-0.00125%): As a result, the MBC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when serine (0.0025-0.00125%) was added to the reaction mixture containing 0.005%-0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0245] Proline (0.0025-0.00125%): As a result, the MBC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when proline (0.0025-0.00125%) was added to the reaction mixture containing 0.005%-0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0246] Increasing the concentration of the enhancing molecule gave the following results (Table 19): Serine (0.01-0.005%): As a result, the MBC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, P. aeruginosa was more susceptible to the addition of serine (0.01-0.005%) to the reaction mixture containing 0.005%-0.000625% (v / v) of the GOX+LPO enzyme solution.

[0247] Proline (0.01-0.005%): As a result, the MBC improved by 2x compared to the reference or the solution containing guaiacol as an enhancer. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, P. aeruginosa was more susceptible to proline (0.01-0.005%) added to the reaction mixture containing 0.005%-0.000625% (v / v) of the GOX+LPO enzyme solution.

[0248] The results showed that the addition of threonine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in a difference in the MBC when compared to that of the reference (without potentiator candidate), indicating a difference in the susceptibility of P. aeruginosa (Table 20):

[0249] Threonine (1%): As a result, the MBC improved by 8x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when threonine (1%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater sensitivity.

[0250] Threonine (0.1%): As a result, the MBC improved by 2x compared to the reference. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when threonine (0.1%) was added to the reaction mixture containing 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater sensitivity.

[0251] The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 21):

[0252] Isoleucine (1%): As a result, the MBC improved by 8x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when isoleucine (1%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater sensitivity.

[0253] Isoleucine (0.1%): As a result, the MBC improved by 2x compared to the reference. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, P. aeruginosa showed greater sensitivity when isoleucine (0.1%) was added to the reaction mixture containing 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution.

[0254] The results showed that adding glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 22):

[0255] Glutamine (1%): As a result, the MBC improved by 8x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when glutamine (1%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater susceptibility.

[0256] Glutamine (0.1%): As a result, the MBC improved by 2x compared to the reference. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, P. aeruginosa was more susceptible to the addition of glutamine (0.1%) to the reaction mixture containing 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution.

[0257] The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Table 23):

[0258] Asparagine (1%): As a result, the MBC improved by 8x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, when asparagine (1%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution, P. aeruginosa showed greater sensitivity.

[0259] Asparagine (0.1%): As a result, the MBC improved by 2x compared to the reference. This means that only 0.000625% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of P. aeruginosa, instead of 0.00125% (v / v). In other words, P. aeruginosa was more sensitive to asparagine (0.1%) added to the reaction mixture containing 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution.

[0260] 2.2.3. Candida albicans The results showed that the addition of serine and isoleucine to the enzyme GOX+LPO combination at a final concentration of 0.01% (v / v) resulted in a difference in the MBC when compared to that of the reference (no potentiator candidate), indicating a difference in the susceptibility of Candida albicans (Table 24):

[0261] Isoleucine (0.01%): As a result, the MBC improved 4x compared to the reference and 2x compared to the potentiating molecule guaiacol. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Candida albicans, instead of 0.000156% (v / v). In other words, when isoleucine (0.01%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, Candida albicans showed greater sensitivity.

[0262] Serine (0.01%): As a result, the MBC improved 4x compared to the reference and 2x compared to the potentiator guaiacol. This means that only 0.0000391% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Candida albicans, instead of 0.000156% (v / v). In other words, when serine (0.01%) was added to the reaction mixture containing 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, Candida albicans showed greater sensitivity.

[0263] 2.2.4. Aspergillus niger The results showed that adding proline to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 25):

[0264] Proline (1% to 0.01%): As a result, the MBC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, Aspergillus niger showed greater sensitivity when proline (1% to 0.01%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution.

[0265] The results showed that adding serine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 26):

[0266] Serine (1% to 0.01%): As a result, the MBC improved 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, Aspergillus niger showed greater sensitivity when serine (1% to 0.01%) was added to the reaction mixture containing 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution.

[0267] The results showed that adding threonine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 27):

[0268] Threonine (1%-0.01%): As a result, the MBC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, Aspergillus niger showed greater sensitivity when threonine (1%-0.01%) was added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution.

[0269] The results showed that adding glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 28):

[0270] Glutamine (1%-0.01%): As a result, the MBC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, Aspergillus niger showed greater sensitivity when glutamine (1%-0.01%) was added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution.

[0271] The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.05% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 29):

[0272] Asparagine (1%-0.05%): As a result, the MBC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, Aspergillus niger showed greater sensitivity when asparagine (1%-0.05%) was added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution.

[0273] The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.005% (v / v) resulted in differences in the MBC when compared to that of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Table 30):

[0274] Isoleucine (1%-0.005%): As a result, the MIC improved by 2x compared to the reference. This means that only 0.000156% (v / v) of the GOX+LPO enzyme solution was required to completely inhibit the growth of Aspergillus niger, instead of 0.000313% (v / v). In other words, Aspergillus niger showed greater sensitivity when isoleucine (1%-0.005%) was added to the reaction mixture containing 0.005%-0.000156% (v / v) of the GOX+LPO enzyme solution.

[0275] [Table 16]

[0276] [Table 17]

[0277] [Table 18]

[0278] Table 19

[0279] Table 20

[0280] Table 21

[0281] Table 22

[0282] Table 23

[0283] Table 24

[0284] Table 25

[0285] Table 26

[0286] Table 27

[0287] Table 28

[0288] Table 29

[0289] [Table 30]

[0290] 2.3. Absolute bacterial counts Absolute microbial counts as a function of the concentration of a tested antimicrobial agent are useful for monitoring the bactericidal and fungicidal efficacy of that agent. Here, the antimicrobial agent is a combination of the natural enzymes GOX+LPO, and absolute microbial counts (as a function of enzyme solution concentration) were investigated by themselves and when GOX+LPO was combined with a potential potentiator (i.e., a molecule that has no antimicrobial effect by itself but can potentiate the antimicrobial effect of the antimicrobial agent).

[0291] A classical broth microdilution assay combined with standard CFU enumeration techniques was used to measure the absolute number of viable microorganisms as a function of the concentrations of the different enzyme solutions tested: (a) the Gram-positive bacterium Staphylococcus aureus ATCC 61444; (登録商標) 6538 TM (The results are shown below.) (b) Pseudomonas aeruginosa ATCC, a gram-negative bacterium (登録商標) 9027 TM (The results are shown below.) (c) Candida albicans ATCC (登録商標) 10231 TM (results shown below), and (d) the fungus Aspergillus niger ATCC (登録商標) 16404 TMThe susceptibilities of a range of microorganisms commonly found in infected wounds, including (results shown below), were determined. Briefly, a broth microdilution assay was set up as previously described (see 2.1). After incubation, absolute microbial counts at the MIC and subsequent higher concentrations (MIC x 2, MIC x 4, etc.) were determined using standard CFU enumeration techniques. This test was repeated at least five times for each combination evaluated. Collected data were further analyzed in MS Excel and expressed as log CFU / ml. Average results were plotted as a function of enzyme solution concentration.

[0292] Staphylococcus aureus The results showed that the addition of serine and isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.00125% (v / v) resulted in differences in the absolute microbial counts when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of S. aureus (Figure 3):

[0293] Serine (0.0025-0.00125%): As a result, the absolute S. aureus count was reduced more strongly at concentrations of 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution compared to the reference and to the solution with guaiacol added as an enhancer. In other words, S. aureus showed a higher sensitivity when serine (0.0025-0.00125%) was added to the reaction mixture.

