WO2021174284A1 - Combination treatment for microorganisms - Google Patents

Abstract

Microbial infections from bacteria and fungi are a growing health concern worldwide. The present invention relates to methods, compositions and their uses for the treatment of bacterial and fungal infections as well as the treatment of biofilm formation. Specifically disclosed are compositions including diethyldithiocarbamate, and copper cations which are useful in the prevention of bacterial and fungal growth. Further disclosed are medical implants and devices treated with diethyldithiocarbamate in combination with copper cations.

Description

Title of Invention

Combination Treatment for Microorganisms

Technical Field

[0001 ] This application claims priority from Australian Provisional Application 2020900675 filed on 5 March 2020, the contents of which is incorporated by reference.

[0002] The present invention relates generally to antimicrobial compositions, solutions and methods for their use. In particular, the compositions comprise a combination of diethyldithiocarbamate and copper which act in coordination to inhibit the growth of, or kill, microorganism such as bacteria and fungi. Further, the present invention relates to combination treatments with antimicrobial agents such as antibiotics, antiseptics, and antifungals.

Background of Invention

[0003] Microbes and higher organisms have coevolved to have reciprocal adaptation. Higher organisms provide microbes with the ideal environment for growth. They provide a source of nutrients, water, protection and in many cases warmth that allow the microbes to replicate. Consequently, most higher organisms, such as humans are colonised internally and externally with a wide variety of microorganism, many of which are harmless or beneficial. However, in some situations, microorganism growth becomes pathogenic to the host. This often happens when non-commensal bacteria colonise the host, or the integrity of the host is compromise such as a laceration or immune compromise, leading to infection.

[0004] Typically, higher organisms have developed complex immune systems to prevent being overrun by microorganisms, and these immune systems will resolve the infection. In response microorganisms have adapted and evolve to develop means to avoid immunological detection and elimination. Consequently, in some situations the immune system is unable to resolve the infection. Therefore, in these situations, it is desirable to restrict the growth of the microorganism using exogenous agents.

[0005] Between the late 1920s to the late 1930s Alexander Fleming, Howard Florey and Ernst Chain discovered, purified and identified the therapeutic action of penicillin. This ushered in the era of antibiotics, which allowed the treatment of serious bacterial infections.

[0006] However, the consistent adaptation of microorganisms and the excessive and prolonged use of antibiotics has led to the emergence of microorganisms resistant to antimicrobials such as antibiotics. Consequently, infectious microorganisms have become increasingly resistant to the existing arsenal of antimicrobial drugs, including resistance to all first-line and many last-resort antimicrobials.

[0007] The most recent World Economic Forum Global Risks report estimated that antimicrobial resistance was responsible for 23,000 deaths annually in the United States, more than 25,000 in Europe, and 700,000 globally. Further, according to a report commissioned by the World Health Organisation this number is expected to rise to 10 million deaths per year by 2050. The associated economic cost is staggering with a current estimated 21 - to 34-billion- dollar burden to the United States alone each year.

[0008] Accordingly, there is a clear need to provide novel approaches to treating microbial infections of to address the issue of antimicrobial resistance. Therefore, the present invention aims to provide an alternative to current antimicrobial treatments. Further, the present invention may supplement or augment current antimicrobial treatments to improve their efficacy.

[0009] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Summary of Invention

[0010] The present invention is predicated, in part, on the work of the Inventor who has surprisingly determined that diethyldithiocarbamate (DDC) forms a working inter-relationship with copper in inhibiting the growth of, or killing, a broad spectrum of microbes. This includes bacterial biofilms which are typically resistant to antibiotic and antiseptic treatments. Further, the Inventor has established that DDC and copper, when combined, potentiate a range of antimicrobial treatments. This enables alternative treatments to those presently available for microbial infections, particularly external infections of a subject or colonisation of objects such as surgical devices and implants.

