Composition

Abstract

To provide a composition for use in the treatment or prevention of pathogenic infections, rosacea, eczema and psoriasis in humans or animals.SOLUTION: There is provided a composition comprising benzalkonium halide; didecyldimethylammonium halide; polyhexamethylene biguanide salt; bronopol; and p-chloro-m-cresol.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for use in treating or preventing pathogenic infections, such as bacterial, fungal, and / or viral infections, in humans or animals. Specific examples of such infections include pathogenic infections within / on the surface of wounds, bacterial foot disease, impetigo, and ringworm. The composition of the present invention can be used at very low concentrations and has been shown to be non-irritating to human or animal skin. The composition of the present invention is used as a disinfectant.

[0002] The compositions of the present invention are also useful for the treatment or prevention of rosacea, eczema and / or psoriasis in humans or animals.

[0003] Furthermore, the compositions of the present invention may be used to heal wounds in humans or animals, either to heal wounds themselves or to heal wounds whilst also treating or preventing pathogenic infections within / on the wound surface.

[0004] Additionally, the present invention relates to methods for killing or inactivating viruses on surfaces by applying the compositions of the present invention to the surfaces. Examples of such viruses include norovirus and coronavirus. [Background technology]

[0005] Pathogenic infections, such as bacterial infections, can become serious infections that, if left untreated, can develop into life-threatening conditions, such as sepsis. Traditional treatments for pathogenic infections include antibiotics and antiseptics, such as chlorhexidine. However, medical professionals attempt to avoid the use of antibiotics whenever possible to prevent the development of antibiotic resistance. Antiseptics are used to destroy or inhibit pathogenic microorganisms on the surface of living tissue without causing harmful effects to such tissue. However, many known antiseptics cause adverse effects, such as allergic reactions, and many pathogenic infections have become resistant to treatment with known antiseptics. Therefore, there is a need for new, non-antibiotic agents for treating pathogenic infections.

[0006] Pathogenic infections inside / on the surface of wounds are particularly problematic because pathogens can lead to local tissue damage and prevent wound healing (leading to chronic wounds such as venous ulcers of the lower extremities). Therefore, effective treatment of pathogenic infections inside / on the surface of wounds can be essential for wound healing. The environment of wounds, especially chronic wounds, is very different from that of hard surfaces and intact skin. Wounds contain a complex biological mixture consisting of damaged tissue, cells necessary for healing, protein-rich exudates, and microorganisms. Therefore, although antiseptics may be used to clean hard surfaces or intact skin, these antiseptics are often inactivated in the wound environment. Therefore, although a drug may prove to be an excellent antiseptic, this does not mean that the antiseptic will be a useful antiseptic for wounds. Indeed, many agents that can be used to disinfect intact skin prior to medical treatment (e.g., alcohol before venipuncture) can prove useless as wound disinfectants, i.e., they are not useful for treating pathogenic infections within / on the wound. The same is true even for some known disinfectants, i.e., some known disinfectants are inactivated in the wound environment.

[0007] Chronic wounds are even more difficult to treat because they usually contain biofilms; in fact, current estimates suggest that approximately 60% to 100% of chronic, non-healing wounds contain biofilms. The presence of a biofilm means that the wound often appears to heal but eventually becomes congested again. Biofilms are complex, invisible structures composed of mixed strains of pathogens, typically formed when a specific type of microorganism attaches itself to the wound surface and then secretes a viscous substance. Pathogens within biofilms are much more difficult to eradicate with known treatments, such as antibiotics and antiseptics, than planktonic cells. The complex structure within biofilms creates a stressful microenvironment, leading to spatio-physiological heterogeneity of the population within the biofilm. This explains why viable-but-non-culturable (VBNC) and persister cells are often found within biofilms. Current recommendations for managing chronic wounds involve removing biofilm by physical debridement or "aggressive" cleaning, acknowledging that these actions are not effective in removing all of the biofilm and therefore require repetition.

[0008] Considering that biofilm-associated infections lead to significant morbidity and mortality, effective treatments for pathogenic infections within / on wounds, especially in chronic wounds containing biofilms, are desirable. In light of this, the development of novel strategies to inhibit and treat biofilms is important.

[0009] Surfaces often come into contact with potentially harmful viruses, providing an environment for them and creating a breeding ground for them. It is common to clean surfaces with agents that work to mitigate and / or destroy potentially harmful viruses. Cleaning surfaces in this manner is beneficial to human and animal health because it prevents the spread of viruses and reduces the chances that a subject or subjects will contract a viral infection through contact with a potentially harmful virus. Particularly important surfaces are those in hospital and veterinary environments, such as veterinary or hospital operating rooms. Therefore, general cleaning is a cornerstone of infection control practices in healthcare settings. However, many of the agents currently used pose serious exposure risks to human / animal health at the concentrations used, which means that clinical areas being decontaminated often must be closed for several days. Similarly, agents often are not sufficiently effective against some of the viruses that cause common problems, lack any residual antimicrobial activity, and recontamination is likely to occur. Therefore, there is a need for effective and non-hazardous agents that can be used in a variety of situations to clean and decontaminate virus-containing surfaces.

[0010] Two specific viruses of particular importance are norovirus and coronavirus. It is estimated that there are over 267 million norovirus infections worldwide each year (including 23 million in the United States and up to 1 million in the United Kingdom). Norovirus outbreaks cost the National Health Service over £184 million (based on 2002-2003 figures) due to hospitalizations, with an estimated annual cost of $2 billion in the United States. While infections are self-limiting, symptoms are unpleasant, and patients shed large numbers of virus particles for days after contracting the illness. Human coronavirus 229E is a human respiratory pathogen that causes mild respiratory illness in healthy individuals but can cause severe disease in immunocompromised patients and those with multiple sclerosis. Human coronavirus 229E is a coronavirus that causes more severe respiratory illnesses, such as severe acute respiratory disease syndrome (SARS) and Middle East respiratory syndrome (MERS), which require higher isolation facilities. It is a hazard group 2 pathogenic virus used as a surrogate for Rus.

[0011] WO 2015 / 028806 describes cleaning solutions and aqueous mixtures comprising cleaning solutions for disinfecting surfaces and water supplies. The cleaning solution and aqueous mixture in WO 2015 / 028806 are compositions according to the present invention. The cleaning solution and aqueous mixture in WO 2015 / 028806 are also described as being useful for disinfecting skin areas. Disinfecting a skin area means that the cleaning solution / aqueous mixture kills or inhibits microorganisms on the surface of intact skin within minutes, for example, to ensure a "clean" area before the skin barrier is disrupted by medical or surgical intervention. The World Health Organization defines disinfectants as chemical agents applied during cleaning to inanimate objects and materials to destroy or inhibit pathogenic microorganisms.

[0012] General definition The term "comprising" is intended to be broadly understood as including and consisting of )" and, for example, a composition "comprising" X can consist exclusively of X or can include something additional, e.g., X+Y. As used herein, the term "comprising" means "consisting essentially of." For example, a composition "comprising" X can consist of X and any other components that do not substantially affect the essential characteristics of the composition.

[0013] The term "about" in reference to a numerical value is optional and means, for example, x+10%.

[0014] The term "animal" means a mammal or a bird.

[0015] A "pathogenic infection of the skin," as described herein, is an infection of the skin caused by a pathogen that invades through the normal skin barrier due to a breakdown in skin integrity caused by an injury (e.g., a wound) or disease (e.g., impetigo) that compromises the skin barrier. This invasion results in an inflammatory response, and if the pathogen is sufficiently virulent, the pathogen can lead to widespread tissue infection, resulting in, for example, sepsis. Treatment of such pathogenic infections of the skin is distinguished from "disinfecting" an area of ​​skin, as disinfection involves killing or inhibiting microorganisms on the surface of intact skin (i.e., simply sterilizing the surface of intact skin) for a short period of time, e.g., to ensure a "clean" area before the skin barrier is disrupted by medical or surgical intervention.

[0016] The term "skin" includes the skin on / inside the mouth.