[0294] Isoleucine (0.00125%): As a result, at concentrations of 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, the absolute S. aureus count was reduced more strongly compared to the reference and to the solution to which guaiacol was added as an enhancer. In other words, S. aureus showed a higher sensitivity when serine (0.00125%) was added to the reaction mixture.

[0295] Increasing the concentration of the enhancer molecule gave the following results (Figure 4): Serine (10% to 0.005%): As a result, the absolute S. aureus count was reduced more strongly at concentrations of 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution compared to the reference and to solutions with guaiacol added as an enhancer. In other words, S. aureus showed a higher sensitivity when serine (10% to 0.005%) was added to the reaction mixture.

[0296] Isoleucine (0.005%): As a result, at concentrations of 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, the absolute S. aureus count was reduced more strongly when compared with the reference solution and with the solution to which guaiacol was added as an enhancer. In other words, S. aureus showed a higher sensitivity when serine (0.005%) was added to the reaction mixture.

[0297] Glutamine (0.1%): As a result, at concentrations of 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution, the absolute S. aureus count was reduced more strongly compared to the reference and to the solution to which guaiacol was added as an enhancer. In other words, S. aureus showed a higher sensitivity when glutamine (0.1%) was added to the reaction mixture.

[0298] Pseudomonas aeruginosa The results showed that the addition of serine and isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.00125% (v / v) resulted in differences in the absolute microbial counts when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 5):

[0299] Serine (0.0025-0.00125%): As a result, at concentrations of 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution, the absolute P. aeruginosa count was reduced more strongly compared to the reference and to the solution with guaiacol added as an enhancer. In other words, P. aeruginosa showed a higher susceptibility when serine (0.0025-0.00125%) was added to the reaction mixture.

[0300] Proline (0.0025-0.00125%): As a result, at concentrations of 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution, the absolute P. aeruginosa count was reduced more strongly compared to the reference and to the solution to which guaiacol was added as an enhancer. In other words, P. aeruginosa showed a higher susceptibility when proline (0.0025-0.00125%) was added to the reaction mixture.

[0301] Increasing the concentration of the enhancer molecule gave the following results (Figure 6): Serine (0.01-0.005%): As a result, the absolute P. aeruginosa count was reduced more strongly at concentrations of 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution compared to the reference and to the solution with guaiacol added as an enhancer. In other words, P. aeruginosa showed a higher susceptibility when serine (0.01-0.005%) was added to the reaction mixture.

[0302] Proline (0.01-0.001%): As a result, at concentrations of 0.005%-0.0000391% (v / v) of the GOX+LPO enzyme solution, the absolute P. aeruginosa count was reduced more strongly compared to the reference and to the solution to which guaiacol was added as an enhancer. In other words, P. aeruginosa showed a higher susceptibility when proline (0.01-0.005%) was added to the reaction mixture.

[0303] The results showed that the addition of threonine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 7):

[0304] Threonine (1%): As a result, the absolute number of P. aeruginosa was reduced more strongly at concentrations of 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution compared to the reference. In other words, P. aeruginosa showed a higher sensitivity when threonine (1%) was added to the reaction mixture.

[0305] Threonine (0.1%): As a result, the absolute number of P. aeruginosa was reduced more strongly at concentrations of 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution compared to the reference. In other words, P. aeruginosa showed a higher sensitivity when threonine (0.1%) was added to the reaction mixture.

[0306] The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 8):

[0307] Isoleucine (1%): As a result, the absolute number of P. aeruginosa was reduced more strongly when the GOX+LPO enzyme solution was added at concentrations of 0.005% to 0.000156% (v / v) compared to the reference. In other words, P. aeruginosa showed a higher sensitivity when isoleucine (1%) was added to the reaction mixture.

[0308] Isoleucine (0.1%): As a result, the absolute number of P. aeruginosa was reduced more strongly at concentrations of 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution compared to the reference. In other words, P. aeruginosa showed a higher sensitivity when isoleucine (0.1%) was added to the reaction mixture.

[0309] The results showed that adding glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 9):

[0310] Glutamine (1%): As a result, the absolute number of P. aeruginosa was reduced more strongly when compared with the reference at concentrations of 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution. In other words, P. aeruginosa showed a higher susceptibility when glutamine (1%) was added to the reaction mixture.

[0311] Glutamine (0.1%): As a result, the absolute number of P. aeruginosa was reduced more strongly when compared with the reference at concentrations of 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution. In other words, P. aeruginosa showed a higher susceptibility when glutamine (0.1%) was added to the reaction mixture.

[0312] The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.1% (v / v) resulted in differences in the absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 10):

[0313] Asparagine (1%): As a result, the absolute number of P. aeruginosa was reduced more strongly at concentrations of 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution compared to the reference. In other words, P. aeruginosa showed a higher susceptibility when asparagine (1%) was added to the reaction mixture.

[0314] Asparagine (0.1%): As a result, the absolute number of P. aeruginosa was reduced more strongly at concentrations of 0.005% to 0.000625% (v / v) of the GOX+LPO enzyme solution compared to the reference. In other words, P. aeruginosa showed a higher susceptibility when asparagine (0.1%) was added to the reaction mixture.

[0315] 2.3.3. Candida albicans The results showed that the addition of serine and isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.00125% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Candida albicans (Figure 11):

[0316] Serine (0.01%): As a result, at concentrations of 0.005% to 0.0000391% (v / v) of GOX+LPO enzyme, the absolute Candida albicans count was reduced more strongly compared to the reference and guaiacol-added solutions. In other words, when serine (0.01%) was added to the reaction mixture, Candida albicans showed a higher sensitivity.

[0317] Isoleucine (0.01%): As a result, the absolute Candida albicans count was reduced more strongly at concentrations of 0.005% to 0.0000391% (v / v) of the GOX+LPO enzyme solution compared to the reference solution and to the solution with guaiacol added. In other words, Candida albicans showed a higher sensitivity when proline (0.01%) was added to the reaction mixture.

[0318] 2.3.4. Aspergillus niger The results showed that adding proline to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 12):

[0319] Proline (1% to 0.01%): As a result, the absolute Aspergillus niger count decreased more strongly when compared with the reference at concentrations of 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution. In other words, Aspergillus niger showed a higher sensitivity when proline (1% to 0.01%) was added to the reaction mixture.

[0320] The results showed that adding serine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in the absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 13):

[0321] Serine (1% to 0.01%): As a result, the absolute number of Aspergillus niger was reduced more strongly when the GOX+LPO enzyme solution was added at concentrations of 0.005% to 0.000156% (v / v) compared to the reference solution. In other words, when serine (1% to 0.01%) was added to the reaction mixture, Aspergillus niger showed a higher sensitivity.

[0322] The results showed that adding threonine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 14):

[0323] Threonine (1% to 0.01%): As a result, the absolute Aspergillus niger count decreased more strongly when compared with the reference solution at concentrations of 0.005% to 0.000156% (v / v) of the GOX+LPO enzyme solution. In other words, Aspergillus niger showed a higher sensitivity when threonine (1% to 0.01%) was added to the reaction mixture.

