Composition

Abstract

The present invention provides an antimicrobial composition comprising: (i) one or more water-soluble organosilanes at a concentration of about 0.01% to about 0.4% w / v; (ii) one or more quaternary ammonium compounds at a concentration of about 0.01% to about 0.5% w / v; and (iii) one or more nonionic or amphoteric or sarcosine anionic surfactants at a concentration of about 0.05% to about 1% w / v. Also provided are methods of making the composition, and methods of using the composition to treat and / or prevent skin and / or wound infections.

Description

[Technical field]

[0001] The present invention relates to antimicrobial compositions, uses thereof and methods for treating or preventing skin and / or wound infections. [Background technology]

[0002] Bacterial resistance is a global problem in which some organisms (e.g., Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus) have developed resistance to multiple different antibiotics. According to the Centers for Disease Control and Prevention (CDC), in 2013, there were more than 2 million infections caused by antibiotic-resistant microorganisms, and subsequently, more than 23,000 deaths from these infections. Antibiotic resistance to antibiotics continues to increase, so their use should be reserved for cases where no effective alternatives are available, such as localized infections or widespread systemic infections. In the treatment of open wounds, continued debridement and the use of topical antimicrobial agents (disinfectants) have been shown to be more effective than antibiotics. Disinfectants, unlike antibiotics, are effective through multiple mechanisms of action, making the development of antiseptic resistance less likely.

[0003] However, even with antiseptics, infection is the single most likely cause of delayed healing in open wounds, especially chronic open wounds. If left untreated, contamination can progress to colonization and local infection, systemic infection, sepsis, and multiple organ dysfunction syndrome, which can be life-threatening. Infection of chronic wounds can be further complicated by the presence of biofilms. Biofilms form from any synthetic trophic consortium of microorganisms in which cells adhere to each other and often to surfaces. These adherent cells are embedded in a slimy extracellular matrix composed of extracellular polymeric substances (EPS). Cells within a biofilm produce EPS components, which are usually macromolecular assemblies of exopolysaccharides, proteins, lipids, and DNA.

[0004] Although there is no diagnosis for the presence of biofilm, it causes excessive inflammation and immune complex and complement activation, leading to delayed healing. Therefore, biofilm control is an important part of chronic wound management, but currently there are relatively few antiseptics that can effectively treat wound biofilms. Furthermore, this problem is complicated by the requirement that antiseptics have little or no cytotoxicity so as not to harm the patient's own cells and tissues. This can be quantified using the therapeutic index, which is the ratio between the safety and efficacy of a drug. Since efficacy can be accompanied by high levels of cytotoxicity, it is important to get this balance right when selecting a treatment for bacteria and biofilms.

[0005] Biofilms have been shown to increase resistance to many antibacterial agents and antibiotics (Non-Patent Document 1). Therefore, it is unlikely that antibacterial agents that are effective against planktonic bacteria will also be effective against biofilms. For this reason, there is a need for antibacterial agents specially formulated for biofilms. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Salisbury AM,Woo K,Sarkar S,Schultz G,Malone M,Mayer DO,Percival SL.Tolerance of Biofilms to Antimicrobials and Significance to Antibiotic Resistance in Wounds.Surg Technol Int.2018 Nov 11;33:59-66.PMID:30326137 Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need for new antimicrobial compositions that are effective against a wide variety of microorganisms, particularly antibiotic-resistant bacteria and bacteria including biofilms, and that also have low cytotoxicity. [Means for solving the problem]

[0008] The present invention provides an antimicrobial composition comprising: (i) one or more water-soluble organosilanes at a concentration of about 0.01% to about 0.4% w / v or about 0.5% w / v; (ii) one or more quaternary ammonium compounds at a concentration of about 0.01% to about 0.5% w / v; and (iii) one or more nonionic or amphoteric or sarcosine anionic surfactants at a concentration of about 0.05% to about 1% w / v. The compositions of the present invention have been demonstrated to have highly effective antimicrobial activity, the ability to inhibit and disrupt established biofilms, and low cytotoxicity.

[0009] The antimicrobial composition can be added to a wound cleansing solution and used to clean all types of wounds. Chronic skin wounds are often covered with slough tissue, necrotic tissue and / or microbial biofilms. The coatings are difficult to remove, resulting in delayed wound healing. The composition of the present invention can remove these barriers to wound healing through its antimicrobial and cleansing activity. Chronic wounds are often infected with microorganisms and contaminants that can delay healing, prolonging this complex process. The components of the composition of the present invention provide a tailored formulation containing antimicrobials, quaternary ammonium compounds and surfactants that work in combination to eliminate and contribute to the prevention of biofilm formation. The antimicrobial properties of the composition are broad-spectrum and have been tested against gram-negative and positive wound microorganisms and multi-species biofilms, and are also effective against fungi such as Candida albicans. The invention will now be described in detail, by way of example only, with reference to the drawings in which: [Brief description of the drawings]