[0017] The term "wound" means damage to living tissue (e.g., skin, muscle, etc.) caused by a cut, bruise, or other impact, typically resulting from a cut or injury to the skin. Examples of wounds include burns, ulcers, cuts, abrasions (rubs), and punctures.

[0018] The term "healing" in relation to wounds means the accelerated closure / repair of the wound.

[0019] By "accelerated wound closure" is meant that the wound closes / repairs faster (when the composition of the present invention is used) than it would close / repair in the absence of the composition of the present invention.

[0020] The term "chronic wound" refers to a wound that does not progress to healing in an orderly and timely manner and does not show significant progression to healing at 30 days.

[0021] "Inactivating" a virus means that the virus enters a dormant state. [Prior art documents] [Patent documents]

[0022] [Patent Document 1] International Publication No. 2015 / 028806 Brochure [Brief explanation of the drawings]

[0023] The invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1-1] ~ [Figure 1-3] 1 is a before and after image of impetigo using a composition of the present invention. [Figure 2a] ~ [Figure 2j] 1A-1C are multispectral before and after images showing the treatment of Gram-positive and Gram-negative viable but non-culturable (VBNC) biofilm wounds with compositions of the present invention and the healing of the wounds. [Figure 3a] ~ [Figure 3b] 1 is a before and after image of a dog bite treated with a composition of the present invention. [Figure 4a] ~ [Figure 4b] 1 is a before and after image of a horse leg wound treated with a composition of the present invention. [Figure 5a] ~ [Figure 5c] 1 is a before and after image of a fungal nail infection treated with a composition of the present invention. [Figure 6] 1 shows the results of a skin irritation test for the composition of the present invention. [Figure 7a] ~ [Figure 7h] FIG. 1 illustrates the killing and inactivation of human coronavirus 229E using compositions of the present invention. [Figure 8a] ~ [Figure 8c] FIG. 1 illustrates the killing and inactivation of murine norovirus using compositions of the present invention. [Figure 9] MICs of various Gram-positive and Gram-negative bacteria using compositions of the present invention. [Figure 10a-1] ~ [Figure 10b-2] Treatment of psoriasis and eczema on human skin using the compositions of the present invention. Summary of the Invention

[0024] These figures are further described in the "Examples" section.

[0025] This application is presented in sections to aid in readability. However, this does not mean that each section is intended to be read in isolation. To the contrary, unless otherwise specified, each section should be read with cross-reference to the other sections, i.e., with the entire application taken as a whole. No artificial separation of embodiments is intended unless expressly stated. Any of the compositions of the present invention described herein may be used for any of the uses described herein.

[0026] If the composition of the present invention is stated to contain, for example, at least 99.2% by weight of water, the other components in the composition should be selected so that the total does not exceed 0.8% by weight, i.e., the total weight percentages in the composition is 100%. Selecting the amounts of components in such a technically rational manner is within the skill of a person skilled in the art. [Means for solving the problem]

[0027] In a first aspect, there is provided a composition of the present invention, i.e., a composition comprising a benzalkonium halide; a didecyldimethylammonium halide; a polyhexamethylene biguanide salt; bronopol; and p-chloro-m-cresol, for use in treating or preventing a pathogenic infection in humans or animals, preferably humans. Thus, the composition of the present invention is used as an antiseptic and is useful for treating or preventing a disease / condition associated with or caused by a pathogenic infection. The pathogenic infection preferably affects the skin, muscle tissue, connective tissue, or the like. Pathogenic infections of tissue and / or skeletal tissue. Examples include pathogenic infections within / on the surface of a wound, and impetigo.

[0028] As shown in the examples, the compositions of the present invention are not only capable of treating or preventing various pathogenic infections, but are also useful for treating or preventing Gram-positive and Gram-negative pathogenic infections containing biofilms containing VBNC cells, such as those in chronic wounds (e.g., chronic burns). The compositions of the present invention can also be used at low concentrations that are non-irritating to the skin.

[0029] In a second aspect, there is provided a composition of the invention for use in the treatment or prevention of rosacea, psoriasis and / or eczema in a human or animal, preferably a human. The composition of the invention may therefore additionally treat or prevent other skin conditions, i.e. skin conditions that are not or are not caused by a pathogenic infection.

[0030] In a third aspect, there is provided a composition of the present invention for use in healing a wound in a human or animal, preferably a human, i.e., in accelerating the time for the wound to close. The composition of the present invention can also heal the wound while simultaneously treating or preventing pathogenic infections inside / on the wound surface, i.e., killing pathogens such as bacteria inside / on the wound surface.

[0031] In a fourth aspect, a method for killing or inactivating viruses on a surface is provided, comprising applying a composition of the present invention to the surface. Preferred viruses are norovirus and coronavirus. It has been found that the composition of the present invention only needs to be used at a low concentration to achieve this effect. Furthermore, the composition of the present invention can be pre-coated on the surface, i.e., the surface can be pre-treated to prevent viral infection of the surface for up to 48 hours.

[0032] In a fifth aspect, the present invention relates to a composition of the present invention for use as a medicament. In a sixth aspect, the present invention relates to a composition of the present invention for use as a disinfectant, i.e., the composition of the present invention is used as a disinfectant on the surface of the human or animal body, preferably the human body. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention relates to a composition of the present invention comprising a benzalkonium halide, a didecyldimethylammonium halide, a polyhexamethylene biguanide salt, bronopol, and p-chloro-m-cresol for use in treating or preventing a pathogenic infection in humans or animals. The composition of the present invention is therefore useful for treating or preventing a disease / condition associated with or caused by a pathogenic infection.

[0034] The compositions of the present invention are also useful for treating or preventing rosacea, psoriasis and / or eczema, for wound healing, ie, accelerating the time it takes for a wound to close, and for killing or inactivating surface viruses.

[0035] Compositions of the Invention The composition of the present invention can be any of the compositions disclosed on page 4, line 11 to page 7, line 2, on page 8, line 7 to page 13, line 31, and in claims 1 to 16 of WO 2015 / 028806 (which is incorporated herein by reference), i.e., the composition of the present invention can be any of the "cleaning solutions" or "aqueous mixtures" disclosed in WO 2015 / 028806. The composition of the present invention can also be any one of the compositions defined herein. .

[0036] The composition of the present invention comprises a benzalkonium halide, a didecyldimethylammonium halide, a polyhexamethylene biguanide salt, bronopol, and p-chloro-m-cresol.

[0037] In one embodiment, the composition of the present invention comprises 0.04 to 0.2 wt % benzalkonium halide; 0.04 to 0.2 wt % decyldimethylammonium halide; 0.04 to 0.2 wt % polyhexamethylene biguanide salt; 0.01 to 0.06 wt % bronopol; and 0.0005 to 0.005 wt % p-chloro-m-cresol.

[0038] In one embodiment, the benzalkonium halide is benzalkonium chloride, and / or the didecyldimethylammonium halide is didecyldimethylammonium chloride, and / or the polyhexamethylene biguanide salt is polyhexamethylene biguanide hydrochloride. In a preferred embodiment, the benzalkonium halide is benzalkonium chloride, the didecyldimethylammonium halide is didecyldimethylammonium chloride, and the polyhexamethylene biguanide salt is polyhexamethylene biguanide hydrochloride.

[0039] The composition may further comprise a solvent. Preferably, the solvent is ethanol. The solvent is preferably present in the composition in an amount of 0.05 to 0.3% by weight.

[0040] The composition may further comprise an alkylene glycol. Suitable alkylene glycols that can be used in the compositions of the present invention include ethylene glycol, propylene glycol, diethylene glycol, block copolymers of ethylene oxide and propylene oxide, any other alkylene glycol formed with alkylene oxides, and / or any combination of alkylene glycols. In a preferred embodiment, the alkylene glycol is ethylene glycol. The alkylene glycol is preferably present in the composition at 0.01 to 0.07 wt. %.

[0041] The alkylene glycols may be substituted in whole or in part with other alkylene glycols, such as ethylene glycol, propylene glycol, diethylene glycol, block copolymers of ethylene oxide and propylene oxide (e.g., various types of Pluronic™ sold by BASF™), any other alkylene glycols formed by combining alkylene oxides, and / or any combination of alkylene glycols.