[0324] The results showed that adding glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.01% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 15):

[0325] Glutamine (1% to 0.01%): As a result, the absolute Aspergillus niger counts were reduced more strongly when compared to the reference GOX+LPO enzyme solution at concentrations of 0.005% to 0.000156% (v / v). In other words, Aspergillus niger showed a higher sensitivity when glutamine (1% to 0.01%) was added to the reaction mixture.

[0326] The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.05% (v / v) resulted in differences in absolute microbial counts when compared to those of the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 16):

[0327] Asparagine (1% to 0.05%): As a result, the absolute Aspergillus niger counts were reduced more strongly when the GOX+LPO enzyme solution was added at concentrations of 0.005% to 0.000156% (v / v) compared to the reference solution. In other words, Aspergillus niger showed a higher sensitivity when asparagine (1% to 0.05%) was added to the reaction mixture.

[0328] The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 1% to 0.005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 17):

[0329] Isoleucine (1% to 0.005%): As a result, the absolute Aspergillus niger count decreased more strongly when the GOX+LPO enzyme solution was added at concentrations of 0.005% to 0.000156% (v / v) compared to the reference solution. In other words, Aspergillus niger showed a higher sensitivity when isoleucine (1% to 0.005%) was added to the reaction mixture.

[0330] 2.4. Challenge testing in an alginate gel environment A microbial challenge test can be performed to measure the ability of an antimicrobial agent to elicit its antimicrobial preservative efficacy in a gel environment. Here, the antimicrobial agent is a combination of the natural enzymes GOX and LPO. The antimicrobial preservative activity of this enzyme combination GOX and LPO in a gel environment was investigated both by itself and when combined with a potentiator (a molecule that has no antimicrobial effect by itself but can enhance the antimicrobial effect of an antimicrobial agent). The microbial challenge test was performed according to the method described in the European Pharmacopoeia current version, chapter 5.1.3, "Efficacy of antimicrobial preservation," except that the time points were changed (0, 10, 20, 30, 40, 50, 60, 70, and 80 minutes instead of 0, 2, 7, 14, and 28 days). Briefly, for each enhancer to be evaluated, lab-scale arginogels containing 0.0075% GOX + LPO (1:1) were prepared according to standard practice (based on the Flaminal® formulation but without the original enhancer, guaiacol). The enhancer was added to the arginogel at final concentrations ranging from 0.01 to 0.00025% (v / w). As a reference, arginogels containing 0.0075% GOX + LPO (1:1) but without the enhancer were included (labeled "enzyme only"). As controls, arginogels lacking both the GOX + LPO (1:1) enzyme solution and the enhancer (labeled "empty gel") and arginogels containing only the enhancer (labeled "enhancer candidate only") were included. After preparation, 10 g of homogenized sample was transferred to a 50 ml conical tube for each arginogel to be evaluated and the microorganism to be tested. For each test microorganism, including Staphylococcus aureus (ATCC® 6538™), Pseudomonas aeruginosa (ATCC® 9027™), Candida albicans (ATCC® 10231™), and Aspergillus niger (ATCC® 16404™), a microbial suspension of approximately 10 CFU / ml in sterile diluent (sodium chloride-peptone buffer solution) was prepared.Next, 50 ml conical tubes containing the alginate gel samples to be evaluated were inoculated with 100 μl of the designated inoculum. At the start (time point 0) and after 0, 10, 20, 30, 40, 50, 60, 70, and 80 minutes, 0.5 g samples of the product were transferred to 15 ml conical tubes containing 4.5 ml of sterile diluent, and the number of viable microorganisms per gram of product (expressed as CFU / g) was determined using standard dilution and plate count techniques. This test was repeated at least one to three times for each combination evaluated. The collected data were further analyzed in MS Excel. The mean absolute counts (expressed as log CFU / g) were visualized graphically.

[0331] 2.4.1. Proline (Staphylococcus aureus) The results showed that adding proline to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.0005% (v / v) resulted in differences in antibacterial efficacy when compared to that of the reference (i.e., enzyme only), indicating differences in the susceptibility of S. aureus (Figure 18):

[0332] Proline (0.0025%): As a result, the absolute number of Staphylococcus aureus was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of the GOX+LPO enzyme solution. In other words, when proline (0.0025%) was added to the alginate gel, Staphylococcus aureus showed the highest sensitivity.

[0333] Proline (0.00125-0.0005%): As a result, the absolute number of Staphylococcus aureus was reduced more quickly and completely when compared to the reference solution at a concentration of 0.0075% (v / v). In other words, when proline (0.00125-0.0005%) was added to the alginate gel, Staphylococcus aureus showed a higher susceptibility.

[0334] Furthermore, the addition of proline at a final concentration of 0.0005% resulted in a more rapid and complete reduction of the absolute S. aureus count compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, S. aureus was more sensitive to the enzyme combination GOX+LPO and arginine gel containing proline than to that containing guaiacol.

[0335] Finally, when S. aureus was exposed to the blank gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that proline does not have an antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0336] 2.4.2. Proline (Pseudomonas aeruginosa) The results showed that the addition of proline to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 19):

[0337] Proline (0.0025%): As a result, the absolute number of P. aeruginosa was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of the GOX+LPO enzyme solution. In other words, P. aeruginosa was most susceptible when proline (0.0025%) was added to the alginate gel.

[0338] Proline (0.00125-0.0005%): As a result, the absolute number of P. aeruginosa was reduced more quickly and completely when compared to the reference solution at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when proline (0.00125-0.0005%) was added to the alginate gel, P. aeruginosa showed a higher susceptibility.

[0339] Furthermore, the addition of proline at a final concentration of 0.0005% resulted in a more rapid and complete reduction in the absolute P. aeruginosa count compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, P. aeruginosa was more sensitive to the enzyme combination GOX+LPO and arginine gel containing proline than to that containing guaiacol.

[0340] Finally, when P. aeruginosa was exposed to the empty gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that proline does not have an antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0341] 2.4.3. Proline (Candida albicans) The results showed that the addition of proline to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Candida albicans (Figure 20):

[0342] Proline (0.0025%): As a result, the absolute number of Candida albicans was immediately and completely reduced when compared with the reference concentration of 0.0075% (v / v) of the GOX+LPO enzyme solution. In other words, when proline (0.0025%) was added to the alginate gel, Candida albicans showed the highest sensitivity.

[0343] Proline (0.00125%): As a result, the absolute number of Candida albicans was reduced more quickly and completely when compared to the reference solution with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when proline (0.00125%) was added to the alginate gel, Candida albicans showed a higher sensitivity.

[0344] Furthermore, the addition of proline at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute C. albicans counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, C. albicans was more sensitive to the enzyme combination GOX+LPO and the alginate gel containing proline than to that containing guaiacol.

[0345] Finally, when Candida albicans was exposed to the blank gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that proline does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0346] 2.4.4. Proline (Aspergillus niger) The results showed that adding proline to the enzyme GOX+LPO combination at a final concentration of 0.0025% (v / v) resulted in a difference in the absolute microbial count when compared to that of the reference (without potentiator candidate), indicating a difference in the susceptibility of Aspergillus niger (Figure 21):

[0347] Proline (0.0025%): As a result, the absolute Aspergillus niger count was reduced more quickly and completely when compared to the reference solution with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when proline (0.0025%) was added to the alginate gel, Aspergillus niger showed a higher sensitivity.