[0010] [Figure 1]FIG. 1 - (A) MBEC (24 hr) of Pseudomonas aeruginosa ATCC 15442 when treated with either Hybrisan Technology or Prontosan® at 1 and 5 minute contact times and (B) MBEC (24 hr) of Staphylococcus aureus ATCC 6538 when treated with either Hybrisan Technology or Prontosan® at 1 and 5 minute contact times. Error bars represent standard deviation and * represents a significant log reduction (p<0.05) compared to control or between time points (N=3). [Diagram 2] Graph of established static (24 hour) biofilms of Pseudomonas aeruginosa ATCC 15442 when treated with either Hybrisan Technology or Prontosan® at 1 minute and 5 minute contact times. Error bars represent standard deviation and * represents significant log reduction (p<0.05) compared to control or between time points (N=3). [Diagram 3] FIG. 1 is a graph of the effect of either Hybrisan Technology or Prontosan® after treatment (5 min) on the regrowth of static (24 hr) biofilms of Pseudomonas aeruginosa ATCC 15442 when treated with either Hybrisan Technology or Prontosan® (N=3). [Figure 4]Graphs for 24 hour biofilms when treated with Hybrisan Technology or Prontosan®: (A) Pseudomonas aeruginosa ATCC 15442 grown in a CDC bioreactor model (ASTM E2871-13) at 1, 5 and 10 minute contact times; (B) Staphylococcus aureus ATCC 29213 grown in a CDC bioreactor model (ASTM E2871-13) at 1, 5 and 10 minute contact times; (C) Candida albicans ATCC 10231 grown in a CDC bioreactor model (ASTM E2871-13) at 1, 5 and 10 minute contact times; and (D) Pseudomonas aeruginosa ATCC 15442 grown in a drip flow reactor (ASTM E2647-13) at 5 minute contact times; (E) Staphylococcus aureus ATCC 29213 grown in a drip flow reactor (ASTM E2647-13) at 5 minute contact times. [Diagram 5] FIG. 1 is a graph showing the % viability of mouse fibroblast L929 cells when treated with Hybrisan technology and Prontosan® for 5 minutes, 10 minutes and 24 hours when tested according to ISO 10993:5-2009. [Figure 6] Graphs of (A) the MBEC (24 hr) of Pseudomonas aeruginosa ATCC 15442 when treated with either v9 or Prontosan® at 1 and 5 minute contact times and (B) the MBEC (24 hr) of Staphylococcus aureus ATCC 6538 when treated with either v9 or Prontosan® at 1 and 5 minute contact times. Error bars represent standard deviation and * represents a significant log reduction (p<0.05) compared to control or between time points (N=3). [Figure 7] Graph of established static (24 hour) biofilms of Pseudomonas aeruginosa ATCC 15442 when treated with either v9 or Prontosan® at 1 minute and 5 minute contact times. Error bars represent standard deviation and * represents significant log reduction (p<0.05) compared to control or between time points (N=3). [Figure 8]FIG. 1 is a graph of the effect of either v9 or Prontosan® after treatment (5 min) on the regrowth of static (24 hr) biofilms of Pseudomonas aeruginosa ATCC 15442 when treated with either Hybridan Technology or Prontosan® (N=3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention provides antimicrobial compositions comprising: (i) one or more water soluble organosilanes at a concentration of about 0.01% to about 0.4% w / v or about 0.5% w / v; (ii) one or more quaternary ammonium compounds at a concentration of about 0.01% to 0.5% w / v; and (iii) one or more nonionic or amphoteric or sarcosine anionic surfactants at a concentration of about 0.05% to about 1% w / v.

[0012] Without being bound by theory, it is believed that the mode of antimicrobial action of each component acts in combination through several mechanisms. It is known that the binding of organosilanes and quaternary ammonium products to cell membranes results in leakage of intracellular components. In particular, organosilanes are known to act only by physical killing mechanisms and do not promote the development of drug-resistant microorganisms. In addition to their physical mechanisms, quaternary ammonium compounds are known to inactivate energy-generating enzymes, denature essential cellular proteins, induce autolytic enzyme activity, and degrade RNA material. Polymeric biguanides are also known to disrupt microbial cell membranes, but also disrupt cellular metabolism, interfere with cellular functions, and can bind to DNA and cause chromosome condensation. These mechanisms are complementary when combined, and it is believed that the bactericidal concentrations of quaternary ammonium compounds and polymeric biguanides are reduced as a result of interference with cell membranes that enhances entry into microbial cells, and the other discussed mechanisms lead to microbial death.

[0013] Organosilanes are known to be hygroscopic (i.e., absorb moisture) and react rapidly when in contact with water, which reduces the efficacy of the organosilanes and clouds the clear compositions. This significantly reduces the biofilm efficacy of the compositions. The inventors have found that nonionic and / or cationic surfactants can stabilize the organosilanes while maintaining the clarity of the compositions for extended periods of time. However, in efficacy testing, the inventors have found that the addition of such surfactants can have significant adverse effects, in some cases completely inhibiting antimicrobial efficacy. Thus, the inventors have determined the minimum concentration range of surfactants that maintains the stability of the compositions while maintaining biofilm efficacy. Based on this work, the inventors have been able to identify concentration ranges for many effective and stable compositions.

[0014] The presence of surfactants and chelating agents enhances efficacy against biofilms through lowering surface tension and binding of metal ions that hold the EPS together. In combination, they can disrupt the EPS and allow the antimicrobial components to penetrate the biofilm and kill microbial cells. This activity also helps to avoid the need for high concentrations of antimicrobial components, thus avoiding cytotoxicity issues. As mentioned above, surfactants also help to stabilize the composition without affecting the efficacy of the antimicrobial agent.

[0015] Hydrolyzed organosilanes, when used in combination with other ingredients, are known to undergo condensation reactions to form water-insoluble products, which reduces their antimicrobial activity and turns clear solutions cloudy. This can be overcome by carefully selecting surfactants, including quaternary ammonium compounds, to stabilize the organosilanes over a wide pH range. Furthermore, it is also known that the inclusion of chelating agents, especially EDTA, is pH-dependent and can interact with surfactants to cause precipitation. Careful selection of EDTA salts and combinations of ingredients can help overcome this.

[0016] The antimicrobial compositions of the present invention can kill and / or inhibit the growth of microorganisms including bacteria, fungi, algae, protozoa, viruses and subviral agents. The compositions may be bactericidal or bacteriostatic and may be bactericidal or disinfectant. The antimicrobial compositions of the present invention may be antibacterial, antifungal or antiparasitic. The antimicrobial compositions of the present invention are preferably antibiofilm.