[0011] The present invention relates to compositions, solutions and methods useful in treating microbial infections and colonization. Accordingly, the present disclosure provides an antimicrobial composition including: copper, or a salt thereof, and diethyldithiocarbamate (DDC), or a salt thereof. DDC in combination with copper acts to effectively inhibit the growth of, or kill, a range of microorganisms and therefore is useful in preventing or treating microbial infections or colonisation. [0012] In some embodiments of the antimicrobial composition the weight to weight ratio of copper, or the salt thereof, to DCC, or the salt thereof, is greater than or equal to 1 :1. Further, in some embodiments, the weight to weight ratio of copper, or the salt thereof, to DCC, or the salt thereof, is less than or equal to 8:1 .

[0013] As will be understood, depending of which (if any) salt of DDC and copper is used, the mole to mole ratio will differ for the same weight. Therefore, in some embodiments, the mole to mole ratio of copper, or the salt thereof, to DCC, or the salt thereof, is less than or equal to 8:1. Further in some embodiments, the mole to mole ratio of copper, or the salt thereof, to DCC, or the salt thereof, is greater than or equal to 1 :1. In some embodiments, the mole to mole ratio of copper, or the salt thereof, to DCC, or the salt thereof, is less than or equal to about 11 :1. Further in some embodiments, the mole to mole ratio of copper, or the salt thereof, to DCC, or the salt thereof, is greater than or equal about 1.5:1 , or is greater than or equal to 1 .4 to 1 . Ratios of copper, or a salt thereof, to DDC, or a salt thereof, in these ranges provides optimal efficacy against a range of microorganism.

[0014] The present invention may be used to inhibit the growth of, or kill, a bacterium. Bacteria can take the form of planktonic bacteria or can form biofilms. Therefore, in some embodiments, the antimicrobial composition is for inhibiting the growth of, or killing, a planktonic bacterium. Further, in some embodiments, the antimicrobial composition is for inhibiting the growth of, or killing, a bacterium in a biofilm.

[0015] In some embodiments, the antimicrobial composition is for inhibiting the growth of, or killing, a gram-positive bacterium. These include bacterium selected from the genus: Enterococcus or Staphylococcus.

[0016] In some embodiments, the antimicrobial composition is for inhibiting bacterial strains selected from the species: Enterococcus faecium, Enterococcis faecalisor, Staphylococcus aureus or Moraxella catarrhalis. In some embodiments, the bacterium is Methicillin-resistant Staphylococcus aureus.

[0017] The present invention may be used to inhibit the growth of or kill, a fungus. Accordingly, in some embodiments, the antimicrobial composition is for inhibiting the growth of, or killing, a fungus. In some embodiments, the fungus is a member of the Candida genus. In some embodiments, the fungus is a strain of Candida albicans, Candida auris, Candida dubliniensis, Candida kruzei or Candida parapsilosis.

[0018] The composition of the present invention may be useful for the treatment of microbial infections in subjects, such as mammalian subjects. As such, in some embodiments, the antimicrobial composition is a pharmaceutical composition and includes a pharmaceutically acceptable excipient. Further, in some embodiments, the antimicrobial composition is suitable for the administration or application to a mammalian subject, preferably a human subject.

[0019] In some embodiment, the composition is for topical treatment. In some embodiment the topical treatment is treatment of a wound, treatment of keratinised skin, treatment of a mucosal surface, treatment of the ear or treatment of the sinuses. Further, in some embodiments, the antimicrobial composition of the present invention is for treatment of a surgical device or implant.

[0020] In some embodiments, the antimicrobial composition further includes an antimicrobial agent. In some embodiments, the antimicrobial agent is an antibiotic, an antiseptic or an antifungal. In some embodiments, the antimicrobial is suitable for administration to a mammalian subject, preferably a human subject

[0021] In some embodiments of the composition including an antibody, the antibiotic is selected from the group consisting of a protein synthesis inhibitor, a cell wall synthesis inhibitor, a beta-lactam antibiotic, a beta-lactamase inhibitor, a lipopeptide, a peptidoglycan synthesis inhibitor, a DNA synthesis inhibitor, a RNA synthesis inhibitor, a mycolic acid synthesis inhibitor, a mechanosensitive channel of large conductance (MscL) or a folic acid synthesis inhibitor, or a combination of the aforementioned antibiotics.