[0348] Furthermore, the addition of proline at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute Aspergillus niger counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, Aspergillus niger was more sensitive to the enzyme combination GOX+LPO and the arginine gel containing proline than to that containing guaiacol.

[0349] Finally, when Aspergillus niger was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself has no antibacterial activity and that proline has no antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0350] 2.4.5. Serine (Staphylococcus aureus) The results showed that adding serine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in antibacterial efficacy when compared to that of the reference (i.e., enzyme only), indicating differences in the susceptibility of S. aureus (Figure 22):

[0351] Serine (0.0025%): As a result, the absolute number of Staphylococcus aureus was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when serine (0.0025%) was added to the alginate gel, Staphylococcus aureus showed the highest sensitivity.

[0352] Serine (0.00125-0.0005%): As a result, the absolute number of Staphylococcus aureus was reduced more quickly and completely when compared to the reference concentration of 0.0075% (v / v) GOX+LPO enzyme solution. In other words, when serine (0.00125-0.0005%) was added to alginate gel, Staphylococcus aureus showed a higher susceptibility.

[0353] Furthermore, the addition of serine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute S. aureus counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, S. aureus was more sensitive to the enzyme combination GOX+LPO and arginine gel containing serine than to that containing guaiacol.

[0354] Finally, when S. aureus was exposed to the empty gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that serine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0355] 2.4.6. Serine (Pseudomonas aeruginosa) The results showed that the addition of serine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in the absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 23):

[0356] Serine (0.0025%): As a result, the absolute number of P. aeruginosa was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when serine (0.0025%) was added to the alginate gel, P. aeruginosa showed the highest sensitivity.

[0357] Serine (0.00125-0.0005%): As a result, the absolute number of P. aeruginosa was reduced more quickly and completely when compared to the reference concentration of 0.0075% (v / v) GOX+LPO enzyme solution. In other words, when serine (0.00125-0.0005%) was added to the alginate gel, P. aeruginosa showed a higher susceptibility.

[0358] Furthermore, the addition of serine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in the absolute P. aeruginosa count compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, P. aeruginosa was more sensitive to the enzyme combination GOX+LPO and the arginine gel containing serine than to that containing guaiacol.

[0359] Finally, when P. aeruginosa was exposed to the empty gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that serine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0360] 2.4.7. Serine (Candida albicans) The results showed that the addition of serine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Candida albicans (Figure 24):

[0361] Serine (0.0025%): As a result, the absolute number of Candida albicans was immediately and completely reduced when compared with the reference concentration of 0.0075% (v / v) GOX+LPO enzyme solution. In other words, when serine (0.0025%) was added to the alginate gel, Candida albicans showed the highest sensitivity.

[0362] Serine (0.00125%): As a result, the absolute number of Candida albicans was reduced more quickly and completely when compared to the reference solution with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when serine (0.00125%) was added to the alginate gel, Candida albicans showed a higher sensitivity.

[0363] Furthermore, the addition of serine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute Candida albicans counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, P. aeruginosa was more susceptible to the enzyme combination GOX+LPO and arginine gel containing serine than to that containing guaiacol.

[0364] Finally, when Candida albicans was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity, and that serine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0365] 2.4.8. Serine (Aspergillus niger) The results showed that the addition of serine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in the absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 25):

[0366] Serine (0.0025-0.0005%): As a result, the absolute Aspergillus niger count was reduced more quickly and completely when compared to the reference solution containing 0.0075% (v / v) of GOX+LPO enzyme. In other words, when serine (0.0025-0.0005%) was added to the alginate gel, Aspergillus niger showed a higher sensitivity.

[0367] Furthermore, the addition of serine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute Aspergillus niger counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, Aspergillus niger was more sensitive to the enzyme combination GOX+LPO and the arginine gel containing serine than to that containing guaiacol.

[0368] Finally, when Aspergillus niger was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity, and that serine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0369] 2.4.9. Threonine (Staphylococcus aureus) The results showed that adding threonine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in antibacterial efficacy when compared to that of the reference (i.e., enzyme only), indicating differences in the susceptibility of Staphylococcus aureus (Figure 26):

[0370] Threonine (0.0025%): As a result, the absolute number of Staphylococcus aureus was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when threonine (0.0025%) was added to the alginate gel, Staphylococcus aureus showed the highest sensitivity.

[0371] Threonine (0.00125-0.0005%): As a result, the absolute number of Staphylococcus aureus was reduced more quickly and completely when compared to the reference concentration of 0.0075% (v / v) of the GOX+LPO enzyme solution. In other words, when threonine (0.00125-0.0005%) was added to the alginate gel, Staphylococcus aureus showed a higher susceptibility.

[0372] Furthermore, the addition of threonine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute S. aureus counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%), indicating that S. aureus was more sensitive to the enzyme combination GOX+LPO and arginine-containing gel than to that containing guaiacol.

[0373] Finally, when S. aureus was exposed to the empty gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that threonine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0374] 2.4.10. Threonine (Pseudomonas aeruginosa) The results showed that the addition of threonine to the enzyme GOX+LPO combination at a final concentration of 0.0025% to 0.0005% (v / v) resulted in a difference in the absolute microbial count when compared to the reference (without potentiator candidate), indicating a difference in the susceptibility of P. aeruginosa (Figure 27):

[0375] Threonine (0.0025%): As a result, the absolute number of P. aeruginosa was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, P. aeruginosa was most susceptible when threonine (0.0025%) was added to the alginate gel.

[0376] Threonine (0.00125-0.0005%): As a result, the absolute number of P. aeruginosa was reduced more quickly and completely when compared to the reference solution with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when threonine (0.00125-0.0005%) was added to the alginate gel, P. aeruginosa showed a higher susceptibility.

[0377] Furthermore, the addition of threonine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute P. aeruginosa counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%), indicating that P. aeruginosa was more sensitive to the enzyme combination GOX+LPO and arginine-containing gel than to that containing guaiacol.

[0378] Finally, when P. aeruginosa was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that threonine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0379] 2.4.11. Threonine (Candida albicans) The results showed that the addition of threonine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Candida albicans (Figure 28):

[0380] Threonine (0.0025%): As a result, the absolute number of Candida albicans was immediately and completely reduced when compared with the reference concentration of 0.0075% (v / v) GOX+LPO enzyme solution. In other words, when threonine (0.0025%) was added to the alginate gel, Candida albicans showed the highest sensitivity.

[0381] Threonine (0.00125-0.0005%): As a result, the absolute number of Candida albicans was reduced more quickly and completely when compared to the reference solution containing 0.0075% (v / v) of GOX+LPO enzyme. In other words, when threonine (0.00125-0.0005%) was added to the alginate gel, Candida albicans showed a higher sensitivity.

[0382] Furthermore, the addition of threonine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute C. albicans counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, C. albicans was more sensitive to the enzyme combination GOX+LPO and arginine-containing gel than to that containing guaiacol.

[0383] Finally, when Candida albicans was exposed to the blank gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that threonine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0384] 2.4.12. Threonine (Aspergillus niger) The results showed that adding threonine at a final concentration of 0.0025% (v / v) to the enzyme GOX+LPO combination resulted in a difference in absolute microbial counts when compared to the reference (without potentiator candidate), indicating a difference in the susceptibility of Aspergillus niger (Figure 29):

[0385] Threonine (0.0025%): As a result, the absolute Aspergillus niger count was reduced more quickly and completely when compared to the reference solution containing 0.0075% (v / v) of GOX+LPO enzyme. In other words, Aspergillus niger was more sensitive to the addition of 0.0025% threonine to the alginate gel.