[0017] Water-soluble organosilanes have silicon-bonded hydrolyzable groups, such as alkoxysilanes, that allow them to be covalently bonded to substrates that contain hydroxyl or other silicon-reactive groups. Thus, organosilanes are often used as coupling agents to improve the bonding of fillers to resins, for example, in the manufacture of fiberglass. Organosilanes may also be used as antimicrobial additives for surfaces and textiles, and are widely formulated into antimicrobial coatings. Lower concentrations of organosilane are desirable because they reduce the concentration of non-ionic or amphoteric surfactants required and also reduce cytotoxicity.Non-ionic and amphoteric surfactants are known to "mask" the antimicrobial effect of quaternary ammonium compounds, so minimal concentrations ensure high efficacy at low concentrations. The water soluble organosilanes have the following general formula: A 3-x B x SiD [chemical formula 1] (In the formula, A is -OH or a hydrolyzable group, e.g., a halide such as -Cl, -Br and I, an alkoxy or an alkoxy ether, e.g., a group of the formula -OR 1 and OR 2A OR 1 (In the formula, R 1 are R 2 or hydrogen, R 2 is an alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, butyl, or -CH 2 CH 2 CH 2 (CH 3 ), preferably methyl, R 2Ais a divalent saturated hydrocarbon radical having 1 to 4 carbon atoms, such as methylene, ethylene, propylene, butyl or -CH 2 CH 2 CH(CH 3 ), with ethylene and propylene being preferred; amino, for example -N(R 1 ) 2 , e.g. -NHCH 3 , -N(CH 3 ) 2 and NCCH 2 CH 2 ) 2 , where the organosilazanes are those in which two organosilanes are linked by an -NH- unit; acetoxy, i.e., -OOCCH 3 ;Acetamide, i.e. -HNOCCH 3 and hydride, i.e. -H; B is an alkyl group having 1 to 4 carbon atoms, preferably methyl. x is 0, 1 or 2; D may be a hydrocarbon group having 1 to 4 carbon atoms, phenyl, or a non-ionic or cationic substituted hydrocarbon group containing at least one oxygen or nitrogen group, or a salt of such a substituted hydrocarbon group. Preferably, the water-soluble organosilane is a quaternary ammonium silane. Quaternary ammonium silanes are known to have low cytotoxicity and broad spectrum antimicrobial activity. Quaternary ammonium silanes have functional terminal -OH groups on their surface, which can be surface-modified (by the use of an acid) to activate the -OH groups. The antimicrobial activity of quaternary ammonium silanes occurs by penetrating and / or binding to bacterial cell walls and membranes, resulting in autolysis, a mechanism known as "contact killing". 18 H 37 Derived from lipophilic alkyl chains, these compounds have demonstrated efficacy in reducing bacterial growth in a wide range of applications including textiles, medical devices, and dental materials. The quaternary ammonium silane may be one or more of dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride and 3-(trihydroxysilyl)propyldimethyloctadecylammonium chloride. Preferably, the quaternary ammonium silane is dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride.

[0018] The antimicrobial composition comprises one or more water-soluble organosilanes at a concentration of about 0.01% to about 0.4% w / v or about 0.5% w / v, preferably about 0.05% to about 0.3% w / v or about 0.4% w / v. The composition may comprise one or more water-soluble organosilanes at a concentration of about 0.1% w / v. The antimicrobial composition further comprises one or more quaternary ammonium compounds. The compounds are preferably water-soluble and / or organic. Preferably, the quaternary ammonium compounds are silicon-free and may have at least one nitrogen-bonded hydrocarbon group with at least 8 carbon atoms. Suitable quaternary ammonium compounds may be selected from one or more of benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDAC), benzethonium chloride, tetradonium bromide, cetrimonium bromide, raultrimonium bromide, cetalkonium chloride, and cetrimonium chloride. The antimicrobial composition comprises one or more quaternary ammonium compounds at a concentration of about 0.01% to 0.5% w / v, preferably about 0.05% to about 0.4% w / v. The composition may comprise one or more quaternary ammonium compounds at a concentration of about 0.2% w / v.

[0019] The antimicrobial composition further comprises one or more nonionic or amphoteric or sarcosine anionic surfactants. Examples of nonionic surfactants include C 8 ~C 18 Alcohol ethoxylates; sorbitan laurate, oleate, stearate, palmitate, anhydrous sorbitan, or polyethoxylated sorbitan C 8 ~C 18Aliphatic esters; PEG-5 cocoate, PEG-15 cocoate, PEG-4 dilaurate, PEG-32 dilaurate, PEG-3 cocamide, PEG-6 cocamide, PEG-11 cocamide, PEG-20 dioleate, PEG-6 isopalmitate, PEG-12 isostearate, PEG-3 lauramide, PEG-8 laurate, PEG-32 laurate, PEG-4 octanoate, PEG-7 oleate, PEG-2 oleate, PEG-14 oleate, PEG-20 palmitate, PEG-14 stearate, PEG-5 tallowamide, etc. 8 ~C 18 Fatty acid esters and amides; C such as capryl alcohol, lauryl alcohol, cetylamide, and stearylamide 8 ~C 18 Fatty esters; C such as caprylic alcohol, lauryl alcohol, cetyl alcohol, and stearyl amide 8 ~C 18 Aliphatic alcohols; tetramethyldecynediol, dimethyloctynediol, etc. 8 ~C 18 Diols; block copolymers of polyethylene oxide and polypropylene oxide; glycerin such as glyceryl caprate, glyceryl isostearate, glyceryl laurate, glyceryl myristate, and glyceryl oleate. 8 ~C 18 Fatty acid esters; ethoxylated and propoxylated C such as ethoxylated and propoxylated lauryl alcohol 8 ~C 18 Fatty alcohols; C such as isododecylamine oxide, cocamine oxide, cocamidopropylamine oxide, myristamine oxide, myristamidopropylamine oxide, palmitamine oxide and stearamine oxide 8 ~C 18 Fatty amines and amidoamine oxides; Cocamide, Cocamide DEA, Cocamide MEA, Stearamide, Stearamide DEA, Stearamide MEA and Stearamide MIPA, etc. 8 ~C 18 Fatty amides and alkanolamides and C 8 ~C 18Fatty alcohol ethoxylates include tetramethyldecyne diol, ethoxylated and propoxylated lauryl alcohol.