[0022] In some embodiments of the composition including an antibiotic, the antibiotic is selected from the group consisting of: a quinolone antibiotic, a tetracycline antibiotic, a glycopeptide antibiotic, an aminoglycoside antibiotic, a penicillin antibiotic, a cephalosporin antibiotic, a cephamycin antibiotic or a macrolide antibiotic. In some embodiments, the antibiotic is ciprofloxacin, doxycycline, vancomycin, gentamycin, amoxicillin, ceftazidime or erythromycin.

[0023] In some embodiments of the composition including an antiseptic, the antiseptic is selected from the group consisting of: an alcohol, benzethonium chloride, chlorhexidine, hexachlorophene, hydrogen peroxide, iodine, octenidine dihydrochloride, oxychlorosene sodium, polyhexanide, povidone iodine, sodium hypochlorite, or triclosan. Preferred alcohols include ethanol or isopropanol.

[0024] In some embodiments of the composition including an antifungal, the antifungal is selected from the group consisting of: azole antifungals, an allylamine, echinocandins or polyenes. In some embodiments, the antifungal is one or more of: amphotericin B, anidulafungin, candicidin, caspofungin, clotrimazole, filipin, fluconazole, flucytosine, griseofulvin, hamycin, isavuconazonium, itraconazole, ketoconazole, micafungin, miconazole, natamycin, nystatin, posaconazole, rimocidin, terbinafine or voriconazole.

[0025] In some embodiments, the composition is a solution. Accordingly, when the composition is a solution the DDC and copper dissolute to anions and cations respectively. Therefore, the present disclosure provides an antimicrobial solution including copper cations and DDC anions.

[0026] Copper can exist in differing oxidative states. Therefore, in some embodiments, the copper is a divalent cation. In some embodiments, the copper is a monovalent cation.

[0027] In some embodiments of the solution, the mol to mole ratio of copper cations to DCC anions in solution is greater than or equal to about 1 :1. In some embodiments, the molar to molar ratio of copper cations to DCC anions in solution is less than or equal to about 8:1

[0028] In some embodiments of the solution, the mole to mole ratio of copper cations to DCC anions in solution is greater than or equal to about 1 .4:1 . In some embodiments, the mole to mole ratio of copper cations to DCC anions in solution is less than or equal to about 11 .2:1

[0029] In some embodiments of the solution, the concentration of DDC, or a salt thereof, in solution is at least about 0.5 pg/ml. In some embodiments of the solution, the concentration of DDC, or a salt thereof, in solution is at least about 0.25 pg/ml.

[0030] In some embodiments of the solution, the concentration of copper, or a salt thereof, in solution is at least about 0.5 pg/ml. In some embodiments of the solution, the concentration of copper, or a salt thereof, in solution is at least about 0.25 pg/ml.

[0031 ] Further provided by the present disclosure is a surgical implant or device including an effective amount of DDC, or a salt thereof, and copper, or a salt thereof. In some embodiments, the surgical implant or device is impregnated with an effective amount of DDC, or a salt thereof, and copper, or a salt thereof. In some embodiments, the surgical implant or device is coated with an effective amount of DDC, or a salt thereof, and copper, or a salt thereof.

[0032] In some embodiments, the surgical device or surgical implant includes, or is formed of, polyester, or polypropylene. In some embodiments, the surgical implant is an implantable mesh.