[0042] In a preferred embodiment, the composition further comprises ethanol and ethylene glycol, preferably 0.05 to 0.3 wt % ethanol and 0.01 to 0.07 wt % ethylene glycol.

[0043] Preferably, the compositions of the present invention do not contain one or more siloxanes.

[0044] Preferably, the compositions of the present invention do not contain antibiotics. Indeed, the advantage of the compositions of the present invention is that they can kill a wide range of pathogenic infections quickly and at low concentrations, without the need to use antibiotics, which are often ineffective. Similarly, the development of antimicrobial resistance can be avoided by using the compositions of the present invention instead of antibiotics.

[0045] In one embodiment, the composition of the present invention further comprises water. In one embodiment, the composition comprises: The composition comprises at least 83.9% by weight of water, or at least 96% by weight of water, or at least 97% by weight of water, or at least 98% by weight of water, or at least 98.7% by weight of water, or at least 99.0% by weight of water, or at least 99.2% by weight of water, or at least 99.5% by weight of water, or at least 99.6% by weight of water, or at least 99.7% by weight of water, or at least 99.76% by weight of water. Preferably, the composition comprises at least 99.2% by weight of water or at least 99.76% by weight of water.

[0046] As shown in the examples, the inventors have found that the compositions of the present invention are capable of treating various pathogenic infections and skin conditions, such as psoriasis, healing wounds, and killing or inactivating viruses when used at surprisingly low concentrations, i.e., even when the water content in the composition is high, for example, at least 99.2% water by weight or even at least 99.76% water by weight.

[0047] Furthermore, as described in Example 7, an additional advantage of the composition of the present invention is that the composition can be effectively used at a very low concentration so that it is non-cytotoxic.For example, when the water content in the composition is greater than about 99.5% by weight, for example, greater than 99.76% by weight, the composition of the present invention is non-cytotoxic, and more surprisingly, as described herein, it is still capable of treating pathogenic infections, psoriasis, eczema, and rosacea in humans or animals, and healing wounds.The combination of non-cytotoxicity while maintaining efficacy for the uses described herein is surprising.

[0048] Thus, in one embodiment, the composition of the present invention comprises at least 99.76% water by weight and is for use in treating impetigo. In one embodiment, the composition of the present invention comprises at least 99.76% water by weight and is for use in treating fungal nail infections. In one embodiment, the composition of the present invention comprises at least 99.76% water by weight and is for use in treating bacterial infections in / on wounds. In one embodiment, the composition of the present invention comprises at least 99.76% water by weight and is for use in wound healing. In one embodiment, the composition of the present invention comprises at least 99.76% water by weight and is for use in treating psoriasis.

[0049] In one embodiment, the composition of the present invention comprises: benzalkonium halides Decyldimethylammonium halide Polyhexamethylene biguanide salt Bronopol p-chloro-m-cresol solvent alkylene glycol, and water Includes.

[0050] In another embodiment, the composition of the present invention comprises: 0.04 to 0.2% by weight of benzalkonium halide 0.04 to 0.2% by weight of decyldimethylammonium halide 0.04 to 0.2% by weight of polyhexamethylene biguanide salt 0.01 to 0.06% by weight of bronopol 0.0005 to 0.005% by weight of p-chloro-m-cresol 0.05 to 0.3 wt% solvent 0.01 to 0.07% by weight of alkylene glycol Including, The remainder is water.

[0051] In a preferred embodiment, the composition of the present invention comprises: Benzalkonium chloride Decyldimethylammonium chloride Polyhexamethylene biguanide hydrochloride Bronopol p-chloro-m-cresol ethanol ethylene glycol, and water Includes.

[0052] In a particularly preferred embodiment, the composition of the present invention comprises: 0.04 to 0.2% by weight of benzalkonium chloride 0.04 to 0.2% by weight of decyldimethylammonium chloride 0.04 to 0.2% by weight of polyhexamethylene biguanide hydrochloride 0.01 to 0.06% by weight of bronopol 0.0005 to 0.005% by weight of p-chloro-m-cresol 0.05 to 0.3% by weight of ethanol 0.01 to 0.07% by weight of ethylene glycol Including, The remainder is water.

[0053] In a more preferred embodiment, the composition of the present invention comprises: 0.15% by weight of benzalkonium chloride 0.15% by weight of decyldimethylammonium chloride 0.165% by weight of polyhexamethylene biguanide hydrochloride 0.045% by weight of bronopol 0.002% by weight of p-chloro-m-cresol 0.245% by weight ethanol 0.05% by weight of ethylene glycol, and 99.2% by weight water Includes.

[0054] In another more preferred embodiment, the composition of the present invention comprises: 0.045% by weight benzalkonium chloride 0.045% by weight decyldimethylammonium chloride 0.0495% by weight of polyhexamethylene biguanide hydrochloride 0.0135% by weight of bronopol 0.0006% by weight of p-chloro-m-cresol 0.0735% by weight ethanol 0.015% by weight of ethylene glycol, and 99.76% by weight water Includes.

[0055] The compositions of the invention may be formulated in any manner suitable for application to humans or animals, preferred examples being liquids, foams, moisturizers and gels.

[0056] The compositions of the present invention may be applied to humans or animals by any suitable means, such as by wipes, sprays, sponges, cloths, towels, mouthwashes, or dressings impregnated with the compositions. Preferably, the compositions are applied to humans or animals by wipes, sprays, or dressings impregnated with the compositions. In another preferred embodiment, the compositions of the present invention are applied to the skin of humans or animals in the oral cavity / on the oral surface by mouthwash, i.e., the compositions of the present invention are used as mouthwashes.

[0057] Uses of the Compositions of the Invention (i) Treatment or prevention of pathogenic infections The compositions of the present invention are useful for treating or preventing pathogenic infections in humans or animals, preferably humans. Most preferably, the compositions of the present invention as defined herein are useful for treating pathogenic infections in humans or animals, preferably humans. Thus, the compositions of the present invention are useful for treating or preventing diseases / conditions associated with or caused by pathogenic infections. The compositions are also useful for preventing the transmission of pathogenic infections from a human or animal to another human or animal, preferably from human to human.

[0058] Pathogenic infections that may be treated or prevented by the compositions of the present invention include bacterial, fungal and viral infections.

[0059] Examples of bacterial infections that can be treated or prevented by the compositions of the present invention include bacterial infections inside / on the wound, impetigo, ulcers (including diabetic foot ulcers and bedsores), boils, leprosy, bacterial foot disease, cellulitis, abscesses, interdigital dermatitis, and erysipelas. Particularly preferred bacterial infections are bacterial infections inside / on the wound (i.e., the wound contains at least one species of bacteria) and impetigo. Treating bacterial infections inside / on the wound means that the bacteria inside / on the wound are killed, i.e., removed in whole or in part, by the compositions of the present invention.

[0060] Other particularly preferred bacterial infections to be treated or prevented by the compositions of the present invention are bacterial infections comprising, i.e., caused at least in part by, Staphylococcus aureus (S. aureus) (e.g., MRSA), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), E. hirae, A. baumannii, Corynebacterium amycolatum, Corynebacterium striatum, and / or Klebsiella sp., preferably Staphylococcus aureus, Pseudomonas aeruginosa and / or Klebsiella sp.

[0061] In a preferred embodiment, the bacterial infection involves rod-shaped (e.g., Klebsiella) and / or coccoid (e.g., Staphylococcus aureus) bacteria. More preferably, the bacterial infection involves rod-shaped bacteria.

[0062] Examples of fungal infections that can be treated or prevented by the compositions of the present invention include fungal infections inside / on the wound, fungal nail infections, fungal infections of the lower extremities, ulcers, yeast infections, athlete's foot, and ringworm. Particularly preferred fungal infections are fungal infections inside / on the wound (i.e., the wound contains at least one fungus) and fungal nail infections. Treating fungal wounds means that the fungi inside / on the wound are killed, i.e., removed (in whole or in part), by the compositions of the present invention.

