FIELD OF THE INVENTION

The present invention generally relates to methods and compositions useful for reducing or eliminating bacterial biofilms on surfaces. The compositions include thermolysin in combination with an antibacterial agent as active ingredients to reduce or eliminate the bacterial biofilms.

BACKGROUND OF THE INVENTION

Bacterial biofilms are populations of bacteria attached to a surface. Bacteria in a biofilm are frequently embedded within a self-produced matrix of an extracellular polymeric substance (EPS), which holds the bacteria together in a mass and firmly attaches the bacterial mass to the underlying surface. The bacterial biofilm EPS, which is often referred to as slime, is a polymeric conglomeration generally composed of extracellular DNA, proteins, polysaccharides and various biopolymers. Biofilms can form on biological or non-biological surfaces and can be prevalent in both industrial and clinical settings.

Evidence has shown that biofilms constitute a significant threat to human health. Biofilms are responsible for more than 80% of microbial infections in the body (“Research on Microbial Biofilms”, National Institutes of Health, PA Number: PA-03-047, Dec. 20, 2002). Biofilms are involved in health conditions such as urinary tract infections, cystitis, lung infections, skin infections, mucous membrane infections, sinus infections, ear infections, acne, dental caries, periodontitis, nosocomial infections, open wounds, and chronic wounds. Additionally, biofilms can form on medical devices such as: urinary tract prostheses; urinary tract catheters; peritoneal membrane catheters, peritoneal dialysis catheters, indwelling catheters for hemodialysis and for chronic administration of chemotherapeutic agents (Hickman catheters); cardiac implants such as pacemakers, prosthetic heart valves, ventricular assist devices, and synthetic vascular grafts and stents; prostheses; percutaneous sutures; and tracheal and ventilator tubing.

Bacteria growing in biofilms exhibit increased tolerance to antibiotics and antibacterial agents and are very difficult to substantially reduce or eliminate. Bacteria within biofilms have increased tolerance (up to 1000-fold higher) to antibacterial compounds than bacteria not within biofilms, even though these same bacteria are sensitive to these agents if grown under planktonic conditions (“Research on Microbial Biofilms”, National Institutes of Health, PA Number: PA-03-047, Dec. 20, 2002). Bacteria grown in biofilms are also physiologically distinct from the same bacteria grown under planktonic conditions. The bacteria in biofilms are stratified into different metabolic states depending on where in the biofilm they reside and thus display different phenotypes compared to their free-living counterparts. Another theory behind the antimicrobial tolerance of bacteria in biofilms is the protective role of the EPS. The EPS can be visualized as a “mesh” or a network that can physically prevent foreign agents (e.g., antibacterial agents) from reaching the bacteria. Because of the EPS, altered metabolic states and acquired resistance factors, biofilms have a multifactorial tolerance to antibacterial agents and antibiotics. Moreover, many antibacterial formulations are water-based preparations which can, in some instances, make it difficult for the antibacterial active to penetrate the biofilm network due to the high surface tension of water molecules.

Wounds, mucous membrane lesions, and skin lesions are especially susceptible to bacterial infection. From a microbiological perspective, the primary function of normal, intact skin is to control microbial populations that live on the skin surface and to prevent underlying tissue from becoming colonized and invaded by potential pathogens. Exposure of subcutaneous tissue, such as a dermal wound or skin lesion, provides a moist, warm and nutritious environment that is conducive to microbial colonization and proliferation. Since wound colonization is mostly polymicrobial, involving numerous microorganisms that are potentially pathogenic, any wound, mucous membrane lesion, or skin lesion is at some risk of becoming infected.

Wounds often have multiple barriers to healing. Wound healing and infection is influenced by the relationship between the ability of bacteria to create a stable, prosperous community within a wound environment and the ability of the host to control the bacterial community. Since bacteria are rapidly able to form their own protective microenvironment, i.e., a biofilm, following their attachment to a surface, the ability of the host to control these organisms is likely to decrease as the biofilm community matures, ultimately affecting the ability of the wound to heal. Wounds in which healing is delayed, i.e., chronic wounds, are of particular concern with respect to biofilm formation. While biofilms are not present in all bacterial infections, some have linked biofilms to chronic wounds (Mertz, 2003, Wounds, 15: 1-9). Wounds such as diabetic foot ulcers, venous ulcers, arterial ulcers, decubitus ulcers, stasis ulcers, pressure ulcers, and burns are examples of wounds which may become chronic wounds. Bacterial biofilms in chronic wounds are generally not resolved by the host's immune system and these biofilms have an increased tolerance to systemic and topical antibacterial/antibiotic agents. Accordingly, bacterial biofilm infections in chronic wounds are very difficult to substantially reduce or eliminate.

Particularly virulent organisms in wounds, mucous membrane lesions, and skin lesions are gram-positive bacteria such asStaphylococcusspp.,Streptococcusspp., andEnterococcusspp. Biofilms ofStaphylococcus aureus, including resistant strains such as methicillin resistantStaphylococcus aureus(MRSA), have become increasingly problematic in wounds, skin lesions, and mucous membrane lesions. These organisms, especially MRSA, can reside in the anterior nares and cause lesions in the nose which can also spread to other parts of the body, causing skin lesions and mucous membrane lesions at those sites. The gram-negative bacteriaPseudomonas aeruginosais also a particularly virulent organism in wounds (Bjarnsholt, 2008, Wound Repair and Regeneration; and Jacobsen, 2011, International Wound Journal).