[0020] Examples of amphoteric surfactants include C sultaine and cocamidopropyl hydroxysultaine. 8 ~C 18 Sultaine; C of amino acids such as cocoamphocarboxyglycinate and lauram-phoglycinate 8 ~C 18 Fatty derivatives and more preferred C betaines such as decyl betaine, coco betaine, lauryl betaine, myristyl betaine and stearyl betaine. 8 ~C 18 Alkyl betaines; and C such as cocoamidoethyl betaine, cocamidopropyl betaine, lauramidopropyl betaine, myristamidopropyl betaine, oleamidopropyl betaine, and stearamidopropyl betaine. 8 ~C 18 Amidoalkyl betaines are examples.

[0021] Examples of sarcosine anionic surfactants include C 8 ~C 18 Alkyl sarcosines and their alkali metal or ammonium salts, such as cocoyl sarcosine, lauroyl sarcosine, sodium lauroyl sarcosine, potassium lauroyl sarcosine, lithium lauroyl sarcosine, ammonium lauroyl sarcosine, sodium cocoyl sarcosine, and potassium cocoyl sarcosine, 8 ~C 18 Examples of the alkyl sarcosine include sodium, potassium, lithium, and ammonium. 8 ~C 18 When alkylsarcosines are used, preferably at least a portion of the acidic carboxyl groups are neutralized, for example with sodium hydroxide, to render the surfactant water-dispersible. Particularly preferred nonionic or amphoteric or sarcosine anionic surfactants include one or more of cocomidopropyl betaine, polyethylene glycol lauryl ether (e.g., Brij 35), Poloxamer 188, polysorbate 80, PEG-7 glyceryl cocoate, PEG-7 oleamide, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., Triton X-100), polysorbate 20, poloxamer 407, cocomidopropylamine oxide, and lauramidopropyl betaine.

[0022] The antimicrobial composition comprises one or more non-ionic or amphoteric or sarcosine anionic surfactants at a concentration of about 0.05% to about 2% w / v, preferably about 0.1% to about 1% w / v. The composition may comprise one or more non-ionic or amphoteric or sarcosine anionic surfactants at a concentration of about 0.4% w / v.

[0023] Optionally, the antimicrobial composition further comprises a chelating agent which may be present at a concentration of about 0.01% to about 0.2% w / v, preferably about 0.01% to about 0.1% w / v. The composition may comprise about 0.05% w / v of the chelating agent. The chelating agent may be selected from one or more of disodium EDTA, trisodium EDTA, and tetrasodium EDTA.

[0024] Optionally, the antimicrobial composition further comprises a polymeric biguanide. Suitable polymeric biguanides include one or more of chlorhexidine and polyhexamethylene biguanide.

[0025] Preferably, the antibacterial composition has a pH of about 4.5 to about 8.5, or about 5 to about 7, or about 5.5 to about 6.5. The antibacterial composition may have a pH of about 5.5 or about 6.5. The pH of the composition can be adjusted to a range of about 4.5 to about 8.5 using a suitable organic or inorganic acid such as citric acid, acetic acid, hydrochloric acid, phosphoric acid, sorbic acid, etc., or an organic or inorganic base such as ammonium hydroxide, sodium hydroxide, potassium hydroxide, ethylamine, dimethylamine, triethylamine, ethanolamine, diethanolamine, and triethanolamine. Preferably, the antimicrobial composition is lipid-free, more preferably, the formulated antimicrobial composition is phospholipid-free.

[0026] Examples of antimicrobial compositions of the present invention include: quaternary ammonium silanes, such as dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride, at a concentration of about 0.01% to about 0.4% or about 0.5% w / v; one or more quaternary ammonium compounds, such as benzalkonium chloride and / or didecyldimethylammonium chloride, at a concentration of about 0.01% to about 0.4% or about 0.5% w / v; optionally biguanides, such as chlorhexidine or polyhexanide; A chelating agent such as disodium EDTA or tetrasodium EDTA at a concentration of about 0.01% to 0.2% w / v; and a nonionic or amphoteric surfactant such as cocomidopropyl betaine, Brij 35, Pluronic® F68, Tween 80, etc., at a concentration of about 0.05% to about 1% w / v; and having a pH of about 4.5 to about 8.5.

[0027] Approximately 0.1% w / v dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride; Approximately 0.1% w / v benzalkonium chloride; Approximately 0.1% w / v didecyldimethylammonium chloride; Approximately 0.1% w / v chlorohexidan digluconate; about 0.18% or about 0.36% or about 0.72% or about 1.08% w / v cocomidopropyl betaine; and Approximately 0.05% tetrasodium EDTA; and a pH of about 5 to about 7.