[0063] Examples of viral infections that can be treated or prevented by the compositions of the present invention include norovirus, coronavirus, herpes, cold sore virus, H. PV, chickenpox, shingles, measles, warts, and molluscum contagiosum. The viruses are norovirus and coronavirus.

[0064] In one embodiment, the compositions of the present invention are used to treat or prevent bacterial and / or fungal infections. In a preferred embodiment, the pathogenic infection is a bacterial infection. In another preferred embodiment, the pathogenic infection is a fungal infection.

[0065] In one embodiment, the pathogenic infection is a pathogenic infection of the skin, muscle tissue, connective tissue and / or skeletal tissue of a human or animal. In a preferred embodiment, the pathogenic infection is a pathogenic infection of the skin of a human or animal.

[0066] In a preferred embodiment, the composition of the present invention is applied topically to humans or animals. If the pathogenic infection is a pathogenic infection of the skin, the composition may be applied directly to the skin of humans or animals. Thus, in a preferred embodiment, the composition of the present invention is applied topically to the skin of humans or animals.

[0067] In a preferred embodiment, the pathogenic infection is within / on the wound, and therefore the compositions of the present invention are intended to treat or prevent, i.e., kill, pathogenic infections within / on the wound. In a preferred embodiment, the wound is a bacterial wound and / or a fungal wound. In a more preferred embodiment, the wound is a bacterial wound, i.e., the wound contains bacteria that are killed, i.e., removed in whole or in part, by the compositions of the present invention. Preferably, the bacteria within / on the wound include one or more of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, E. hirae, A. baumannii, Corynebacterium amycolatum, Corynebacterium striatum, and / or Klebsiella sp., more preferably one or more of Staphylococcus aureus, Pseudomonas aeruginosa, and / or Klebsiella sp. This example demonstrates that the compositions of the present invention have bactericidal activity against these common wound bacteria when tested under "contaminated conditions" and in real-life wounds. In another preferred embodiment, the bacterial infection within / on the wound surface comprises rod-shaped and / or sphere-shaped bacteria, more preferably rod-shaped bacteria. In some embodiments, the wound is a cut, burn, puncture wound, or ulcer.

[0068] The wound may be a chronic wound.As shown in the examples, the composition of the present invention can treat chronic wounds because its activity spectrum covers Gram-positive and Gram-negative bacteria, and has rapid bactericidal and fungicidal action with excellent residual activity.The composition of the present invention acts quickly and exhibits broad-spectrum bactericidal action, which reduces the possibility of resistance development, and is therefore suitable for treating or preventing chronic wounds.

[0069] This example also demonstrates that the compositions of the present invention are capable of treating pathogenic infections containing biofilms, particularly biofilms comprising VBNC cells, i.e., pathogenic infections containing pathogens in a VBNC state. In a preferred embodiment, the compositions of the present invention are for use in treating bacterial infections containing Gram-positive and / or Gram-negative VBNC biofilms. In a particularly preferred embodiment, the compositions of the present invention are for use in treating bacterial infections within / on a wound, wherein the wound contains a biofilm, and the biofilm contains Gram-positive and / or Gram-negative bacteria, which may be in a VBNC state.

[0070] The compositions of the present invention may also be used to heal the wound and simultaneously treat pathogenic infections in the wounds described herein, which may be done simultaneously or sequentially. By "wound healing" it is meant that the compositions of the present invention promote closure of the wound, i.e., accelerate healing.

[0071] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: For use in the treatment or prevention of bacterial and / or fungal infections of the skin of humans or animals, Benzalkonium chloride Decyldimethylammonium chloride Polyhexamethylene biguanide hydrochloride Bronopol p-chloro-m-cresol ethanol ethylene glycol, and water a composition for topical application to human or animal skin, comprising: a composition as defined above for use in the treatment or prevention of bacterial and / or fungal infections in / on a wound, optionally further healing the wound; or The present invention relates to the composition for use in the treatment or prevention of bacterial infection inside / on the surface of a wound, wherein the wound is a chronic wound containing a biofilm that may include VBNC cells.

[0072] In another embodiment, the present invention provides a composition comprising: 0.04 to 0.2% by weight of benzalkonium chloride 0.04 to 0.2% by weight of decyldimethylammonium chloride 0.04 to 0.2% by weight of polyhexamethylene biguanide hydrochloride 0.01 to 0.06% by weight of bronopol 0.0005 to 0.005% by weight of p-chloro-m-cresol 0.05 to 0.3% by weight of ethanol 0.01 to 0.07% by weight of ethylene glycol and the balance is water, a composition to be applied topically to the skin of a human or animal; or a composition as defined above for use in the treatment or prevention of bacterial and / or fungal infections in / on a wound, optionally further healing the wound; or The present invention relates to the composition for use in the treatment or prevention of bacterial infection inside / on the surface of a wound, wherein the wound is a chronic wound containing a biofilm that may include VBNC cells.

[0073] In another embodiment, the present invention provides a composition comprising: 0.15% by weight of benzalkonium chloride 0.15% by weight of decyldimethylammonium chloride 0.165% by weight of polyhexamethylene biguanide hydrochloride 0.045% by weight of bronopol 0.002% by weight of p-chloro-m-cresol 0.245% by weight ethanol 0.05% by weight of ethylene glycol, and 99.2% by weight water A composition comprising: a composition to be applied topically to the skin of a human or animal; or a composition as defined above for use in the treatment or prevention of bacterial and / or fungal infections in / on a wound, optionally further healing the wound; or The above composition for use in the treatment or prevention of bacterial infection inside / on the wound, The present invention relates to the above composition, wherein the wound is a chronic wound containing a biofilm that may include VBNC cells.

[0074] In another embodiment, the present invention provides a composition comprising: 0.045% by weight benzalkonium chloride 0.045% by weight decyldimethylammonium chloride 0.0495% by weight of polyhexamethylene biguanide hydrochloride 0.0135% by weight of bronopol 0.0006% by weight of p-chloro-m-cresol 0.0735% by weight ethanol 0.015% by weight of ethylene glycol, and 99.76% by weight water A composition comprising: a composition to be applied topically to the skin of a human or animal; or a composition as defined above for use in the treatment or prevention of bacterial and / or fungal infections in / on a wound, optionally further healing the wound; or The present invention relates to the composition for use in the treatment or prevention of bacterial infection inside / on the surface of a wound, wherein the wound is a chronic wound containing a biofilm that may include VBNC cells.

[0075] (ii) treating or preventing rosacea, eczema, and / or psoriasis Any one of the compositions defined herein may be used to treat or prevent, most preferably treat, rosacea, eczema or psoriasis.Therefore, the present invention also relates to the compositions defined herein for use in treating rosacea, eczema or psoriasis, preferably psoriasis and / or eczema.As shown in examples, the inventors have surprisingly found that the compositions of the present invention also treat skin infections that are not caused by or are not caused by pathogenic infections.

[0076] (iii) Wound healing Any one of the compositions of the present invention described herein can also be used to heal wounds, i.e., accelerate wound closure.Thus, the compositions of the present invention can be used to heal wounds (without necessarily treating pathogenic infections inside / on the surface of the wound).

[0077] However, treating pathogenic infections inside / on the wound surface contributes to wound healing, i.e., successful treatment of pathogenic infections inside / on the wound surface using the compositions of the present invention contributes to wound healing.Therefore, in a preferred embodiment, the compositions of the present invention are used to treat pathogenic infections inside / on the wound surface as described herein while simultaneously healing the wound, i.e., promoting wound healing / closure, which may be performed simultaneously or sequentially.In a particularly preferred embodiment, the compositions of the present invention are used to treat or prevent bacterial and / or fungal infections inside / on the wound surface and to heal the wound.

[0078] The wound can be any wound described herein, such as a cut, burn, puncture wound, or ulcer. In a preferred embodiment, the wound is a chronic wound. In a more preferred embodiment, the wound is a chronic wound containing a biofilm. In another preferred embodiment, the composition of the present invention used to heal a wound comprises at least 99.76% by weight of water.