[0386] Furthermore, the addition of threonine at a final concentration of 0.0025% resulted in a more rapid and complete reduction in absolute Aspergillus niger counts compared to the addition of guaiacol at the same concentration (i.e., 0.0025%). In other words, Aspergillus niger was more sensitive to the enzyme combination GOX+LPO and arginine-containing gel than to that containing guaiacol.

[0387] Finally, when Aspergillus niger was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that threonine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0388] 2.4.13. Glutamine (Staphylococcus aureus) The results showed that adding glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in antibacterial efficacy when compared to that of the reference (i.e., enzyme only), indicating differences in the susceptibility of Staphylococcus aureus (Figure 30):

[0389] Glutamine (0.0025%): As a result, the absolute number of Staphylococcus aureus was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when glutamine (0.0025%) was added to the alginate gel, Staphylococcus aureus showed the highest susceptibility.

[0390] Glutamine (0.00125-0.0005%): As a result, the absolute number of Staphylococcus aureus was reduced more quickly and completely when compared to the reference solution with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when glutamine (0.00125-0.0005%) was added to the alginate gel, Staphylococcus aureus showed a higher susceptibility.

[0391] Furthermore, the addition of glutamine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute S. aureus counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%), indicating that S. aureus was more susceptible to the enzyme combination GOX+LPO and arginine gel containing glutamine than to that containing guaiacol.

[0392] Finally, when S. aureus was exposed to the empty gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that glutamine has no antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0393] 2.4.14. Glutamine (Pseudomonas aeruginosa) The results showed that adding glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025% to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 31):

[0394] Glutamine (0.0025%): As a result, the absolute number of P. aeruginosa was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when glutamine (0.0025%) was added to the alginate gel, P. aeruginosa showed the highest susceptibility.

[0395] Glutamine (0.00125-0.0005%): As a result, the absolute number of P. aeruginosa was reduced more quickly and completely when compared to the reference solution with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when glutamine (0.00125-0.0005%) was added to the alginate gel, P. aeruginosa showed a higher susceptibility.

[0396] Furthermore, the addition of glutamine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute P. aeruginosa counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%), indicating that P. aeruginosa was more sensitive to the enzyme combination GOX+LPO and arginine gel containing glutamine than to that containing guaiacol.

[0397] Finally, when P. aeruginosa was exposed to the empty gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity, and that glutamine does not have an antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0398] 2.4.15. Glutamine (Candida albicans) The results showed that adding glutamine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Candida albicans (Figure 32):

[0399] Glutamine (0.0025%): As a result, the absolute number of Candida albicans was immediately and completely reduced when compared with the reference concentration of GOX+LPO enzyme solution at 0.0075% (v / v). In other words, when glutamine (0.0025%) was added to the alginate gel, Candida albicans showed the highest sensitivity.

[0400] Glutamine (0.00125-0.0005%): As a result, the absolute number of Candida albicans was reduced more quickly and completely when compared to the reference solution containing 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when glutamine (0.00125-0.0005%) was added to the alginate gel, Candida albicans showed a higher susceptibility.

[0401] Furthermore, the addition of glutamine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute C. albicans counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, C. albicans was more sensitive to the enzyme combination GOX+LPO and arginine gel containing glutamine than to that containing guaiacol.

[0402] Finally, when Candida albicans was exposed to the blank gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity, and that glutamine does not have an antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0403] 2.4.16. Glutamine (Aspergillus niger) The results showed that adding glutamine to the enzyme GOX+LPO combination at a final concentration of 0.0025% (v / v) resulted in a difference in the absolute microbial count when compared to the reference (without potentiator candidate), indicating a difference in the susceptibility of Aspergillus niger (Figure 33):

[0404] Glutamine (0.0025%): As a result, the absolute Aspergillus niger count was reduced more quickly and completely when compared to the reference solution containing 0.0075% (v / v) of GOX+LPO enzyme. In other words, when glutamine (0.0025%) was added to the alginate gel, Aspergillus niger showed a higher sensitivity.

[0405] Furthermore, the addition of glutamine at a final concentration of 0.0025% resulted in a more rapid and complete reduction in absolute Aspergillus niger counts compared to the addition of guaiacol at the same concentration (i.e., 0.0025%). In other words, Aspergillus niger was more sensitive to the enzyme combination GOX+LPO and the glutamine-containing alginate gel than to that containing guaiacol.

[0406] Finally, when Aspergillus niger was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity, and that glutamine does not have an antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0407] 2.4.17. Asparagine (Staphylococcus aureus) The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.00125% (v / v) resulted in differences in antibacterial efficacy when compared to that of the reference (i.e., enzyme only), indicating differences in the susceptibility of Staphylococcus aureus (Figure 34):

[0408] Asparagine (0.0025%): As a result, the absolute number of Staphylococcus aureus was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of the GOX+LPO enzyme solution. In other words, when asparagine (0.0025%) was added to the alginate gel, Staphylococcus aureus showed the highest susceptibility.

[0409] Asparagine (0.00125%): As a result, the absolute number of S. aureus was reduced more quickly and completely when compared to the reference with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, S. aureus showed a higher susceptibility when asparagine (0.00125%) was added to the alginate gel.

[0410] Finally, when S. aureus was exposed to the empty gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that asparagine does not have an antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0411] 2.4.18. Asparagine (Pseudomonas aeruginosa) The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.00125% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 35):

[0412] Asparagine (0.0025%): As a result, the absolute number of P. aeruginosa was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of the GOX+LPO enzyme solution. In other words, when asparagine (0.0025%) was added to the alginate gel, P. aeruginosa showed the highest sensitivity.

[0413] Asparagine (0.00125%): As a result, the absolute number of P. aeruginosa was reduced more quickly and completely when compared to the reference at a concentration of 0.0075% (v / v) of the GOX+LPO enzyme solution. In other words, when asparagine (0.00125%) was added to the alginate gel, P. aeruginosa showed a higher susceptibility.

[0414] Finally, when P. aeruginosa was exposed to the empty gel or the potentiating candidate alone (tested at the highest concentration), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that asparagine does not have an antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0415] 2.4.19. Asparagine (Candida albicans) The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.00125% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Candida albicans (Figure 36):

[0416] Asparagine (0.0025%): As a result, the absolute number of Candida albicans was immediately and completely reduced when compared with the reference concentration of 0.0075% (v / v) GOX+LPO enzyme solution. In other words, when asparagine (0.0025%) was added to the alginate gel, Candida albicans showed the highest sensitivity.

[0417] Asparagine (0.00125%): As a result, the absolute Candida albicans count was reduced more quickly and completely when compared to the reference solution with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when asparagine (0.00125%) was added to the alginate gel, Candida albicans showed a higher sensitivity.

[0418] Finally, when Candida albicans was exposed to the blank gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that asparagine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0419] 2.4.20. Asparagine (Aspergillus niger) The results showed that the addition of asparagine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 37):

[0420] Asparagine (0.0025-0.0005%): As a result, the absolute Aspergillus niger count was reduced more quickly and completely when compared to the reference solution containing 0.0075% (v / v) of GOX+LPO enzyme. In other words, Aspergillus niger was more sensitive to the addition of asparagine (0.0025-0.0005%) to the alginate gel.