[0028] Approximately 0.1% w / v dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride; Approximately 0.1% w / v benzalkonium chloride; About 0.1% w / v didecyldimethylammonium chloride; and Approximately 0.4% w / v Cocomidopropyl Betaine 1. An antimicrobial composition comprising:

[0029] Approximately 0.1% w / v dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride; Approximately 0.1% w / v benzalkonium chloride; Approximately 0.1% w / v didecyldimethylammonium chloride; About 0.05% w / v disodium EDTA; and Approximately 0.4% w / v Cocomidopropyl Betaine 1. An antimicrobial composition comprising:

[0030] The antimicrobial composition may further comprise one or more pharma- ceutically acceptable carriers and / or excipients, such as diluents, adjuvants, excipients, vehicles, fillers, binders, disintegrants, wetting agents, emulsifiers, suspending agents, flavoring agents, buffers, dispersants, thickeners, solubilizers, lubricants and dispersing agents, depending on the mode of administration and the nature of the dosage form. The antimicrobial composition is preferably in the form of a liquid preparation, such as a gel, lotion, spray or / and solution. Preferably, the composition is in the form of a solution. The composition is preferably formulated for topical administration, such as in the form of a wound irrigation solution. The antimicrobial composition may be used in therapy to treat and / or prevent skin and / or wound infections. The infection may be a bacterial infection which may involve a biofilm. The antimicrobial composition may be an antimicrobial composition, an antifungal composition, an antiparasitic composition or an antibiofilm composition, which may be used to treat or prevent a bacterial infection, such as a gram-negative or gram-positive bacterial infection, a fungal infection, a parasitic infection or a biofilm, as appropriate.

[0031] Gram-positive bacteria include, for example, streptococci such as S. viridans, staphylococci such as Staphylococcus aureus, and Bacillus species such as B. subtilis, B. anthracis, and B. cereus.

[0032] Examples of gram-negative bacteria include E. coli, Pseudomonas species such as P. aeruginosa, and Klebsiella species such as K. pneumoniae, K. aerogenes, and K. oxytoca.

[0033] The compositions of the present invention may be used to treat or prevent a fungal infection, including a Candida infection, such as C. albicans, C. parapsilosis, or C. tropicalis, or a combination thereof.

[0034] The wound may be an open wound and may be acute or chronic. The antimicrobial compositions described herein may be used to cleanse, moisturize, and decontaminate acute wounds, chronic wounds, burn wounds, chemical and radiation induced wounds, and superficial burns. The compositions may also be used for wound cleansing and irrigation during surgery.

[0035] The present invention also provides a method for treating or preventing a skin and / or wound infection in a patient, the method comprising administering to the skin and / or wound an antimicrobial composition as described herein. Applying an antimicrobial composition to the skin and / or wound generally refers to contacting the skin and / or wound with the antimicrobial composition. The antimicrobial composition may be placed in contact with the skin and / or wound for about 1 minute, or about 5 minutes, or about 10 minutes.

[0036] In certain embodiments, the patient is a human, a primate, a cow, a sheep, a horse, a pig, a bird, a rodent (such as a mouse or a rat), a cat, or a dog. Preferably, the patient is a human. The patient may also be a production animal such as a cow, a bull, a deer, a goat, a sheep, and a pig, a farm animal and a sport animal such as a dog, a horse, and a pony, a pet animal such as a dog and a cat, and an experimental animal such as a rabbit, a rat, a mouse, a hamster, a gerbil, or a guinea pig.

[0037] The invention further provides a method of making an antimicrobial composition as described herein, comprising: (a) combining the following to form a solution: (i) a water-soluble organosilane at a concentration of about 0.01% to about 0.4% w / v or about 0.5% w / v; (ii) one or more quaternary ammonium compounds at a concentration of about 0.01% to 0.5% w / v; (iii) a nonionic or amphoteric surfactant at a concentration of about 0.05% to about 1% w / v; and (iv) water; and (b) adjusting the pH of the solution to about 4.5 to about 8.5. EXAMPLES

[0038] Exemplary formulations of the invention, referred to herein as "Hybrisan Technology," can be found in Table 1 below.

[0039] [Table 1] Biocompatibility Testing Cytotoxicity Testing Cytotoxicity testing refers to an in vitro assay to evaluate the ability of a test substance to cause cell death or inhibit cell proliferation. Testing for in vitro cytotoxicity specifies a procedure for testing liquids by direct contact, and the Hybrisan technology is evaluated both qualitatively and quantitatively for cell morphology changes. The Hybrisan technology was tested by exposure to cell culture media according to the ISO standard, Biological Evaluation of Medical Devices - Part 5: In Vitro Cytotoxicity Testing (ISO 10993-5:2009), and compared to an untreated control, PBS, and a positive control of Prontosan®, a commercial wound cleansing solution. In addition to this, two shorter time points (5 minutes and 10 minutes) were also tested, which are closer to the clinical application of the Hybrisan technology. The tests were carried out at an independent laboratory of 5D Health Protection Group Ltd.

[0040] Cytotoxicity Testing - Qualitative Evaluation Mouse fibroblast L929 cells were used to assess viability and proliferation after treatment with Hybrisan technology, Prontosan®, or PBS compared to untreated controls and analyzed by microscopy. After all treatments, cell morphology showed a decrease in size. Treatment with Prontosan® showed increased cell lysis, vacuolation, and floating cells. PBS did not affect cell morphology, but more cell detachment was observed after 24 hours. Overall, a qualitative assessment concluded that Hybrisan technology had less effect on cell morphology than Prontosan®.

[0041] Cytotoxicity Testing - Quantitative Evaluation Quantitative cytotoxicity testing was performed using CyQUANT® viability dye to test the wound irrigation solutions at neat concentrations. Viability is shown in Table 2 and Figure 5 (higher ratios indicate more viable cells). As expected, the Hybridsan technology is cytotoxic to L929 cells. Hybridsan technology was less cytotoxic than Prontosan® at all time points. Table 2 - Viability of Hybrisan Technology, Prontosan® and PBS compared to untreated control (N=5)

[0042] [Table 2] Antibacterial Testing The Hybrisan technology was systematically tested in-house and independently to evaluate its antibacterial and antibiofilm efficacy against chronic wound infection pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. In-house testing has demonstrated that Hybrisan Technology is an effective antimicrobial agent, which was confirmed by antimicrobial susceptibility testing according to ISO 20776-1:2019 and anti-biofilm efficacy testing according to ASTM E2799.