[0079] (iv) Killing or inactivating viruses on surfaces Any one of the compositions of the invention described herein can be used to kill or inactivate viruses on a surface. Accordingly, the invention also relates to a method of killing or inactivating viruses on a surface, the method comprising applying a composition of the invention to the surface.

[0080] As shown in Examples 8 and 9, the compositions of the present invention can kill or inactivate viruses on surfaces even at low concentrations, i.e., even when the compositions contain large amounts of water, e.g., greater than 99% water by weight.

[0081] The surface may be any surface that contains viruses. Non-limiting examples of surfaces include floors, tabletops, veterinary operating rooms, hospital operating rooms, and kitchen sideboards, and may be at any angle to the ground and have any shape, i.e., reference to a surface is not limited to flat surfaces. The surface may be made of any material, such as plastic, wood, metal, etc. In a preferred embodiment, the surface is a plastic or metal, such as steel, surface.

[0082] In one embodiment, the method of killing or inactivating a virus is not a method for treating the human or animal body by therapy.

[0083] Viruses that can be killed or inactivated on surfaces by the compositions of the invention include coronaviruses, such as human coronavirus 229E (HuCoV-229E), noroviruses, such as murine norovirus 1 (MNV-1) strain CW1, and herpes. Preferred viruses that can be killed or inactivated on surfaces by the compositions of the invention are coronavirus 229E (HuCoV-229E) and murine norovirus 1 (MNV-1) strain CW1.

[0084] As shown in Examples 8 and 9, the compositions of the present invention kill or inactivate even the most hardy viruses on surfaces, such as murine norovirus 1 (MNV-1) strain CW1.

[0085] In one embodiment, the surface is pre-treated with the composition, meaning that the composition is applied to the surface before viruses are identified on the surface, i.e., the composition of the present invention is used as a preventative measure against viruses on surfaces.

[0086] When applied to a surface, the compositions of the present invention prevent the proliferation / activation of viruses on the surface for an extended period of time after application of the composition to the surface. In one embodiment, the compositions kill or inactivate viruses on the surface for at least 1 hour after application of the composition to the surface. In a preferred embodiment, the compositions kill or inactivate viruses on the surface for at least 24 hours after application of the composition to the surface. In another preferred embodiment, the compositions kill or inactivate viruses on the surface for at least 48 hours after application of the composition to the surface.

[0087] Many modifications and variations of the embodiments described herein may be made without departing from the spirit and scope of the invention, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only. [Example]

[0088] The compositions of the present invention used in this example contained the following components:

[0089] Composition of the present invention (83.9% by weight of water): 3% by weight of benzalkonium chloride 3% by weight decyldimethylammonium chloride 3.3% by weight of polyhexamethylene biguanide hydrochloride 0.9% by weight of bronopol 0.04% by weight of p-chloro-m-cresol 4.9% by weight ethanol 1.0% by weight of ethylene glycol, and 83.9% water by weight.

[0090] Composition of the present invention (99.2% by weight water): 0.15% by weight of benzalkonium chloride 0.15% by weight of decyldimethylammonium chloride 0.165% by weight of polyhexamethylene biguanide hydrochloride 0.045% by weight of bronopol 0.02% by weight of p-chloro-m-cresol 0.245% by weight ethanol 0.05% by weight of ethylene glycol, and 99.2% water by weight.

[0091] Composition of the present invention (99.76% by weight water): 0.045% by weight benzalkonium chloride 0.045% by weight decyldimethylammonium chloride 0.0495% by weight of polyhexamethylene biguanide hydrochloride 0.0135% by weight of bronopol 0.0006% by weight of p-chloro-m-cresol 0.0735% by weight ethanol 0.015% by weight of ethylene glycol, and 99.76% water by weight.

[0092] All examples presented herein involved experiments performed under confidentiality agreements. [Example]

[0093] Treatment of widespread acute impetigo A 55-year-old female patient developed widespread acute impetigo. Initially, the patient was misdiagnosed by a local pharmacist as having a tinea pedis infection, which allowed the infection to grow and spread uncontrollably for two weeks. The pharmacist prescribed clotrimazole / Canestin cream twice daily along with thorough cleaning and drying procedures. The patient adhered to these and applied dressings and cleansing four times daily for several days. The condition continued to worsen, and the patient's legs swelled to the point where she was unable to fit into closed shoes. Furthermore, similar symptoms of infection, such as a rash, began to appear on the patient's arms, chest, neck, and face, accompanied by a slight fever.

[0094] The patient then attended University College Hospital A&E, where he was promptly diagnosed with impetigo. Due to the acute and widespread nature of the infection, oral antibiotics were advised and prescribed. The patient had a history of anaphylactic reactions, primarily to penicillin class antibiotics, and therefore asked for a parenteral alternative. The resident practitioner stated that although the antibiotic cream was not an issue related to the patient's allergy, it was probably insufficient to adequately treat the patient's widespread infection. Clindamycin ointment was prescribed, and if symptoms did not improve within 48 hours, he was advised to return to A&E for oral clindamycin. I received the message.

[0095] Clindamycin ointment initially appeared to be effective, slightly reducing the exudation. The treated area appeared inflamed, but the infection appeared to be reducing, and application was continued. Subsequently, the area became increasingly red / inflammed and swollen. Topical treatment was discontinued after the first morning application. The swelling persisted around the clock, accompanied by difficulty breathing and swallowing, and chest pain. This is a classic anaphylactic reaction. The National Health Service (NHS) was contacted directly, and the patient was treated by ambulance, but I was told I did not own an epinephrine pen.

[0096] The patient then made an appointment with her local general practitioner, who immediately administered epinephrine, which stopped any further allergic reaction. However, the impetigo infection still persisted. The impetigo infection was located on the patient's tops of her feet, ankles, forearms, chest, neck, and face. Corticosteroids were prescribed by the general practitioner, which helped to reduce some of the swelling, but did not reduce the infection.

[0097] Wipes containing the composition of the present invention (99.2% water by weight) were received from the applicant under a confidentiality agreement, and all infected areas were immediately wiped. The patient felt an immediate cooling / calming sensation, and the itching significantly subsided. The raised, oozing rash began to dry and subside. The composition was applied to the entire area several times a day after thoroughly cleaning and drying the affected area.

[0098] There was a marked improvement, with the previous oozing visibly drying up, but swelling still present, and the redness and itching exponentially diminished. There was no longer any uncontrollable spread of infection. After one week of application, some redness and swelling were still present, but the oozing had significantly diminished and there was no itching.

[0099] Use of the composition of the present invention was continued for four weeks, decreasing the frequency of application from three to four times per day, to two times per day, to one time per day, and after four weeks the patient was free of all impetigo infections.

[0100] In summary, the composition of the present invention stopped and controlled the widespread impetigo bacterial infection within one week and promoted complete recovery within three to four weeks. However, the patient did not experience improvement with known medications, such as antibiotic and corticosteroid treatment, thus demonstrating the superiority of the composition of the present invention over known medications. Photographs of the patient's leg before, during, and after treatment with the composition of the present invention are presented in Figure 1. [Example]

[0101] Treatment of Gram-positive and Gram-negative VBNC bacterial (biofilm) wounds and wound healing Testing was conducted in an approved clinical study in India under a confidentiality agreement, utilizing a non-invasive, fluorescence-based, multispectral imaging device to capture images of wounds before and after treatment with the composition of the present invention (99.2% water by weight). This method enabled automated detection of the presence (or absence) of specific Gram-positive and Gram-negative VBNC (biofilm) pathogens and wound size and clinical classification within two minutes of imaging, without the addition of any external reagents. The device uses multispectral imaging in combination with advanced computational algorithms and a proprietary, state-of-the-art ML engine for spatial mapping and detection of pathogens.

[0102] The results show that before the composition of the present invention was applied to the wound (by wipe), the wound contained VBNC Gram-negative bacteria (in this case, Klebsiella) in the form of a biofilm (Figure 2a). However, 2 minutes after application of the composition of the present invention, the bacteria and biofilm were The rhesus monkeys were completely absent (Fig. 2b).