In recent years, there have been numerous efforts to use various antibiotics and antibacterial agents for the treatment of mucous membrane lesions, skin lesions, and chronic wounds, many of which are infected or contaminated with bacterial biofilms. These agents are of varying chemical compounds and include, among others, antibacterial agents such as mupirocin (and other antibiotics), iodine compounds, and silver/silver ions. However, many bacteria have become increasingly resistant to some antibacterial compounds.

Thus, there is a need for safe and effective compositions which can reduce or eliminate bacterial biofilms in wounds, mucous membrane lesions, and skin lesions, and on other biological and non-biological surfaces.

SUMMARY OF THE INVENTION

The present invention provides a solution to the aforementioned limitations and deficiencies in the art relating to bacterial biofilms. The solution is premised on the combination of thermolysin with an antibacterial agent to reduce or eliminate bacterial biofilms on surfaces, including biological and non-biological surfaces, when administered directly onto the surfaces. In particular, the combination of thermolysin with an aminoglycoside antibacterial agent surprisingly produces a synergistic antibacterial effect against bacterial biofilms. Stated another way, the synergistic effect means the total antibacterial activity against bacterial biofilms of the combination of the two components, i.e., the thermolysin plus the aminoglycoside antibacterial agent, is greater than the sum of the antibacterial activity against biofilms of each component when measured separately. This combination of thermolysin and an aminoglycoside antibacterial agent can be used to produce a composition capable of: treating wounds, mucous membrane lesions, skin lesions, and/or other biological surfaces infected or contaminated with bacterial biofilms; reducing bacteria in and/or eliminating bacterial biofilms on biological surfaces; and/or reducing bacteria in and/or eliminating bacterial biofilms on non-biological surfaces such as on medical devices when the compositions are administered directly onto the surfaces. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside. Non-limiting examples of aminoglycoside antibacterial agents that are 4,6-disubstituted deoxystreptamine aminoglycosides include kanamycin, amikacin, arbekacin, tobramycin, dibekacin, gentamicin, isepamicin, sisomicin, and netilmicin; and salt forms thereof. In some embodiments, the aminoglycoside antibacterial agent that is a 4,6-disubstituted deoxystreptamine aminoglycoside is gentamicin or a salt form thereof, e.g., gentamicin sulfate. In one instance, the compositions of the present invention can include active agents against biofilms (anti-biofilm active agents that disrupt biofilms) that consist essentially of or consist of only thermolysin and an aminoglycoside antibacterial agent. Stated another way, and in certain embodiments, the compositions of the present invention can include a variety of ingredients but be limited to a combination of thermolysin and an aminoglycoside antibacterial agent as the sole anti-biofilm active agents. In certain other non-limiting aspects, however, the compositions of the present invention can include other anti-biofilm active agents in addition to thermolysin and an aminoglycoside antibacterial agent.

In one aspect of the invention, disclosed is a composition comprising a combination of thermolysin and an aminoglycoside antibacterial agent. In some embodiments, the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm on a biological and/or a non-biological surface. In some embodiments, the combination of thermolysin and the aminoglycoside antibacterial agent exhibits synergistic antibacterial activity against a bacterial biofilm on a biological and/or a non-biological surface. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

In another aspect of the invention, disclosed is a method for treating a wound, mucous membrane lesion, or skin lesion, infected or contaminated with a bacterial biofilm, the method comprising topically administering to the wound, mucous membrane lesion, or skin lesion a composition comprising a combination of thermolysin and an aminoglycoside antibacterial agent, wherein the bacterial biofilm is reduced or eliminated. In some embodiments, the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm in a wound, mucous membrane lesion, or skin lesion infected or contaminated with the bacterial biofilm. In some embodiments, the combination of thermolysin and the aminoglycoside antibacterial agent exhibits synergistic antibacterial activity against a bacterial biofilm in a wound, mucous membrane lesion, or skin lesion infected or contaminated with the bacterial biofilm. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

In another aspect of the invention, disclosed is a method of reducing or eliminating a bacterial biofilm on a biological surface, the method comprising administering to the biological surface a composition comprising a combination of thermolysin and an aminoglycoside antibacterial agent. In some embodiments, the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm on a biological surface. In some embodiments, the combination of thermolysin and the aminoglycoside antibacterial agent exhibits synergistic antibacterial activity against a bacterial biofilm on a biological surface. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

In still another aspect of the invention, disclosed is a method of reducing or eliminating a bacterial biofilm on a non-biological surface, the method comprising administering to the non-biological surface a composition comprising a combination of thermolysin and an aminoglycoside antibacterial agent. In some embodiments, the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm on a non-biological surface. In some embodiments, the combination of thermolysin and the aminoglycoside antibacterial agent exhibits synergistic antibacterial activity against a bacterial biofilm on a non-biological surface. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

In another aspect of the invention, disclosed is an article of manufacture comprising a surface coated with a composition comprising a combination of thermolysin and an aminoglycoside antibacterial agent. In preferred embodiments, the aminoglycoside antibacterial agent is an aminoglycoside antibacterial agent having 4,6-disubstituted deoxystreptamine.