[0043] Susceptibility testing The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the Hybrisan technology were determined for P. aeruginosa and S. aureus using methods recommended by the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.Eucast.org), and ISO 20776-1:2019. The MICs for P. aeruginosa and S. aureus by the Hybrisan technology were 23.4 and 0.72, and the MBCs for P. aeruginosa and S. aureus by the Hybrisan technology were 46.89 and 4.39. Table 3 - MIC and MBC results for Hybrisun Technology using EUCAST test method

[0044] [Table 3] Minimum Biofilm Elimination Concentration (MBEC) (ASTM E2799) Further in-house testing confirmed that Hybrisan Technology is an effective anti-biofilm product using the MBEC ASTM E2799 protocol with wound microorganisms. Pseudomonas aeruginosa ATCC 15442 biofilms grown for 24 hours and then treated with Hybrisan Technology had a 6.6 and 7.2 log reduction, whereas Prontosan® had a 3.2 and 5.6 log reduction at 1 and 5 minutes, respectively (Figure 1A). Both were significant reductions in biofilm biomass. Staphylococcus aureus ATCC 6538 biofilms grown for 24 hours and then treated with Hybrisan Technology had a 1.1 and 6.3 log reduction, whereas Prontosan® had a 1.1 and 1.9 log reduction at 1 and 5 minutes, respectively (Figure 1B). Both products showed significant reductions in biofilm biomass with improved efficacy as a function of contact time. However, Hybrisan Technology consistently outperformed Prontosan®.

[0045] Effect of Hybridan Technology on Static Biofilms; Establishment (24 hours), and Regrowth Established biofilms of P. aeruginosa ATCC 15442 were grown for 24 hours and then treated with either Hybrisan technology or Prontosan® for 1 or 5 minutes and compared to untreated controls. Significant reductions were observed with both Hybrisan technology and Prontosan®. At 1 and 5 minutes, there was greater reduction with Hybrisan technology (94 and 96%) than with Prontosan® (59 and 57%), respectively (Figure 2).

[0046] Further studies were performed by growing biofilms (24 hours) and treating them with either Hybrisan Technology or Prontosan® for 5 minutes, followed by incubation in the presence of growth medium for an additional 24 hours. This was done to evaluate the potential efficacy of either treatment. Growth was significantly reduced with Hybrisan Technology and Prontosan®, but Hybrisan Technology resulted in less regrowth compared to the untreated control. Growth in the untreated control was 50% of what is typically seen in 24 hour static biofilms. Regrowth after treatment with Hybrisan Technology was minimal (Figure 3).

[0047] Independent Antibacterial Testing The 5D Health Protection Group has demonstrated that Hybrisan Technology exhibits superior anti-biofilm efficacy when tested against similar commercial products such as Prontosan®. CDC Bioreactor Models - Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans An in vitro method was used to generate robust, clinically similar biofilms using a Centers for Disease Control (CDC) biofilm reactor according to ASTM E2871-13. Good adherence to the method demonstrated that the Hybrisan technology worked faster and removed more microbial biofilm than Prontosan® over clinically significant periods (1 min, 5 min, 10 min) for all strains tested for Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans (Figures 5A, 5B, and 5C). Hybrisan Technology completely eliminated P. aeruginosa after 5 minutes, while Prontosan® only reduced it by 2 logs (Figure 5A). Again, Staphylococcus aureus was completely eliminated after 5 minutes, while Prontosan® only reduced it by 2.3 logs (Figure 5B). As expected, Hybrisan Technology and Prontosan® were unable to completely eliminate Candida albicans biofilms after 10 minutes (Figure 5C), but Hybrisan Technology consistently performed better than Prontosan®, demonstrating greater anti-biofilm efficacy.

[0048] Drip flow biofilm reactor model - Pseudomonas aeruginosa and Staphylococcus aureus A drip-flow biofilm reactor was used according to ASTM E2647-13 (Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilms Grown in a Low Shear and Continuous Flow Drip-Flow Biofilm Reactor). This validated model showed that for both P. aeruginosa and S. aureus test strains, the Hybrisan technology worked faster and removed more microbial biofilm over a clinically significant period (5 minutes), again more quickly than Prontosan® (Figures 5D and 5E). The Hybrisan technology reduced P. aeruginosa by 4.26 logs after 5 minutes of contact time, while Prontosan® reduced it by only 3.62 logs (Figure 5D). Staphylococcus aureus was completely eliminated after 5 minutes, while Prontosan® reduced it by only 3.8 logs (Figure 5E). Table 4 - CDC biofilm counts and log reduction (Woundsan = Hybrisun Technology)

[0049] [Table 4] Table 5 - Drip flow counts and log reduction - 5 minutes (Woundsan = Hybrisun Technology)

[0050] [Table 5] These data clearly demonstrate that the Hybrisan Technology is an effective antibacterial and antibiofilm product that performs better than market leading products. Our specialized independent testing laboratory suggests that the Hybrisan Technology could be used in hospitals to treat biofilms that have formed in chronic wounds, etc. EXAMPLES