[0421] Furthermore, the addition of asparagine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute Aspergillus niger counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, Aspergillus niger was more sensitive to the enzyme combination GOX+LPO and the asparagine-containing alginate gel than to that containing guaiacol.

[0422] Finally, when Aspergillus niger was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that asparagine does not have an antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0423] 2.4.21. Isoleucine (Staphylococcus aureus) The results showed that adding isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in antibacterial efficacy when compared to that of the reference (i.e., enzyme only), indicating differences in the susceptibility of S. aureus (Figure 38):

[0424] Isoleucine (0.0025%): As a result, the absolute number of Staphylococcus aureus was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when isoleucine (0.0025%) was added to the alginate gel, Staphylococcus aureus showed the highest sensitivity.

[0425] Isoleucine (0.00125-0.0005%): As a result, the absolute number of Staphylococcus aureus was reduced more quickly and completely when compared to the reference concentration of 0.0075% (v / v) of the GOX+LPO enzyme solution. In other words, when isoleucine (0.00125-0.0005%) was added to the alginate gel, Staphylococcus aureus showed a higher susceptibility.

[0426] Furthermore, the addition of isoleucine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute S. aureus counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, S. aureus was more sensitive to the enzyme combination GOX+LPO and arginine-containing gel than to the one containing guaiacol.

[0427] Finally, when S. aureus was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that isoleucine has no antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0428] 2.4.22. Isoleucine (Pseudomonas aeruginosa) The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of P. aeruginosa (Figure 39):

[0429] Isoleucine (0.0025%): As a result, the absolute number of P. aeruginosa was immediately and completely reduced when compared with the reference at a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, P. aeruginosa was most susceptible when isoleucine (0.0025%) was added to the alginate gel.

[0430] Isoleucine (0.00125-0.0005%): As a result, the absolute number of P. aeruginosa was reduced more quickly and completely when compared to the reference solution with a concentration of 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when isoleucine (0.00125-0.0005%) was added to the alginate gel, P. aeruginosa showed a higher susceptibility.

[0431] Furthermore, the addition of isoleucine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute P. aeruginosa counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%), indicating that P. aeruginosa was more sensitive to the enzyme combination GOX+LPO and arginine-containing gel than to the one containing guaiacol.

[0432] Finally, when P. aeruginosa was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that isoleucine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0433] 2.4.23. Isoleucine (Candida albicans) The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Candida albicans (Figure 40):

[0434] Isoleucine (0.0025%): As a result, the absolute number of Candida albicans was immediately and completely reduced when compared with the reference concentration of 0.0075% (v / v) GOX+LPO enzyme solution. In other words, Candida albicans was most sensitive when isoleucine (0.0025%) was added to the alginate gel.

[0435] Isoleucine (0.00125-0.0005%): As a result, the absolute Candida albicans count was reduced more quickly and completely when compared to the reference solution containing 0.0075% (v / v) of GOX+LPO enzyme solution. In other words, when isoleucine (0.00125-0.0005%) was added to the alginate gel, Candida albicans showed a higher sensitivity.

[0436] Furthermore, the addition of isoleucine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute Candida albicans counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, P. aeruginosa was more susceptible to the enzyme combination GOX+LPO and arginine gel containing isoleucine than to that containing guaiacol.

[0437] Finally, when Candida albicans was exposed to the blank gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity, and that isoleucine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0438] 2.4.24. Isoleucine (Aspergillus niger) The results showed that the addition of isoleucine to the enzyme GOX+LPO combination at final concentrations ranging from 0.0025 to 0.0005% (v / v) resulted in differences in absolute microbial counts when compared to the reference (without potentiator candidate), indicating differences in the susceptibility of Aspergillus niger (Figure 41):

[0439] Isoleucine (0.0025-0.0005%): As a result, the absolute Aspergillus niger count was reduced more quickly and completely when compared to the reference solution containing 0.0075% (v / v) of GOX+LPO enzyme. In other words, Aspergillus niger was more sensitive to the addition of isoleucine (0.0025-0.0005%) to the alginate gel.

[0440] Furthermore, the addition of isoleucine at a final concentration of 0.0005% resulted in a more rapid and complete reduction in absolute Aspergillus niger counts compared to the addition of guaiacol at the same concentration (i.e., 0.0005%). In other words, Aspergillus niger was more sensitive to the enzyme combination GOX+LPO and the arginine gel containing isoleucine than to the one containing guaiacol.

[0441] Finally, when Aspergillus niger was exposed to the empty gel or the potentiator alone (the highest concentration tested), there was no antibacterial activity, confirming that the arginine gel itself does not have any antibacterial activity and that isoleucine does not have any antibacterial effect by itself, but instead can potentiate the antibacterial effect of the GOX+LPO enzyme solution.

[0442] Example 3. Comparison of ethanolamine and proline The purpose of the experiment described here was to compare the efficacy of ethanolamine and proline in enhancing the antibacterial activity of the enzyme combination GOX+LPO (1:1) in a microbial challenge test using Staphylococcus aureus. The test was set up as described in the "Challenge Test" section of Example 2.4. The test was repeated at least three times.

[0443] The results showed that adding proline to a final concentration of 0.0005% (v / v) to arginine gel containing the enzyme combination of GOX and LPO (1:1) resulted in a difference in antibacterial efficacy against Staphylococcus aureus when compared with that of the reference (i.e., arginine gel containing only the enzyme combination of GOX and LPO) (Figure 42). Proline (0.0005%) resulted in a faster and complete reduction in the absolute number of Staphylococcus aureus (average 60 minutes) compared with that of the reference without the addition of the potentiating molecule (average 70 minutes). In other words, when proline (0.0005%) was added to the arginine gel, Staphylococcus aureus was more susceptible.

[0444] However, the addition of ethanolamine at a final concentration of 0.0005% (v / v) to argininogel containing the GOX+LPO (1:1) enzyme combination did not result in any difference in antibacterial efficacy, indicating no difference in the susceptibility of S. aureus, when compared with the reference (i.e., argininogel containing only the GOX+LPO enzyme combination) (Figure 42). Both required similar exposure times (average 70 minutes) to completely reduce the absolute S. aureus count. In other words, the susceptibility of S. aureus was comparable whether or not ethanolamine was added to the argininogel.

[0445] Based on the present experimental setup, it can be concluded that proline was found to be a more effective molecule for enhancing the antibacterial activity of the GOX+LPO enzyme combination than ethanolamine.

[0446] Example 4. Comparison of different halides The purpose of the experiment described here was to compare the efficacy of the antibacterial activity of the enzyme combination GOX + LPO (1:1) in the presence of potassium chloride / potassium bromide and potassium iodide. A microbial challenge test was performed using Staphylococcus aureus. The test was set up as described in the "Challenge Test" section of Example 2.4.

[0447] The results showed that the addition of KI to arginine gel containing the enzyme combination of GOX and LPO (1:1) at final concentrations of 0.03–0.003% (v / v) resulted in a difference in antibacterial efficacy against S. aureus when compared with the addition of KBr or KCl at concentrations of 0.03–0.003% (v / v) (Figure 43). Compared with the addition of KBr and KCl (0.03–0.003%), which did not result in complete kill within the 80-minute time frame, KI (0.03–0.003%) resulted in a more rapid and complete reduction in absolute S. aureus counts (70–80 min, respectively). In other words, S. aureus was more susceptible when KI (0.03–0.003%) was added to the arginine gel.