[0103] Similar results are shown in Figures 2c-h. In Figures 2c-e, a visible reduction in the rod-shaped bacteria Klebsiella and Pseudomonas aeruginosa can be seen after application of the composition of the present invention to the wound, and in Figures 2f-h, a visible reduction in Klebsiella can be seen after application of the composition of the present invention to a different wound. The bacteria in these examples appear to initially reside in the muscle and connective tissue within / on the surface of the wound, and can then be dramatically reduced after treatment with the composition of the present invention.

[0104] Figures 2i-j show that MRSA, Staphylococcus aureus, is reduced in wounds after application of the composition of the present invention.

[0105] These surprising results are significant because even if a wound is culturally "clean," it may still fail to heal due to the presence of biofilm. However, the compositions of the present invention completely removed the biofilm (e.g., Figures 2a-b), thus promoting improved healing without the use of systemic antibiotics.

[0106] Thus, because the compositions of the present invention are capable of eliminating Gram-positive and Gram-negative bacteria-containing biofilms in a VBNC state, the compositions of the present invention can be utilized to facilitate the treatment of chronic wounds (which are prone to biofilm formation), as well as traumatic wounds, ulcers, burns, etc. The compositions of the present invention are believed to be particularly important given that biofilm-associated infections lead to significant morbidity and mortality. [Example]

[0107] Treatment of dog bites and equine leg wounds: healing and prevention of pathogenic infections An 82-year-old man has dog bite puncture on his finger (that is, wound).The composition of the present invention (99.2% by weight water) is applied immediately and every day (under confidentiality agreement), and the wound heals quickly without infection, and then completely heals in 3 weeks.This male subject strongly claims that the use of the composition of the present invention significantly contributes to the acceleration of healing process.

[0108] Before and after photographs of the wound and healed wound are shown in Figure 3a and Figure 3b (after 1 month), respectively.

[0109] A 24-year-old horse suffered a traumatic injury to the tendon sheath of its front leg. Based on past experience, the likelihood of infection was high, and the veterinarian recommended immediate euthanasia, based on the poor prognosis for recovery in a horse of that age. The owner refused, instead opting to suture the injury and (under a confidentiality agreement) apply the composition of the present invention daily. To the veterinarian's surprise, the horse made a full recovery with no apparent infection.

[0110] Before and after photographs (5 week intervals) of the horse's leg are presented in Figure 4a and Figure 4b, respectively. [Example]

[0111] Treating Fungal Nail Infections The patient had tried various treatments for her fungal nail infection for three years, and after numerous visits to doctors, nothing worked.

[0112] The patient was introduced to the composition of the present invention (99.2% water by weight) under a confidentiality agreement and began applying it to her nails. After just one year, her nails were clear, despite previous treatments having failed. The patient rated the composition a "10 out of 10" and also commented that it had no odor or greasy feel.

[0113] Photographs of the patient's thumb before, during and after treatment with the composition of the present invention are shown in Figures 5a, 5b and 5c, respectively. [Example]

[0114] Treatment of psoriasis and eczema on human skin within 48 hours The patient was 20 years old and had suffered from a long-term skin condition diagnosed as psoriasis and eczema. The patient had used various over-the-counter and over-the-counter medications with limited success. Following the insult, the patient, under a confidentiality agreement, chose to undergo an experiment using the composition of the present invention (in this case, containing 99.76% water by weight). The patient applied it only to the rash on her hands and compared the results with the rash on her face. The first application was at time 0, and at 24 hours, the patient reported that the irritation of the skin on her hands had decreased, and that the pain was no longer significant, accompanied by reduced redness. The patient applied the composition of the present invention again at that time (24 hours), and after this, at 48 hours, observed that the symptoms on her hands had significantly improved, the rash was no longer sore or itchy, and the skin color had returned to almost normal. The untreated area, i.e., the patient's face, still experienced soreness, pain, itching, and redness.

[0115] Before and after photographs of the patient's hands and face are shown in Figures 10a and 10b, respectively, which show that the untreated skin (i.e., face) deteriorated over a 48 hour period, while the skin treated with the composition of the present invention (i.e., hands) showed a dramatic improvement over the same period.

[0116] Thus, the compositions of the present invention not only treat pathogenic infections (eg, of the skin), but also treat skin conditions (eg, psoriasis and / or eczema) that are not caused by pathogenic infections. [Example]

[0117] Skin irritation test An in vitro evaluation of the irritation potential of the compositions of the present invention using a tissue-engineered human skin model was conducted under confidentiality agreement by Dr. David Voegeli, Associate Professor of Nursing, Skin Health Research Group.

[0118] The overall aim of this project was to investigate the effects of several compositions of the invention on a recognised tissue engineered human skin model (European Centre for the Validation of Alternatives to Animal Experiments, EURL-ECVAM).

[0119] The objectives of this study are to: 1. Determining the degree of skin irritation caused by various compositions of the present invention. 2. Determination of the in vitro toxicity of various compositions of the invention to human keratinocytes.

[0120] result Macroscopic and microscopic inspection of inserts All tissue received was found to be intact and viable upon visual inspection and was used. After tissue administration, a random selection of inserts was examined by low-power optical microscopy to ensure no damage had been caused during the procedure. No cellular damage was observed in any of the inserts examined.

[0121] MTT assay The raw optical density of each well at 570 nm is shown in Table 1 below:

[0122] [Table 1]

[0123] Relative survival rate The relative survival calculations for the controls and compositions tested are shown below (Table 2) and summarized in Figure 6:

[0124] [Table 2]

[0125] conclusion This study was designed to investigate the potential skin irritation of various compositions of the present invention intended for topical application in humans / animals. A total of four products were tested, as well as a positive control and a negative control. Calculation of RV% as a measure of irritation showed that all of the tested compositions of the present invention had an RV% greater than 50, and the majority (75%) had an RV% greater than 100.

[0126] These data demonstrate that, unlike many known disinfectants in the art, none of the compositions tested are likely to cause skin irritation, even at higher concentrations, such as compositions according to the present invention containing 16% water by weight. [Example]

[0127] non-cytotoxic The compositions of the present invention have been tested for cytotoxicity (according to ISO 10993-5) under confidentiality agreements. It is not essential that the compositions of the present invention be non-cytotoxic according to this test, but this is an advantageous property.

[0128] The composition according to the invention (containing 99.2% culture medium) was found to be cytotoxic, i.e., resulting in complete cell death, whereas the composition according to the invention containing 99.76% culture medium was found to be non-cytotoxic (26% reduction in cell density compared to the negative control; ISO 10993-5 allows for 30% inhibition of cell growth).

[0129] Thus, when the compositions of the present invention comprise at least about 99.5% by weight water (e.g., at least 99.76% by weight water), the compositions of the present invention are non-cytotoxic. As shown in Examples 5 and 11, at this non-cytotoxic concentration, the compositions of the present invention are surprisingly capable of treating skin conditions such as psoriasis and eczema, and also have bactericidal activity against various bacteria in contaminated conditions (i.e., in vitro conditions simulating conditions inside / on the surface of human / animal wounds), and therefore are capable of treating pathogenic infections, such as pathogenic infections inside / on the surface of wounds, and infections such as those exemplified in Examples 1-4. [Example]

[0130] Killing and inactivating coronaviruses on surfaces protocol Two methods were performed under confidentiality agreement at 21°C: i) A stainless steel (S30400) surface was pre-coated with a composition of the invention (in this case containing 99.2% water by weight) and then challenged with virus. ii) The virus was dried onto a stainless steel surface and then challenged with a composition of the invention (in this case containing 99.2% water by weight).

[0131] Virus was then recovered from all surfaces and tested to see how much infectious virus (i.e., capable of causing disease) remained after treatment.

[0132] Preparation of various reagents HuCoV-229E virus stock solution (crude infected cell lysate): This virus was originally isolated from the upper respiratory tract of a human with bronchitis in the 1960s. It has been used as a surrogate for the highly pathogenic coronaviruses that cause SARS and MERS, which require specialized, higher-level containment facilities. Unlike other surrogates, such as TGEV, HuCoV-229E is a hazard group 2 (HG2) virus that causes respiratory infections in humans and affects the intestinal mucosa. In the majority of individuals, the immune response is unable to eradicate the virus over time, so infections with HuCoV-229E occur every few years. The virus preparation contains killed human lung cells and a complex mixture of biomolecules (including growth medium components) to mimic viral contamination in sputum and natural respiratory secretions.