[0051] method Formulations were prepared according to Tables 6-13 and tested for stability and minimum biofilm eliminating concentration (MBEC). MBEC: The MBEC model was adapted from ASTM E2799. Briefly, overnight cultures of P. aeruginosa or S. aureus were prepared in Mueller Hinton broth (MHB) and approximately 1 × 10 5 CFU / ml. Wells of a 96-well plate (Nunc, Thermo Fisher, UK) were inoculated with 100 μl of the test inoculum. A 96-peg grid (Nunc, Thermo Fisher, UK) was added to the plate and incubated for 24 h at 37 °C in a humidified container with shaking at 110 rpm to allow biofilm formation. After incubation, the biofilm was washed in a sterile 0.85% sodium chloride (Thermo Fisher, UK) solution in deionized water. After rinsing, the lid was transferred to a new 96-well plate containing 150 μl of test solution. Prontosan® (B. Braun, Germany) was used as a positive control and a 0.85% sodium chloride solution in deionized water was used as a negative control. The pegs were treated for 5 min. After treatment, the pegs were removed and placed in wells containing the appropriate neutralizing medium in an ultrasonic bath for 30 min at maximum power. Each well was serially diluted and the total viable count (TVC) was determined. The log reduction was calculated using the following formula:

[0052] Logarithmic reduction = log10(A / B) (where A is the number of surviving organisms before treatment and B is the number of surviving organisms after treatment) Stability: For initial stability testing, a 100 ml sample of the composition was prepared and 10 ml was transferred to each of three 15 ml tubes. The tubes were placed at 4° C., 20° C. and 37° C. The samples were then monitored for stability and graded weekly over a four week period: 1=clear, 2=cloudy, 3=precipitation, 4=cloudy and precipitate. Accelerated aging was performed on selected samples to ensure their long-term stability. For this purpose, a protocol was established based on ASTM F1980. Briefly, the required test period was determined using the Arrhenius equation.

[0053]

number

[0054] [Table 6] Table 6 shows the effect of cocamidopropyl betaine and pH on stability and minimum biofilm eliminating concentration (MBEC). (Amounts are in % w / v; - indicates MBEC was not tested.) v11.3.1 and v11.4.1 showed good antimicrobial efficacy, but were significantly less effective against biofilms.

[0055] [Table 7] Table 7 shows the effect of increasing concentrations of organosilanes (v12.2-v12.3), no BAC (v13.1), no DAC (v13.2), and no BAC and no DAC (v13.3) on stability and minimum biofilm eliminating concentration (MBEC). (Amounts are in % w / v; - indicates MBEC was not tested).

[0056] [Table 8] Table 8 shows the effect of compositions without chlorhexidane digluconate (v13.4 and v13.4.1), without EDTA (v13.5), without chlorhexidane digluconate and EDTA (v13.6.1), and without cocodigluconate and EDTA, but with reduced chlorhexidane betaine (v13.7.1) (amounts are % w / v; - indicates MBEC not tested).

[0057] [Table 9] Table 9 shows the effect of increasing F-68 concentration (0.1%; v8.8) (0.5%; v8.9) (1%; v8.2) on stability. (Amounts are in % w / v; - indicates MBEC was not tested).

[0058] [Table 10] Table 10 shows the effect of increasing concentrations of glycerol on stability (amounts are in % w / v; - indicates MBEC was not tested).

[0059] [Table 11] Table 11 shows the effect of Brij35 concentrations (0.1%; v7.3) (1%; v7.1) (2%; v7.2) on stability and minimum biofilm eliminating concentration (MBEC). (Amounts are in % w / v; - indicates MBEC was not tested).

[0060] [Table 12] Table 12 shows the effect of dipropylene glycol concentration (10%; v15.1) (20%; v15.2) (40%; v15.3) (50%; v15.4) (60%; v15.5) on stability. (Amounts are in % w / v; - indicates MBEC was not tested).

[0061] [Table 13] Table 13 shows the effect of increasing concentrations of dipropylene glycol (0.1%; v15.6), (1%; v15.7) (5%; v15.8), and glycerol (0.1%; v14.6), (1%; v14.7), and (5%; v14.8). (Amounts are % w / v; - indicates MBEC was not tested).

[0062] result Stable formulations with sufficient efficacy against biofilms were v11.1.1, v11.1.3, v11.2.1, v11.2.3, v11.3.3, v13.4, v13.4.1, v13.5, v14.1, v7.1, v7.2, and v14.8. These formulations completely eliminated biofilms and were stable over the test period when tested using the MBEC assay. Formulations that were stable but had poor efficacy against biofilms were v11.3.1 and v11.4.1. These formulations were stable over the study period but did not completely eliminate biofilms when tested using the MBEC assay. The more potent but unstable solutions were v12.3, v15.1, and v15.8, which completely removed the biofilm but were not stable over the duration of the study. The poorly performing and unstable solutions were v13.1, v13.2, and v13.3. These solutions were not stable over the test period and did not completely remove biofilm when tested using the MBEC assay. EXAMPLES

[0063] Further formulation v9 of the invention herein can be found in Table 14 below.

[0064] [Table 14] Antibacterial Testing v9 was systematically tested in-house to evaluate its antibacterial and antibiofilm efficacy against chronic wound infection pathogens Pseudomonas aeruginosa and Staphylococcus aureus. In-house testing has demonstrated that v9 is an effective antimicrobial agent, which was confirmed by antimicrobial susceptibility testing according to ISO 20776-1:2019 and antibiofilm efficacy testing according to ASTM E2799.

[0065] Susceptibility testing The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of v9 were determined for P. aeruginosa and S. aureus using methods recommended by the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.Eucast.org), and ISO 20776-1:2019. The MICs for P. aeruginosa and S. aureus with v9 were 15.6 and 0.79, and the MBCs for P. aeruginosa and S. aureus were 45.1 and 1.44. Table 15 - MIC and MBC results for v9 using EUCAST test method

[0066] [Table 15] Minimum Biofilm Elimination Concentration (MBEC) (ASTM E2799) Further in-house testing confirmed that v9 was an effective anti-biofilm product using the MBEC ASTM E2799 protocol with wound microorganisms. P. aeruginosa ATCC 15442 biofilms grown for 24 hours were reduced by 5.2 and 6.8 logs with v9 treatment, whereas Prontosan® reduced by 3.2 and 5.3 logs at 1 and 5 minutes, respectively (Figure 6A). Both were significant reductions in biofilm biomass. Staphylococcus aureus ATCC 6538 biofilms grown for 24 hours were reduced by 1.5 and 6.15 logs with v9 treatment, whereas Prontosan® reduced by 1.1 and 2.3 logs at 1 and 5 minutes, respectively (Figure 6B). Both products showed significant reductions in biofilm biomass with improved efficacy as a function of contact time. However, v9 was consistently superior to Prontosan®.