In another aspect of the invention, disclosed is a method of treating a surface of an article of manufacture to prevent or reduce the likelihood of biofilm formation on said surface, the method comprising coating the surface with a composition comprising a combination of thermolysin and an aminoglycoside antibacterial agent. In preferred embodiments, the aminoglycoside antibacterial agent is an aminoglycoside antibacterial agent having 4,6-disubstituted deoxystreptamine.

In still another aspect of the invention, disclosed is a method for treating a wound infected or contaminated with a bacterial biofilm and in need of debridement, the method comprising topically administering to the wound a composition comprising a combination of thermolysin and an aminoglycoside antibacterial agent, wherein the bacterial biofilm is reduced or eliminated, and wherein the wound is debrided. In some embodiments, the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm in a wound infected or contaminated with the bacterial biofilm. In some embodiments, the concentration of thermolysin is at an amount effective to debride a wound in need of debridement. In some embodiments, the combination of thermolysin and the aminoglycoside antibacterial agent exhibits synergistic antibacterial activity against a bacterial biofilm in a wound infected or contaminated with the bacterial biofilm. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

In another aspect of the invention, disclosed is a method of reducing or eliminating a bacterial biofilm on a biological surface, the method comprising administering to the biological surface a first composition comprising thermolysin and a second composition comprising an aminoglycoside antibacterial agent, wherein the first and second compositions are combined. In some embodiments, the first and second compositions are combined prior to administration to a biological surface. In other embodiments, the first and second compositions are combined after administration to a biological surface. In some embodiments, the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm on a biological surface when the first and second compositions are combined. In some embodiments, the combination of the first and second compositions exhibits synergistic antibacterial activity against a biofilm on a biological surface. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

In another aspect of the invention, disclosed is a method of treating a wound, mucous membrane lesion, or skin lesion infected or contaminated with a bacterial biofilm, the method comprising topically administering to the wound, mucous membrane lesion, or skin lesion a first composition comprising thermolysin and a second composition comprising an aminoglycoside antibacterial agent, wherein the first and second compositions are combined, and wherein the bacterial biofilm is reduced or eliminated. In some embodiments, the first and second compositions are combined prior to administration to a wound, mucous membrane lesion, or skin lesion. In other embodiments, the first and second compositions are combined after administration to a wound, mucous membrane lesion, or skin lesion. In some embodiments, the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm on a wound, mucous membrane lesion, or skin lesion infected or contaminated with the bacterial biofilm when the first and second compositions are combined. In some embodiments, the combination of the first and second compositions exhibits synergistic antibacterial activity against a biofilm on a wound, mucous membrane lesion, or skin lesion infected or contaminated with the bacterial biofilm. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

In still another aspect of the invention, disclosed is a method of treating a wound infected or contaminated with a bacterial biofilm and in need of debridement, the method comprising topically administering to the wound a first composition comprising thermolysin and a second composition comprising an aminoglycoside antibacterial agent, wherein the first and second compositions are combined, wherein the bacterial biofilm is reduced or eliminated, and wherein the wound is debrided. In some embodiments, the first and second compositions are combined prior to administration to a wound. In other embodiments, the first and second compositions are combined after administration to a wound. In some embodiments, the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm on a wound when the first and second compositions are combined. In some embodiments, the concentration of thermolysin is at an amount effective to debride a wound in need of debridement. In some embodiments, the combination of the first and second compositions exhibits synergistic antibacterial activity against a biofilm on a wound infected or contaminated with the bacterial biofilm. In preferred embodiments, the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

In another aspect of the invention, disclosed is a kit comprising (a) a first composition comprising thermolysin, and (b) a second composition comprising an aminoglycoside antibacterial agent. In preferred embodiments, the aminoglycoside antibacterial agent in the second composition is a 4,6-disubstituted deoxystreptamine aminoglycoside. In some embodiments, the kit further comprises instructions for administering the first and second compositions to a biological surface, wound, mucous membrane lesion, and/or skin lesion infected or contaminated with a bacterial biofilm. In some embodiments, the instructions include combining the first and second compositions. In other embodiments, the wound is further in need of debridement.

As disclosed herein, compositions that comprise a combination of thermolysin and gentamicin sulfate (a 4,6-disubstituted deoxystreptamine aminoglycoside) exhibited synergistic antibacterial activity against bacterial biofilms when administered directly onto surfaces that were contaminated with bacterial biofilms. This is an unexpected and surprising result because as the data disclosed herein show, the MIC value of gentamicin sulfate in a bacteria suspension is the same as the MIC value of a combination of gentamicin sulfate and thermolysin in a bacteria suspension. Thus, the addition of thermolysin does not increase the bactericidal effectiveness of gentamicin sulfate against bacteria (not present in a biofilm). This is the case for both gram-positive and gram-negative bacteria. However, the combination of gentamicin sulfate and thermolysin does show a synergistic antibacterial effect when the combination is administered directly onto surfaces contaminated with bacterial biofilms. Stated another way, the total antibacterial activity against bacterial biofilms of the combination of thermolysin and gentamicin sulfate is greater than the sum of the antibacterial activity against bacterial biofilms of each component when measured separately. This is the case for both gram-positive and gram-negative bacterial biofilms. The data also show that thermolysin alone has some antibacterial activity against gram-positive bacterial biofilms, but has little to no antibacterial activity against gram-negative bacterial biofilms. Thus, the synergistic antibacterial effect of the combination of thermolysin and gentamicin sulfate seen on gram-negative bacterial biofilms is even more surprising.