[0448] Addition of KI, KBr, and KCl alone (without enzymes) to a final concentration of 0.03% (v / v) to alginate gel containing the GOX+LPO (1:1) enzyme combination did not result in any difference in antibacterial efficacy over the 80-minute time frame when comparing the 0-minute and 80-minute time points, indicating no difference in the susceptibility of S. aureus (Figure 43). This further confirmed that the halides used were not antibacterial in themselves, but merely served as substrates for the peroxidase complex.

[0449] Based on the present experimental setup, it can be concluded that the antibacterial activity of the enzyme combination of GOX+LPO (1:1) was found to be more effective in the presence of KI than in the presence of KBr and KCl.

[0450] Example 5. Characterization of the anti-inflammatory effects of the composition To compare the inflammation-modulating activity of the GOX+LPO (1:1) enzyme combination with and without increasing concentrations of the potentiator molecule isoleucine, we investigated three pro-inflammatory mediators: IL-6 (an interleukin), TNFα (a non-interleukin cytokine), and MMP-9 (an enzyme). First, we evaluated the effect on the secretion of these mediators by macrophages. Second, we evaluated the direct neutralization of already secreted mediators. Both were measured by modern ELISA techniques.

[0451] First, the secretion of IL-6, TNFα, and MMP9, representative pro-inflammatory mediators, in cell culture supernatants was measured by THP-1 (a monocytic cell line) after their differentiation into macrophages. Briefly, to induce macrophage differentiation, THP-1 cells were plated at 3 × 10 cells / cm in 6-well plates in the presence of 160 nM phorbol-12-myristate-13-acetate (PMA). 2 Macrophages were seeded at a density of 1000 μg / ml. Differentiation was allowed to proceed for another 48 hours. Macrophages were then treated with the enzyme combination of GOX and LPO (1:1), their substrates (i.e., glucose and potassium iodide), and different concentrations (i.e., 0.0025%, 0.05%, and 0.5%) of the enhancer molecule isoleucine in serum-free medium for 24 hours at 37°C. The supernatant was collected, centrifuged at 150 g for 5 minutes to remove cell debris, and kept at -20°C before being processed for ELISA.

[0452] Second, direct neutralization of secreted pro-inflammatory mediators, IL-6, TNFα, and MMP9, was measured in parallel. Briefly, 48 hours after macrophage differentiation, cell culture supernatants were collected and centrifuged at 150 g for 5 minutes to remove cell debris. The cell culture supernatants were then treated with the enzyme combination of GOX and LPO (1:1), their substrates (i.e., glucose and potassium iodide), and 0.05% potentiator isoleucine for 3 hours at room temperature. The treated supernatants were kept frozen until processed for ELISA.

[0453] IL-6, TNFα, and MMP9 ELISA kits were purchased from BioTechne, and experiments were performed according to the manufacturer's instructions. Briefly, 96-well plates were coated with capture antibodies overnight at room temperature. After incubating the plates with blocking solution for 1 hour, the supernatant was added to the wells for 2 hours at room temperature. After several washing steps, the corresponding detection antibodies conjugated with biotin were incubated for 2 hours. Streptavidin conjugated to horseradish peroxidase (HRP) was added for 20 minutes, and HRP substrate solution was added for another 20 minutes. The reaction was stopped by adding sulfuric acid, and the absorbance at 450 nm was read using a plate reader. The absorbance was proportional to the concentration of the analyte present in the medium, which was calculated based on standard curves performed with recombinant IL-6, TNFα, and MMP9. The test was repeated at least once to gain insight into the preliminary results.

[0454] IL-6 mediator The results showed that the addition of the potentiating molecule isoleucine to the GOX+LPO (1:1) enzyme combination at final concentrations ranging from 0.5 to 0.0025% (v / v) resulted in further differences in IL-6 secretion by macrophages (Figure 44A). When compared to macrophages treated with the GOX+LPO (1:1) enzyme combination alone, further decreases of 7.6, 20.6, and 33.6% were observed when 0.0025, 0.05, and 0.5% isoleucine was added, respectively (Figure 44A).

[0455] The results further demonstrated that adding the potentiating molecule isoleucine at a final concentration of 0.05% (v / v) to the GOX + LPO (1:1) enzyme combination resulted in a further difference in the direct neutralization of previously secreted IL-6 (Figure 44B). Compared with the supernatant treated with the GOX + LPO (1:1) enzyme combination alone, a further decrease of 29.4% was observed when 0.05% isoleucine was added (Figure 44B).

[0456] Therefore, these results reveal that there is a difference in the IL-6 regulatory activity of the enzyme combination GOX+LPO (1:1) with or without increasing concentrations of the potentiator molecule isoleucine.

[0457] TNFα mediator For TNFα, the results showed that adding the potentiating molecule isoleucine to the GOX+LPO (1:1) enzyme combination at a final concentration of 0.05% (v / v) resulted in a further difference in the direct neutralization level of already secreted TNFα (Figure 45). Compared with supernatants treated with the GOX+LPO (1:1) enzyme combination alone, a further reduction of 42.5% was observed when 0.05% isoleucine was added (Figure 45).

[0458] Therefore, these results reveal that there is a difference in the TNFα-regulating activity of the enzyme combination GOX+LPO (1:1) with or without increasing concentrations of the potentiator molecule isoleucine.

[0459] MMP9 mediator The results showed that the addition of the potentiating molecule isoleucine to the GOX+LPO (1:1) enzyme combination at final concentrations ranging from 0.05 to 0.0025% (v / v) resulted in further differences in MMP9 secretion by macrophages (Figure 46A). Compared to macrophages treated with the GOX+LPO (1:1) enzyme combination alone, further decreases of 12.7 and 21.5% were observed when 0.0025 and 0.05% isoleucine was added, respectively (Figure 46A).

[0460] The results further demonstrated that adding the potentiating molecule isoleucine at a final concentration of 0.05% (v / v) to the GOX+LPO (1:1) enzyme combination resulted in a further difference in the direct neutralization of previously secreted MMP9 (Figure 46B). Compared with the supernatant treated with the GOX+LPO (1:1) enzyme combination alone, a further decrease of 27.1% was observed when 0.05% isoleucine was added (Figure 46B).

[0461] Thus, these results reveal differences in the MMP9-regulating activity of the enzyme combination GOX+LPO (1:1) with or without increasing concentrations of the potentiating molecule isoleucine.

[0462] Based on the present experimental setup, it can be concluded that the enzyme combination GOX + LPO (1:1) was found to be sensitive to the enhancement of regulatory activity by isoleucine against three representatives of pro-inflammatory mediators: IL-6 (interleukin), TNFα (non-interleukin cytokine), and MMP9 (enzyme). For IL-6 and MMP9, both inhibition of secretion by macrophages and direct neutralization of already secreted mediators were observed. For TNFα, a direct neutralization effect of already secreted mediators was observed.

[0463] Example 6. Effect of Enhancers on the Use of Eosinophil Peroxidase in Compositions In further experiments, the potency of isoleucine-enhanced antibacterial activity of GOX in combination with eosinophil peroxidase (EPO) was evaluated. To this end, the test was set up as described in the "Challenge Test" section above, except that EPO was used instead of LPO.