[0067] Effect of v9 on static biofilms; establishment (24 h) and regrowth (D) Established biofilms of P. aeruginosa ATCC 15442 were grown for 24 hours and then treated with v9 or Prontosan® for 1 and 5 minutes and compared to untreated controls. v9 caused greater reductions (77 and 88%) than Prontosan® (54 and 57%) at 1 and 5 minutes, respectively (Figure 7).

[0068] Further studies were performed by growing biofilms (24 hours) and treating with either v9 or Prontosan® for 5 minutes, followed by incubation for an additional 24 hours in the presence of growth medium. This was done to evaluate the potential efficacy of either treatment. Growth was significantly reduced with v9 and Prontosan®, but v9 resulted in less regrowth compared to the untreated control. Regrowth after treatment with v9 and Prontosan® compared to the untreated control was 10% and 13%, respectively (Figure 8). References Salisbury AM,Woo K,Sarkar S,Schultz G,Malone M,Mayer DO,Percival SL.Tolerance of Biofilms to Antimicrobials and Significance to Antibiotic Resistance in Wounds.Surg Technol Int.2018 Nov 11;33:59-66.PMID:30326137

Claims

1. below: (i) one or more water-soluble organosilanes at a concentration of about 0.01% to about 0.4% w / v; (ii) one or more quaternary ammonium compounds at a concentration of about 0.01% to 0.5% w / v; and (iii) An antimicrobial composition comprising one or more nonionic or amphoteric or sarcosine anionic surfactants at a concentration of about 0.05% to about 1% w / v.

2. 10. The antimicrobial composition of claim 1, wherein the water-soluble organosilane is a quaternary ammonium silane.

3. 3. The antimicrobial composition of claim 2, wherein the quaternary ammonium silane is one or more of dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride and 3-(trihydroxysilyl)propyldimethyloctadecylammonium chloride.

4. 2. The antimicrobial composition of claim 1, wherein the quaternary ammonium compound is selected from one or more of benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDAC), benzethonium chloride, tetradonium bromide, cetrimonium bromide, raultrimonium bromide, cetalkonium chloride, and cetrimonium chloride.

5. 2. The antimicrobial composition of claim 1, wherein the nonionic or amphoteric or sarcosine anionic surfactant is selected from one or more of cocomidopropyl betaine, polyethylene glycol lauryl ether, Poloxamer 188, Polysorbate 80, PEG-7 glyceryl cocoate, PEG-7 oleamide, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Polysorbate 20, Poloxamer 407, cocomidopropylamine oxide, and lauramidopropyl betaine.

6. The antimicrobial composition of claim 1 further comprising a chelating agent.

7. 7. The antimicrobial composition of claim 6, wherein the concentration of the chelating agent is from about 0.01% to about 0.2% w / v.

8. 7. The antimicrobial composition of claim 6, wherein the chelating agent is selected from one or more of disodium EDTA, trisodium EDTA, and tetrasodium EDTA.

9. The antimicrobial composition of claim 1 further comprising a polymeric biguanide.

10. 10. The antimicrobial composition of claim 9, wherein the polymeric biguanide is selected from one or more of chlorhexidine and polyhexamethylene biguanide.

11. 10. The antimicrobial composition of claim 1, wherein the pH is from about 4.5 to about 8.

5.

12. 10. The antimicrobial composition of claim 1, further comprising one or more pharmaceutically acceptable carriers and / or excipients.

13. 10. The antimicrobial composition of claim 1 formulated for topical administration.

14. The antimicrobial composition of claim 1 which is antimicrobial.

15. The antimicrobial composition of claim 1 which is anti-biofilm.

16. 10. A method of making the antimicrobial composition of claim 1, comprising: (a) the following: (i) a water-soluble organosilane at a concentration of about 0.01% to about 0.4% w / v; (ii) one or more quaternary ammonium compounds at a concentration of about 0.01% to 0.5% w / v; (iii) a nonionic or amphoteric surfactant at a concentration of about 0.05% to about 1% w / v; and (iv) water to form a solution; and (b) adjusting the pH of the solution to about 4.5 to about 8.5; A method comprising:

17. 10. The antibacterial composition of claim 1 for use in therapy.

18. 10. The antimicrobial composition of claim 1 for use in the treatment and / or prevention of skin and / or wound infections.

19. 19. The antimicrobial composition of claim 18, wherein the infection is a bacterial infection.

20. 20. The antimicrobial composition of claim 19, wherein the bacterial infection comprises a biofilm.

21. 19. The antimicrobial composition of claim 18, wherein the wound is an open wound.

22. 19. The antimicrobial composition of claim 18, wherein the wound is acute or chronic.

23. A pharmaceutical composition for use in a method for treating and / or preventing skin and / or wound infections in a patient, the method comprising administering to the skin and / or wound an antimicrobial composition according to claim 1.

24. 24. The pharmaceutical composition of claim 23, wherein the infection is a bacterial infection.

25. 25. The pharmaceutical composition of claim 24, wherein the bacterial infection comprises a biofilm.

26. 24. The pharmaceutical composition of claim 23, wherein the wound is an open wound.

27. 24. The pharmaceutical composition of claim 23, wherein the wound is acute or chronic.

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