Also disclosed in the context of the present invention are embodiments 1 to 142:

Embodiment 1 is a composition comprising a combination of thermolysin and an aminoglycoside antibacterial agent.

Embodiment 2 is the composition of embodiment 1, wherein the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate a bacterial biofilm on a biological and/or a non-biological surface.

Embodiment 3 is the composition of embodiment 2, wherein the combination of thermolysin and the aminoglycoside antibacterial agent exhibits synergistic antibacterial activity against the biofilm on the biological and/or the non-biological surface.

Embodiment 4 the composition of any one of embodiments 1 to 3, wherein the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

Embodiment 5 is the composition of embodiment 4, wherein the aminoglycoside antibacterial agent is kanamycin, amikacin, arbekacin, tobramycin, dibekacin, gentamicin, isepamicin, sisomicin, or netilmicin; or salt forms thereof.

Embodiment 6 is the composition of embodiment 5, wherein the aminoglycoside antibacterial agent is gentamicin sulfate.

Embodiment 7 is the composition of embodiment 6, wherein the concentration of thermolysin is 0.1 to 1% w/w and the concentration of gentamicin sulfate is equivalent to a concentration of gentamicin of 0.1 to 1% w/w.

Embodiment 8 is the composition of any one of embodiments 1 to 7, wherein the composition further comprises a carrier suitable for application to a biological and/or a non-biological surface.

Embodiment 9 is the composition of embodiment 8, wherein the carrier is a lotion, solution, suspension, liquid, emulsion, cream, gel, ointment, paste, aerosol spray, aerosol foam, non-aerosol spray, non-aerosol foam, film, or sheet.

Embodiment 10 is the composition of embodiment 9, wherein the carrier is suitable for topical administration.