[0133] The host cell line is MRC-5 normal human lung fibroblasts (HuCov-229E can attach to and replicate in this cell line).

[0134] Throughout, stainless steel specimens (1 cm 2 × 0.5 mm) was used as an example of a common surface material.

[0135] Phosphate buffered saline (PBS), cell culture growth medium, and supplements trypsin / EDTA, low melting point agarose solution for overlay, neutral red dye.

[0136] · consumables Disposable tubes (6 mL, 30 mL and 50 mL), glass beads, tissue culture flasks and 6-well trays, loops and spreaders, Petri dishes, culture boxes, pipettes, tips, pastettes, syringes and syringe filters.

[0137] · Device Microbiological Safety Cabinet Microbiological incubator (37°C, carbon dioxide was pumped into the incubator and maintained at 5% for optimal cell growth). Microcentrifuge inverted microscope Lightbox and magnifying lens

[0138] Testing procedures The efficacy of the compositions of the present invention in inactivating HuCoV-229E was determined by two methods: 1. Non-biocidal surfaces were pre-coated with the compositions of the present invention for 1, 24 and 48 hours to determine whether viruses were inactivated on contact, i.e., if a surface was cleaned with the compositions of the present invention, would an untreated layer inactivate any subsequent viral contamination of that surface in respiratory secretions, for example during coughing and sneezing? 2. A non-biocidal surface was pre-coated with a virus and it was determined whether a composition of the present invention applied for 0, 30 seconds, 5 minutes or 10 minutes would inactivate the virus, i.e. whether cleaning an already contaminated surface with a composition of the present invention would inactivate the virus.

[0139] Experimental Protocols Preliminary experiments and preparation: - Passage cell line MRC-5 to sufficient quantities for the experiment. This involves passage every 3 days for each increasing number of flasks (cells adhere to plastic and need to be removed using trypsin / EDTA). The day before the experiment, place the cells into approximately 10 tissue culture flasks (75cm bottom). 2 ), resuspended in fresh culture medium, and seeded into six-well trays, where the cells settle and attach to the bottom of each well, forming a monolayer of cells. 24 hours and 48 hours before the experiment, the test specimens are coated with the composition of the invention.

[0140] On the day of the experiment: - Ensure cells appear healthy and sub-confluent before starting the experiment. - Inoculate stainless steel specimens (in triplicate) with one of the following: (1) 1 hour, the composition of the present invention (20 μL of the composition of the present invention (99.2 wt % water) was spread over the entire surface of the test piece) - Method 1 (2) 1 hour, coronavirus (20 μL of HuCo-229E, approximately 10 plaque-forming units, on the surface of the test piece (1 cm 2 )Spread it all over) - Method 2 o Test strips for method 1 are loaded with virus for 0, 30 seconds, 5 minutes and 10 minutes, or test strips for method 2 are loaded with a composition of the invention for the same times. o Virus is removed in the same way for each method by placing each specimen in a separate tube of 5 mL of growth medium (5% fetal bovine serum) / glass beads (2 mm). Vortex each for 15 seconds. Prepare a 10-fold dilution of each sample in growth medium. Remove the medium from the MRC-5 cells in each well of a 6-well tray, add 1 mL of test dilution, and incubate at 37°C, 5% CO2 for 90 minutes (this allows the virus to attach to the cells). Carefully remove the virus and add the overlay. Allow to cool for 15 minutes to ensure the overlay has hardened, then transfer to an incubator. Incubate at 37°C, 5% CO2 for 6 days.

[0141] Observations and calculations of infectious virus recovered from each specimen: Add 3 mL of a 3% solution of sterile-filtered neutral red vital stain to each well and incubate for 2 hours. Remove the liquid stain from the top of the well and incubate for an additional hour. This stain is taken up by viable cells, staining them red. Dead cells do not take up the stain. If the virus replicates inside the cells, plaques are visualized as unstained areas. These are actually holes in the cell monolayer, where infected cells burst and die (lyse), surrounded by remnants of the dead infected cells. The overlay prevents the virus from spreading beyond the attachment time, resulting in clearer plaques that are easier to count.

[0142] - Count plaques * The amount of infectious virus recovered from each specimen is calculated. Photographs are taken if necessary. For plaque counts, virus numbers are often expressed as plaque-forming units (pfu) (not all viruses produce plaques), which is an approximation of the amount of infectious virus present, as it is difficult to express the results as an actual virus count. - Analyze the data using graphics software.

[0143] result The results are shown in Figure 7 and summarized below: The tables in Figures 7a and 7b contain the raw data (mean pfu recovered per surface of the test strip) for each contact time, and the Log10 reduction in infectious human coronavirus 229E (HuCoV-229E). 2 The maximum amount of infectious virus per well is 2500 plaque-forming units (pfu).

[0144] Figures 7a and 7c to 7e (Method (1)): 1 cm 48 hours, 24 hours, or 1 hour before virus application 2 The surface is pre-coated with 10 μL of the composition of the present invention per sample.

[0145] Figures 7b and 7f-7g (Method (2)): The surface is pre-coated with 20 μL of HuCoV-229E (2500 pfu) 1 hour before applying the composition of the invention.

[0146] Figure 7h = Inactivation rate at 30 seconds for methods (1) and (2)

[0147] The Log reduction in infectious virus was calculated according to the following formula: Log reduction = log10(pfu before treatment) - log10(pfu after treatment) 1 log reduction = 90% reduction 2 log reduction = 99% reduction 3 log reduction = 99.9% reduction 4 log reduction = 99.99% reduction 5 log reduction = 99.999% reduction 6 log reduction = 99.9999% reduction

[0148] The summarized results show that the compositions of the present invention, even when used at very low concentrations, can effectively prevent or alleviate the symptoms of rheumatoid arthritis in humans. It has been shown to be highly effective against coronavirus 229E.

[0149] Furthermore, these results demonstrate that coating surfaces with the compositions of the present invention continues to inactivate viruses for extended periods of time. There was no difference between 1-hour and 24-hour coatings, and only a slight decrease in efficacy was observed for surfaces coated for 48 hours. All viruses were inactivated within 30 seconds to 5 minutes.

[0150] The compositions of the present invention were highly effective against dried viruses, again inactivating dried viruses within 30 seconds to 5 minutes.

[0151] Thus, the compositions of the present invention are capable of keeping any surface inactive for extended periods of time, which is useful, for example, for contamination that occurs between cleaning regimens. [Example]

[0152] Killing and inactivating noronaviral viruses on surfaces The purpose of these experiments was to determine whether a composition of the present invention (in this case, containing 99.2% water by weight) could kill or inactivate murine norovirus. Three experiments were conducted under confidentiality agreement, as summarized below.

[0153] Experimental protocol Virus and cell lines Murine norovirus 1 (MNV-1) strain CW1 and murine monocyte-macrophage strain RAW264.7 were kindly provided by Dr. Herbert Virgin IV (Washington University) Cells were provided by the University of California, San Diego, CA, USA. Semi-adherent cell lines were maintained at subconfluence to prevent loss of characteristic phenotypes and maintained at 37°C in 5% CO2 in HEPES-buffered Dulbecco's modified Eagle's medium (DMEM) containing GlutaMAX, 25 mM D-glucose, and 10% fetal bovine serum, but without sodium pyruvate. Cells adhere to tissue culture-grade plastic via cation-dependent and -independent receptors and can be easily detached by scraping.

[0154] Sample surface preparation Stainless steel specimens (10 x 10 x 0.5 mm) were degreased in acetone, stored in absolute ethanol, and flamed before use.