[0464] Briefly, microbial challenge testing was performed according to the method described in Chapter 5.1.3 "Efficacy of Antimicrobial Preservatives" of the current edition of the European Pharmacopoeia, but with modified time points (0, 10, 20, 30, 40, 50, 60, 70, and 80 minutes instead of 0, 2, 7, 14, and 28 days). Laboratory-scale argininogels (based on the Flaminal® formulation) containing 0.00001875% (w / w) GOX and 0.000005% (w / w) EPO (Creative enzymes NATE-0228) were prepared. Isoleucine was added to the argininogel at a final concentration of 0.0025% (w / w). As a reference, an argininogel containing 0.00001875% GOX + 0.000005% EPO without added isoleucine was included. As a negative control, an arginine gel (empty gel) without both the GOX+EPO enzyme combination and the potentiator candidate was included. After preparation, for each arginine gel to be evaluated, 10 g of homogenized sample was transferred to a 50 ml conical tube. For the test microorganism, i.e., Staphylococcus aureus (ATCC® 6538™), approximately 10 8 A microbial suspension of CFU / ml was prepared in sterile diluent (0.9% (w / v) NaCl). Next, 100 μl of the designated inoculum was inoculated into a 50 ml conical tube containing the alginate gel sample to be evaluated and incubated at 25°C for 80 minutes. At the start (time point 0) and after 0, 10, 20, 30, 40, 50, 60, 70, and 80 minutes, 0.5 g of the product sample was transferred to a 15 ml conical tube containing 4.5 ml of sterile diluent, and the number of viable microorganisms per gram of product (expressed as CFU / g) was determined using standard dilution and plate counting techniques. For each combination evaluated, the absolute counts (expressed as log(CFU / g)) at each time point were visualized graphically. The test was repeated once to gain insight into the preliminary results.

[0465] The results showed that adding isoleucine to an arginine gel containing the GOX + EPO enzyme combination at a final concentration of 0.0025% (w / w) did not result in any difference in antibacterial efficacy against Staphylococcus aureus when compared with that of the reference (i.e., arginine gel containing only the GOX + EPO enzyme combination) (Figure 47). Isoleucine (0.0025%) did not result in a faster or more complete reduction in absolute Staphylococcus aureus counts (80 minutes) when compared with the reference arginine gel without the addition of the enhancing molecule (80 minutes). In other words, isoleucine does not enhance the antibacterial activity of GOX + EPO.

Claims

1. A pharmaceutical and / or cosmetic composition comprising: ・Peroxide generation system; Lactoperoxidase; - halides or pseudohalides; and an enhancer; wherein the enhancer has a free amino functional group (-NH ３ +) and a free carboxylic acid functional group (-CO ２ H) and (i) -OH group; (ii) -(CH ２ ) ｎ an OH group, where n is 1, 2, or 3; (iii) -(CH ２ ) ｎ CONH ２ group, wherein n is 1, 2, or 3; (iv) - cyclic C ４ H ９ an N group; or (v) branched alkyl group and a side chain comprising:

2. 2. The composition of claim 1, wherein the enhancer is a hydrophilic amino acid, amino acid mimic, or amino acid derivative having an octanol / water partition coefficient of about -1 to about -5.

3. 3. The composition of claim 1 or 2, wherein the enhancer is a hydrophilic natural or unnatural amino acid.

4. 10. The composition of any one of the preceding claims, wherein the enhancer is a naturally occurring amino acid having an octanol / water partition coefficient of -1.72 to -3.82, preferably wherein the enhancer is a naturally occurring amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, isoleucine, proline, or any combination thereof.

5. 10. A composition according to any one of the preceding claims, wherein the halide or pseudohalide is a water-soluble iodide salt.

6. 10. A composition according to any one of the preceding claims, wherein the potentiator is present in the composition in a concentration from 0.0001 mg / ml to 500 mg / ml, preferably from 0.005 mg / ml to 100 mg / ml, most preferably from 0.1 mg / ml to 100 mg / ml.

7. 10. A composition according to any preceding claim, wherein the peroxide generating system comprises an oxidase, preferably the peroxide generating system is glucose oxidase.

8. 10. The composition of any one of the preceding claims, wherein the lactoperoxidase comprises an amino acid sequence having at least 90%, preferably 95%, more preferably 100% sequence identity to SEQ ID NO:

1.

9. The composition comprises: about 150 to about 4000 U / kg, preferably about 200 to about 3000 U / kg, more preferably about 300 to about 2500 U / kg of a peroxide generating system; - about 10 to about 100,000 U / kg, preferably about 10 to 4,000 U / kg, more preferably about 10 to 100 U / kg of lactoperoxidase; about 0.01 to about 500 mg / kg, preferably about 0.1 to about 200 mg / kg, more preferably about 1 mg to about 100 mg / kg, even more preferably about 2 mg to about 75 mg / kg, and most preferably 5 to 50 mg / kg of a halide or pseudohalide; and 0.0005 mg / ml to 200 mg / ml, preferably 0.005 mg / ml to 10 mg / ml, most preferably 0.05 to 1 mg / ml of an enhancer The composition according to any one of claims 1 to 8, comprising:

10. 10. A composition according to any one of the preceding claims for use as a medicine.

11. The composition according to any one of claims 1 to 9 for use in treating or preventing skin disorders.

12. A composition according to any one of claims 1 to 9 for use in wound healing.

13. A composition according to any one of claims 1 to 9 for use as an antibacterial composition.

14. A composition according to any one of claims 1 to 9 for use as an anti-inflammatory composition.

15. Use of a composition according to any one of claims 1 to 9 for the manufacture of a medicament.

16. 16. Use according to claim 15 for the manufacture of a medicament for the treatment or prevention of skin disorders.

17. 16. Use according to claim 15 for the manufacture of a medicament for wound healing.

18. 16. The use according to claim 15 for the manufacture of an antibacterial drug.

19. 16. The use according to claim 15 for the preparation of an anti-inflammatory drug.

20. A method of treating or preventing a skin disorder in a subject, comprising administering to said subject a composition according to any one of claims 1 to 9.

21. A method of healing one or more wounds in a subject, comprising administering to said subject a composition according to any one of claims 1 to 9.

22. A method of treating or preventing a microbial infection in a subject, comprising administering to said subject a composition according to any one of claims 1 to 9.

23. 10. A method of treating or preventing inflammation in a subject, comprising administering to said subject a composition according to any one of claims 1 to 9.

24. Use of an enhancer to improve the minimum inhibitory concentration (MIC) and / or minimum bactericidal concentration (MBC) and / or absolute number and / or enzyme activity of a pharmaceutical composition comprising a peroxide generating system, lactoperoxidase, and a halide or pseudohalide, wherein the enhancer is a compound having a free amino functional group (-NH ３ +) and a free carboxylic acid functional group (-CO ２ H) and (i) -OH group; (ii) -(CH ２ ) ｎ an OH group, where n is 1, 2, or 3; (iii) -(CH ２ ) ｎ CONH ２ group, wherein n is 1, 2, or 3; (iv) - cyclic C ４ H ９ an N group; or (v) branched alkyl group and a side chain comprising:

25. 25. The use according to claim 24, wherein the use is an in vitro use.

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