Embodiment 11 is a method of treating a wound, mucous membrane lesion, or skin lesion infected or contaminated with a bacterial biofilm, the method comprising topically administering to the wound, mucous membrane lesion, or skin lesion the composition of any one of embodiments 1 to 10, wherein the bacterial biofilm is reduced or eliminated.
Embodiment 12 is the method of embodiment 11, wherein the bacterial biofilm comprises at least one gram-positive bacterial species.
Embodiment 13 is the method of embodiment 12, wherein the gram-positive bacterial species is aStaphylococcussp.
Embodiment 14 is the method of embodiment 13, wherein theStaphylococcussp. isStaphylococcus aureus.
Embodiment 15 is the method of embodiment 13, wherein theStaphylococcussp. is methicillin resistantStaphylococcus aureus(MRSA).
Embodiment 16 is the method of embodiment 11, wherein the bacterial biofilm comprises at least one gram-negative bacterial species.
Embodiment 17 is the method of embodiment 16, wherein the gram-negative bacterial species is aPseudomonassp.
Embodiment 18 is the method of embodiment 17, wherein thePseudomonassp. isPseudomonas aeruginosa.
Embodiment 19 is the method of embodiment 11, wherein the bacterial biofilm comprises at least one gram-positive bacterial species and at least one gram-negative bacterial species.
Embodiment 20 is the method of any one of embodiments 11 to 19, wherein the wound is a chronic wound, acute wound, or burn.
Embodiment 21 is the method of embodiment 20, wherein the chronic wound is a diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer, stasis ulcer, or pressure ulcer.
Embodiment 22 is the method of any one of embodiments 11 to 19, wherein the skin lesion or mucous membrane lesion is a blister, ulceration, abrasion, wart, scrape, or infection.
Embodiment 23 is a method of reducing or eliminating a bacterial biofilm on a biological surface, the method comprising administering to the biological surface the composition of any one of embodiments 1 to 10.
Embodiment 24 is the method of embodiment 23, wherein the bacterial biofilm comprises at least one gram-positive bacterial species.
Embodiment 25 is the method of embodiment 24, wherein the gram-positive bacterial species is aStaphylococcussp.
Embodiment 26 is the method of embodiment 25, wherein theStaphylococcussp. isStaphylococcus aureus.
Embodiment 27 is the method of embodiment 25, wherein theStaphylococcussp. is methicillin resistantStaphylococcus aureus(MRSA).
Embodiment 28 is the method of embodiment 23 wherein the bacterial biofilm comprises at least one gram-negative bacterial species.
Embodiment 29 is the method of embodiment 28, wherein the gram-negative bacterial species is aPseudomonassp.
Embodiment 30 is the method of embodiment 29, wherein thePseudomonassp. isPseudomonas aeruginosa.
Embodiment 31 is the method of embodiment 23, wherein the bacterial biofilm comprises at least one gram-positive bacterial species and at least one gram-negative bacterial species.
Embodiment 32 is the method of any one of embodiments 23 to 31, wherein the biological surface is a wound.
Embodiment 33 is the method of embodiment 32, wherein the wound is a chronic wound, acute wound, or burn.
Embodiment 34 is the method of embodiment 33, wherein the chronic wound is a diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer, stasis ulcer, or pressure ulcer.
Embodiment 35 is the method of any one of embodiments 23 to 31, wherein the biological surface is a skin lesion or mucous membrane lesion.
Embodiment 36 is the method of embodiment 35, wherein the skin lesion or mucous membrane lesion is a blister, ulceration, abrasion, wart, abscess, scrape, or infection.
Embodiment 37 is the method of any one of embodiments 23 to 31, wherein the biological surface is an internal organ, a body cavity, an oral cavity, a bone tissue, a muscle tissue, a nerve tissue, an ocular tissue, a urinary tract tissue, a lung tissue, a trachea tissue, a sinus tissue, an ear tissue, a dental tissue, a gum tissue, a nasal tissue, a vascular tissue, a cardiac tissue, an epithelium tissue, an epithelial lesion, a vaginal tissue, or a peritoneal tissue.
Embodiment 38 is a method of reducing or eliminating a bacterial biofilm on a non-biological surface, the method comprising administering to the non-biological surface the composition of any one of embodiments 1 to 10.
Embodiment 39 is the method of embodiment 38, wherein the non-biological surface is the surface of a medical device.
Embodiment 40 is the method of embodiment 39, wherein the medical device is a urinary tract prosthesis, urinary tract catheter, peritoneal membrane catheter, peritoneal dialysis catheter, indwelling catheter for hemodialysis, indwelling catheter for administration of chemotherapeutic agents, cardiac implant, pacemaker, prosthetic heart valve, ventricular assist device, synthetic vascular graft, synthetic vascular stent, prosthesis, percutaneous suture, tracheal tubing, or ventilator tubing.
Embodiment 41 is an article of manufacture comprising a surface coated with the composition of any one of embodiments 1 to 10.
Embodiment 42 is the article of manufacture of embodiment 41, wherein the article of manufacture is a medical device.
Embodiment 43 is the article of manufacture of embodiment 42, wherein the medical device is a urinary tract prosthesis, urinary tract catheter, peritoneal membrane catheter, peritoneal dialysis catheter, indwelling catheter for hemodialysis, indwelling catheter for administration of chemotherapeutic agents, cardiac implant, pacemaker, prosthetic heart valve, ventricular assist device, synthetic vascular graft, synthetic vascular stent, prosthesis, percutaneous suture, tracheal tubing, or ventilator tubing.
Embodiment 44 is the article of manufacture of any one of embodiments 41 to 43, wherein a biofilm is not present on the surface of the article of manufacture prior to coating with the composition.
Embodiment 45 is the article of manufacture of any one of embodiments 41 to 43, wherein a biofilm is present on the surface of the article of manufacture prior to coating the surface with the composition.
Embodiment 46 is a method of treating a surface of an article of manufacture to prevent or reduce the likelihood of biofilm formation on said surface, the method comprising coating the surface with the composition of any one of embodiments 1 to 10.
Embodiment 47 is the method of embodiment 46, wherein the article of manufacture is a medical device.
Embodiment 48 is the method of embodiment 47, wherein the medical device is a urinary tract prosthesis, urinary tract catheter, peritoneal membrane catheter, peritoneal dialysis catheter, indwelling catheter for hemodialysis, indwelling catheter for administration of chemotherapeutic agents, cardiac implant, pacemaker, prosthetic heart valve, ventricular assist device, synthetic vascular graft, synthetic vascular stent, prosthesis, percutaneous suture, tracheal tubing, or ventilator tubing.