[0155] Inoculation of metal coupons with MNV-1 (to mimic contamination by wet contaminated intermediaries) and assessment of infectious virus by detection of cytopathic effects in mouse cell lines (plaque assay) To represent the wet contamination intervention, 20 μL (6 × 10 6 PFU / ml) in MNV-1 1.2 × 10 5 Plaque-forming units (pfu) were inoculated (drying at 22°C for 30-40 minutes). Drying time was included in the exposure time. Virus was removed from the specimens for 2 hours by vortexing for 30 seconds in 2 ml of complete DMEM containing glass beads approximately 50 x 2 mm in diameter. Various dilutions were immediately prepared in complete DMEM, and 1 ml aliquots were plated at 10 per well in a 6-well plate (3.5 cm diameter) 3 hours prior to inoculation. 6 The cells were seeded onto a monolayer of seeded RAW264.7 cells and incubated for 90 minutes at 37°C and 5% CO2. The inoculum was aspirated and 3% low melting point (LMP) agarose in complete medium was added per well to prevent virus spread to other cells. A 3 mL overlay of 1 mL of lysosomes was added. The plates were incubated at 4°C for 15 minutes until hardened, then at 37°C and 5% CO2 for 72 hours. The monolayers were incubated in PBS for 2 hours at 37°C and 5% CO2 with filtered 0.0 mL of Neutral Red, a supravital stain that is pinocytosed by viable cells and accumulates in lysosomes, staining cells red. Staining was performed using 2 mL of a 1% solution per well. Excess stain was removed and the plates were reincubated for an additional hour. Plates were stored overnight at 4°C to enhance the resolution of plaques, which were counted and used to calculate the pfu recovered per specimen.

[0156] Modifications to the above protocol 1. Add virus to a test strip that has been pre-coated in a composition of the present invention. Twenty hours prior to the experiment, steel specimens were coated with 20 μl of the composition of the present invention or sterile distilled water. These liquids were spread over the entire surface and allowed to dry. The specimens were then stored at room temperature until required for the assay, at which time the virus was added on top of this pre-coated layer. The assay was continued as detailed above.

[0157] 2. Add the composition of the present invention to a test strip pre-coated with virus. Forty minutes before starting the assay, 1.2 × 10 MNV-1 in 20 μL was applied to the surface of the test strip. 5 Plaque-forming units (pfu) were inoculated. Once the surface had dried, it was then coated with a composition of the present invention or sterile distilled water. After a 2-hour incubation, the assay continued as described above.

[0158] 3. Pre-coating the test piece with a composition of the present invention, then applying the virus, then the composition of the present invention. Two hours before the start of the assay, steel specimens were coated with 20 μl of a composition of the present invention or sterile distilled water. Forty minutes before the start of the assay, 1.2×10 MNV-1 in 20 μl was applied to the surface of the specimen. 5Plaque-forming units (pfu) were inoculated. Then, once the surface had dried, it was coated with the composition of the present invention or sterile distilled water. After a 2-hour incubation, the assay continued as described above. The specimens received only one type of treatment, with two doses of the composition of the present invention or distilled water.

[0159] Experiments and Results Effect of the composition of the present invention pre-coated on a surface on murine norovirus A 20 μl aliquot of the inventive composition was applied to a stainless steel surface. This was allowed to dry overnight at room temperature to mimic the routine cleaning of the surface. A virus suspension was then applied to the pre-coated surface to mimic the droplet spread caused by human infection during a norovirus outbreak. After 2 hours of incubation on the steel surface pre-coated with the inventive composition, the infectivity of the virus was reduced by 1 log and 2 log, respectively (Table 3 and Figure 8a). This indicates that the inventive composition remained active while drying on the surface for some time, as it had been applied to the steel surface 20 hours prior.

[0160] [Table 3]

[0161] Effect of the composition of the present invention when applied directly to murine norovirus A 20 μl aliquot of the virus suspension was allowed to dry on a steel surface to mimic the virus-containing droplets spread during human infection. This was then treated with 20 μl of the composition of the present invention to mimic a post-outbreak surface cleaning procedure. After 2 hours of incubation, virus infectivity was reduced by 2 logs (Table 3 and Figure 8b).

[0162] Effect of the composition of the present invention applied directly to dry surfaces and murine norovirus A stainless steel surface was pre-coated with a composition of the present invention, a virus suspension was applied to the surface, and then the composition of the present invention was re-applied to the surface and incubated for 2 hours. This study was intended to mimic a routine cleaning procedure, a norovirus outbreak, and then a second surface decontamination. After 2 hours of incubation at room temperature with these applications of the composition of the present invention, a reduction in virus infectivity of at least 3 logs was observed with the composition of the present invention (Table 3 and Figure 8b).

[0163] Summary of Results The composition of the present invention (containing 99.2% water by weight) was able to kill or inactivate murine norovirus (MNV-1) by 2 logs by directly treating contaminated surfaces. When surfaces were pre-treated with the composition of the present invention, a 1-log reduction in surface viral load was achieved due to the long-lasting effect of the composition on the surface, and a greater than 3-log reduction in viral load was observed when surfaces were treated both before and after a viral contamination event (Figure 8c). [Example]

[0164] MICs of various Gram-positive and Gram-negative bacteria for the compositions of the invention. The compositions of the present invention have a broad spectrum of activity at low concentrations, encompassing a wide range of Gram-positive and Gram-negative bacteria, as shown in Figure 9. Figure 9 shows the MICs of the compositions of the present invention against a variety of bacteria in standard bacteriological culture environments.

[0165] Thus, this example illustrates that the compositions of the present invention have a broad spectrum of activity against a range of Gram-positive, Gram-negative, and spore-forming bacteria, which is of interest, for example, in the treatment of chronic wounds. This example, in combination with Examples 1-7 and 11, illustrates that the compositions of the present invention have a broad spectrum of activity against bacteria in the treatment of pathogenic infections, such as pathogenic infections in / on wounds, in humans or animals. [Example]

[0166] Bactericidal activity of the compositions of the present invention under contaminated conditions Tests were conducted by Abbott Analytical Ltd. under confidentiality agreement to evaluate the activity of compositions of the present invention against various bacteria under contaminated conditions (defined below).

[0167] Test methods and their verification Method = Dilution-Neutralization Neutralizer = 30.0 g / l Polysorbate 80 + 3.0 g / l Lecithin + 1.0 g / l L-Histidine + 1.0 g / l L-Cysteine ​​(Neutralizer A) Neutralizer verification = verified according to EN 13727:2012+A2:2015(5.5.2) Experimental conditions Contact time = 5 minutes ± 10 seconds Test temperature: 20±1℃ Interfering substances = 3.0 g / l bovine albumin + 3.0 ml / l sheep red blood cells ("contaminated condition") Incubation temperature Requirements Bactericidal activity was defined as at least a 5.00 log (lg) reduction.

[0168] result

[0169] [Table 4]

[0170] conclusion The composition of the present invention exhibits bactericidal activity against various bacteria under contaminated conditions according to EN 13727:2012+A2:2015. This contaminated condition (as defined above) was selected because it corresponds to the condition found inside / on the surface of human / animal wounds. The range of bacteria tested in this study includes the bacteria most commonly found in wounds, especially chronic wounds. For example, some studies highlight that the majority of chronic wounds contain Staphylococcus aureus, and more than half also contain Pseudomonas aeruginosa. Both of these bacteria, as well as other bacteria commonly found in wounds, were also tested in this study.

[0171] These results demonstrate that the compositions of the present invention, when tested in an environment that mimics a real-life wound environment, have a greater than 5 lg reduction in bacteria most commonly found in wounds. Furthermore, the compositions of the present invention have a greater than 5 lg reduction in bacteria even at very low concentrations, i.e., when the compositions , have been shown to be active against these bacteria even when containing at least 99.2% water by weight (e.g., 99.76% water by weight).

[0172] Thus, it has surprisingly been found that the compositions of the present invention are capable of treating a variety of bacterial infections in / on the wound, even when used in very low concentrations.

Claims

[Claim 1] For use in the treatment or prevention of pathogenic infections in humans or animals, Benzalkonium halides; Didecyldimethylammonium halide; Polyhexamethylene biguanide salts; Bronopol; and p-chloro-m-cresol; A composition comprising:

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