Embodiment 49 is a method of treating a wound infected or contaminated with a bacterial biofilm and in need of debridement, the method comprising topically administering to the wound the composition of any one of embodiments 1 to 10, wherein the concentration of thermolysin is at an amount effective to debride the wound, wherein the bacterial biofilm is reduced or eliminated, and wherein the wound is debrided.
Embodiment 50 is the method of embodiment 49, wherein the bacterial biofilm comprises at least one gram-positive bacterial species.
Embodiment 51 is the method of embodiment 50, wherein the gram-positive bacterial species is aStaphylococcussp.
Embodiment 52 is the method of embodiment 51, wherein theStaphylococcussp. isStaphylococcus aureus.
Embodiment 53 is the method of embodiment 51, wherein theStaphylococcussp. is methicillin resistantStaphylococcus aureus(MRSA).
Embodiment 54 is the method of embodiment 49, wherein the bacterial biofilm comprises at least one gram-negative bacterial species.
Embodiment 55 is the method of embodiment 54, wherein the gram-negative bacterial species is aPseudomonassp.
Embodiment 56 is the method of embodiment 55, wherein thePseudomonassp. isPseudomonas aeruginosa.
Embodiment 57 is the method of embodiment 49, wherein the bacterial biofilm comprises at least one gram-positive bacterial species and at least one gram-negative bacterial species.
Embodiment 58 is the method of any one of embodiments 49 to 57, wherein the wound is a chronic wound, acute wound, or burn.
Embodiment 59 is the method of embodiment 58, wherein the chronic wound is a diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer, stasis ulcer, or pressure ulcer.
Embodiment 60 is a method of reducing or eliminating a bacterial biofilm on a biological surface, the method comprising administering to the biological surface a first composition comprising thermolysin and a second composition comprising an aminoglycoside antibacterial agent, wherein the first and second compositions are combined.
Embodiment 61 is the method of embodiment 60, wherein the first and second compositions are combined prior to administration to the biological surface.
Embodiment 62 is the method of embodiment 60, wherein the first and second compositions are combined after administration to the biological surface.
Embodiment 63 is the method of any of embodiments 60 to 62, wherein the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate the bacterial biofilm on the biological surface when the first and second compositions are combined.
Embodiment 64 is the method of any one of embodiment 60 to 63, wherein the combination of the first and second compositions exhibits synergistic antibacterial activity against the biofilm on the biological surface.
Embodiment 65 is the method of any one of embodiments 60 to 64, wherein the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.
Embodiment 66 is the method of embodiment 65, wherein the aminoglycoside antibacterial agent is kanamycin, amikacin, arbekacin, tobramycin, dibekacin, gentamicin, isepamicin, sisomicin, or netilmicin; or salt forms thereof.
Embodiment 67 is the method of embodiment 66, wherein the aminoglycoside antibacterial agent is gentamicin sulfate.
Embodiment 68 is the method of embodiment 67, wherein the concentration of thermolysin is 0.1 to 1% w/w and the concentration of gentamicin sulfate is equivalent to a concentration of gentamicin of 0.1 to 1% w/w.
Embodiment 69 is the method of any one of embodiments 60 to 68, wherein the first composition and the second composition further comprises a carrier suitable for application to a biological surface.
Embodiment 70 is the method of embodiment 69, wherein the carrier is a lotion, solution, suspension, liquid, emulsion, cream, gel, ointment, paste, aerosol spray, aerosol foam, non-aerosol spray, non-aerosol foam, film, or sheet.
Embodiment 71 is the method of embodiment 70, wherein the carrier is suitable for topical administration.
Embodiment 72 is the method of any of embodiments 60 to 71, wherein the bacterial biofilm comprises at least one gram-positive bacterial species.
Embodiment 73 is the method of embodiment 72, wherein the gram-positive bacterial species is aStaphylococcussp.
Embodiment 74 is the method of embodiment 73, wherein theStaphylococcussp. isStaphylococcus aureus.
Embodiment 75 is the method of embodiment 73, wherein theStaphylococcussp. is methicillin resistantStaphylococcus aureus(MRSA).
Embodiment 76 is the method of any one of embodiments 60 to 71, wherein the bacterial biofilm comprises at least one gram-negative bacterial species.
Embodiment 77 is the method of embodiment 76, wherein the gram-negative bacterial species is aPseudomonassp.
Embodiment 78 is the method of embodiment 77, wherein thePseudomonassp. isPseudomonas aeruginosa.
Embodiment 79 is the method of any one of embodiments 60 to 71, wherein the bacterial biofilm comprises at least one gram-positive bacterial species and at least one gram-negative bacterial species.
Embodiment 80 is the method of any one of embodiments 60 to 79, wherein the biological surface is a wound.
Embodiment 81 is the method of embodiment 80, wherein the wound is a chronic wound, acute wound, or burn.
Embodiment 82 is the method of embodiment 81, wherein the chronic wound is a diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer, stasis ulcer, or pressure ulcer.
Embodiment 83 is the method of any one of embodiments 60 to 79, wherein the biological surface is a skin lesion or mucous membrane lesion.
Embodiment 84 is the method of embodiment 83, wherein the skin lesion or mucous membrane lesion is a blister, ulceration, abrasion, wart, abscess, scrape, or infection.
Embodiment 85 is the method of any one of embodiments 60 to 79, wherein the biological surface is an internal organ, a body cavity, an oral cavity, a bone tissue, a muscle tissue, a nerve tissue, an ocular tissue, a urinary tract tissue, a lung tissue, a trachea tissue, a sinus tissue, an ear tissue, a dental tissue, a gum tissue, a nasal tissue, a vascular tissue, a cardiac tissue, an epithelium tissue, an epithelial lesion, a vaginal tissue, or a peritoneal tissue.
Embodiment 86 is a method of treating a wound, mucous membrane lesion, or skin lesion infected or contaminated with a bacterial biofilm, the method comprising topically administering to the wound, mucous membrane lesion, or skin lesion a first composition comprising thermolysin and a second composition comprising an aminoglycoside antibacterial agent, wherein the first and second compositions are combined, and wherein the bacterial biofilm is reduced or eliminated.
Embodiment 87 is the method of embodiment 86, wherein the first and second compositions are combined prior to administration to the wound, mucous membrane lesion, or skin lesion.
Embodiment 88 is the method of embodiment 86, wherein the first and second compositions are combined after administration to the wound, mucous membrane lesion, or skin lesion.
Embodiment 89 is the method of any of embodiments 86 to 88, wherein the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate the bacterial biofilm on the wound, mucous membrane lesion, or skin lesion when the first and second compositions are combined.
Embodiment 90 is the method of any one of embodiments 86 to 89, wherein the combination of the first and second compositions exhibits synergistic antibacterial activity against the biofilm on the wound, mucous membrane lesion, or skin lesion.
Embodiment 91 is the method of any one of embodiments 86 to 90, wherein the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.
Embodiment 92 is the method of embodiment 91, wherein the aminoglycoside antibacterial agent is kanamycin, amikacin, arbekacin, tobramycin, dibekacin, gentamicin, isepamicin, sisomicin, or netilmicin; or salt forms thereof.
Embodiment 93 is the method of embodiment 92, wherein the aminoglycoside antibacterial agent is gentamicin sulfate.
Embodiment 94 is the method of embodiment 93, wherein the concentration of thermolysin is 0.1 to 1% w/w and the concentration of gentamicin sulfate is equivalent to a concentration of gentamicin of 0.1 to 1% w/w.
Embodiment 95 is the method of any one of embodiments 86 to 94, wherein the first composition and the second composition further comprises a carrier suitable for topical administration.
Embodiment 96 is the method of embodiment 95, wherein the carrier is a lotion, solution, suspension, liquid, emulsion, cream, gel, ointment, paste, aerosol spray, aerosol foam, non-aerosol spray, non-aerosol foam, film, or sheet.
Embodiment 97 is the method of any one of embodiment 86 to 96, wherein the bacterial biofilm comprises at least one gram-positive bacterial species.
Embodiment 98 is the method of embodiment 97, wherein the gram-positive bacterial species is aStaphylococcussp.
Embodiment 99 is the method of embodiment 98, wherein theStaphylococcussp. isStaphylococcus aureus.
Embodiment 100 is the method of embodiment 98, wherein theStaphylococcussp. is methicillin resistantStaphylococcus aureus(MRSA).
Embodiment 101 is the method of any one of embodiments 86 to 96, wherein the bacterial biofilm comprises at least one gram-negative bacterial species.
Embodiment 102 is the method of embodiment 101, wherein the gram-negative bacterial species is aPseudomonassp.
Embodiment 103 is the method of embodiment 102, wherein thePseudomonassp. isPseudomonas aeruginosa.
Embodiment 104 is the method of any one of embodiments 86 to 96, wherein the bacterial biofilm comprises at least one gram-positive bacterial species and at least one gram-negative bacterial species.
Embodiment 105 is the method of any one of embodiments 86 to 104, wherein the wound is a chronic wound, acute wound, or burn.
Embodiment 106 is the method of embodiment 105, wherein the chronic wound is a diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer, stasis ulcer, or pressure ulcer.
Embodiment 107 is the method of any one of embodiments 86 to 104, wherein the skin lesion or mucous membrane lesion is a blister, ulceration, abrasion, wart, scrape, or infection.
Embodiment 108 is a method of treating a wound infected or contaminated with a bacterial biofilm and in need of debridement, the method comprising topically administering to the wound a first composition comprising thermolysin and a second composition comprising an aminoglycoside antibacterial agent, wherein the first and second compositions are combined, wherein the bacterial biofilm is reduced or eliminated, and wherein the wound is debrided.
Embodiment 109 is the method of embodiment 108, wherein the first and second compositions are combined prior to administration to the wound.
Embodiment 110 is the method of embodiment 108, wherein the first and second compositions are combined after administration to the wound.
Embodiment 111 is the method of any of embodiments 108 to 110, wherein the concentrations of thermolysin and the aminoglycoside antibacterial agent are at amounts effective to reduce or eliminate the bacterial biofilm on the wound when the first and second compositions are combined.
Embodiment 112 is the method of any of embodiments 108 to 110, wherein the concentration of thermolysin is at an amount effective to debride the wound.
Embodiment 113 is the method of any one of embodiments 108 or 112, wherein the combination of the first and second compositions exhibits synergistic antibacterial activity against the biofilm on the wound.
Embodiment 114 is the method of any one of embodiments 108 to 113, wherein the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.
Embodiment 115 is the method of embodiment 114, wherein the aminoglycoside antibacterial agent is kanamycin, amikacin, arbekacin, tobramycin, dibekacin, gentamicin, isepamicin, sisomicin, or netilmicin; or salt forms thereof.
Embodiment 116 is the method of embodiment 115, wherein the aminoglycoside antibacterial agent is gentamicin sulfate.
Embodiment 117 is the method of embodiment 116, wherein the concentration of thermolysin is 0.1 to 1% w/w and the concentration of gentamicin sulfate is equivalent to a concentration of gentamicin of 0.1 to 1% w/w.
Embodiment 118 is the method of any one of embodiments 108 to 117, wherein the first composition and the second composition further comprises a carrier suitable for topical administration.
Embodiment 119 is the method of embodiment 118, wherein the carrier is a lotion, solution, suspension, liquid, emulsion, cream, gel, ointment, paste, aerosol spray, aerosol foam, non-aerosol spray, non-aerosol foam, film, or sheet.
Embodiment 120 is the method of any one of embodiments 108 to 119, wherein the bacterial biofilm comprises at least one gram-positive bacterial species.
Embodiment 121 is the method of embodiment 120, wherein the gram-positive bacterial species is aStaphylococcussp.
Embodiment 122 is the method of embodiment 121, wherein theStaphylococcussp. isStaphylococcus aureus.
Embodiment 123 is the method of embodiment 121, wherein theStaphylococcussp. is methicillin resistantStaphylococcus aureus(MRSA).
Embodiment 124 is the method of any one of embodiments 108 to 119, wherein the bacterial biofilm comprises at least one gram-negative bacterial species.
Embodiment 125 is the method of embodiment 124, wherein the gram-negative bacterial species is aPseudomonassp.
Embodiment 126 is the method of embodiment 125, wherein thePseudomonassp. isPseudomonas aeruginosa.
Embodiment 127 is the method of any one of embodiments 108 to 119, wherein the bacterial biofilm comprises at least one gram-positive bacterial species and at least one gram-negative bacterial species.
Embodiment 128 is the method of any one of embodiments 108 to 127, wherein the wound is a chronic wound, acute wound, or burn.
Embodiment 129 is the method of embodiment 128, wherein the chronic wound is a diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer, stasis ulcer, or pressure ulcer.
Embodiment 130 is a kit comprising:

(a) a first composition comprising thermolysin, and

(b) a second composition comprising an aminoglycoside antibacterial agent.

Embodiment 131 is the kit of embodiment 130, wherein the aminoglycoside antibacterial agent is a 4,6-disubstituted deoxystreptamine aminoglycoside.

Embodiment 132 is the kit of embodiment 131, wherein the aminoglycoside antibacterial agent is kanamycin, amikacin, arbekacin, tobramycin, dibekacin, gentamicin, isepamicin, sisomicin, or netilmicin; or salt forms thereof.

Embodiment 133 is the kit of embodiment 132, wherein the aminoglycoside antibacterial agent is gentamicin sulfate.

Embodiment 134 is the kit of embodiment 133, wherein the concentration of thermolysin is 0.1 to 1% w/w and the concentration of gentamicin sulfate is equivalent to a concentration of gentamicin of 0.1 to 1% w/w.

Embodiment 135 is the kit of any one of embodiments 130 to 134, wherein the first composition and the second composition further comprise a carrier suitable for application to a biological surface.

Embodiment 136 is the kit of embodiment 135, wherein the carrier is a lotion, solution, suspension, liquid, emulsion, cream, gel, ointment, paste, aerosol spray, aerosol foam, non-aerosol spray, non-aerosol foam, film, or sheet.

Embodiment 137 is the kit of embodiment 136, wherein the carrier is suitable for topical administration.

Embodiment 138 is the kit of any one of embodiments 130 to 137, further comprising instructions for administering the first and second compositions to a biological surface, wound, mucous membrane lesion, and/or skin lesion; wherein the wound, mucous membrane lesion, and/or skin lesion is infected or contaminated with a bacterial biofilm.
Embodiment 139 is the kit of embodiment 138, wherein the instructions include combining the first and second compositions.
Embodiment 140 is the kit of embodiment 139, wherein the instructions include combining the first and second compositions prior to administration to the wound, mucous membrane lesion, and/or skin lesion.
Embodiment 141 is the kit of embodiment 139, wherein the instructions include combining the first and second compositions after administration to the wound, mucous membrane lesion, and/or skin lesion.
Embodiment 142 is the kit of any one of embodiments 138 to 141, wherein the wound is further in need of debridement.

Unless otherwise specified, the percent values expressed herein are weight by weight and are in relation to the weight of the total composition. By way of example, 10 grams of component in 100 grams of material is 10% w/w of component.

For purposes of this application, a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e. round up if the number being rounded is 5, 6, 7, 8, or 9; and round down if the number being rounded is 0, 1, 2, 3, or 4. For example, 0.42 can be rounded to 0.4.

The terms “reduce”, “reduced”, “reducing”, or “reduction” in the context of a bacterial biofilm means a reduction in the count of bacteria in the biofilm.

The terms “treat,” “treated,” “treatment”, or “treating,” in the context of treating a bacterial biofilm on a biological surface, or treating a mucous membrane lesion, a wound, or a skin lesion, means any measurable reduction or complete elimination of the bacterial biofilm, and/or a therapeutic improvement of the mucous membrane lesion, wound, or skin lesion.

The term “effective,” in the context of treating a bacterial biofilm or treating a wound, mucous membrane lesion, or skin lesion means adequate to accomplish a desired, expected, or intended result, including a therapeutic improvement.

The terms “eliminate”, “eliminated”, “eliminating”, or “elimination” in the context of a bacterial biofilm means total eradication of the bacteria present in the biofilm.

The terms “prevent”, “prevented”, or “preventing” in the context of a bacterial biofilm means reduced likelihood or complete prevention of bacterial biofilm formation on a surface such as a biological or a non-biological surface that has been coated with a composition of the present invention.

The term “wound” as used herein means an external wound of the skin or mucous membranes and includes chronic and acute wounds.

The term “lesion” as used herein means a region on a bodily tissue that has suffered damage through injury or disease.

As used herein, the terms “administer”, “administration”, “administering” in the context of applying a composition to a biological surface means physically placing the composition directly onto the surface, i.e., not systemic, intravenous, or oral administration.

The terms “about” or “approximately” are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The use of the word “a” or “an” when used in conjunction with the terms “comprising”, “having”, “including”, or “containing” (or any variations of these words) may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. With respect to the transitional phase “consisting essentially of,” in one non-limiting aspect, a basic and novel characteristic of the compositions and methods disclosed in this specification includes the ability of the combination of thermolysin and an aminoglycoside antibacterial agent to disrupt bacterial biofilms.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa.

Furthermore, compositions of the invention can be used to achieve methods of the invention.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