[0033] Also provided by the present disclosure is a method of inhibiting the growth of a bacterium or a fungus, or killing a bacterium or a fungus, or preventing the formation of a bacterial or fungal biofilm, or treating a bacterial or fungal biofilm, the method comprising, providing, or exposing the bacterium or fungus or biofilm to, an effective amount of the antimicrobial composition or solution as disclosed herein.

[0034] In some embodiments of the method, the bacterium is a gram-positive bacterium. In some embodiments, the bacterium is a strain selected from the species: Enterococcus faecium, Enterococcis faecalisor, Staphylococcus aureus or Moraxella catarrhal is.

[0035] In some embodiments of the method, the fungus is a member of the candita genus. In some embodiments, the fungus is a strain of Candida albicans, Candida auris, Candida dubliniensis, Candida kruzei or Candida parapsilosis.

[0036] In some embodiments, the method is performed on a surgical device or implant. In some embodiments, the surgical device or surgical implant includes or is formed of polyester, or polypropylene. In some embodiments, the surgical implant is an implantable mesh.

[0037] Further provided by the present disclosure is a method of preventing or treating an infection in a subject, the method comprising providing to the site of infection an effective amount of an antimicrobial composition or an antimicrobial solution as described herein.

[0038] In some embodiments of the method of preventing or treating an infection in a subject, the method further includes providing, or exposing the infection to, an antimicrobial agent. In such embodiments, the antimicrobial agent is potentiated by the antimicrobial composition or antimicrobial solution, or vice versa. In some embodiments, the antimicrobial agent and antimicrobial composition or antimicrobial solution synergistically interact to prevent or treat the infection.

[0039] In some embodiments the antimicrobial agent is an antibiotic, an antiseptic or an antifungal.

[0040] In embodiments where the antimicrobial agent is an antibiotic, the antibiotic is suitable for administration to a mammalian subject, preferably a human subject. In some embodiments, the antibiotic is selected from the group consisting of: a protein synthesis inhibitor, a cell wall synthesis inhibitor, a beta-lactam antibiotic, a beta-lactamase inhibitor, a lipopeptide, a peptidoglycan synthesis inhibitor, a DNA synthesis inhibitor, a RNA synthesis inhibitor, a mycolic acid synthesis inhibitor, a mechanosensitive channel of large conductance (MscL) or a folic acid synthesis inhibitor, or a combination of the aforementioned antibiotics.

[0041] In some embodiments, the antibiotic is: a quinolone antibiotic, a tetracycline antibiotic, a glycopeptide antibiotic, an aminoglycoside antibiotic, a penicillin antibiotic, a cephalosporin antibiotic, a cephamycin antibiotic or a macrolide antibiotic. [0042] In some embodiments, the antibiotic is: ciprofloxacin, doxycycline, vancomycin, gentamycin, amoxicillin, ceftazidime or erythromycin.

[0043] In embodiments where the antimicrobial agent is an antiseptic, the antiseptic is suitable for administration to a mammalian subject, preferably a human subject. In some embodiments, the antiseptic is: an alcohol, benzethonium chloride, chlorhexidine, hexachlorophene, hydrogen peroxide, iodine, octenidine dihydrochloride, oxychlorosene sodium, polyhexanide, povidone iodine, sodium hypochlorite or triclosan. Preferred alcohols include ethanol and isopropanol.

[0044] In embodiments where the antimicrobial agent is an antifungal, the antifungal is suitable for administration to a mammalian subject, preferably a human subject. In some embodiments, the antifungal is selected from the group consisting of: azole antifungals, echinocandins or polyenes. In some embodiments, the antifungal is one or more of: amphotericin B, anidulafungin, candicidin, caspofungin, clotrimazole, filipin, fluconazole, flucytosine, griseofulvin, hamycin, isavuconazonium, itraconazole, ketoconazole natamycin, micafungin, miconazole, nystatin, posaconazole, rimocidin, terbinafine or voriconazole.

[0045] Also provided by the present disclosure is a kit including: diethyldithiocarbamate (DDC), or a salt thereof, and copper, or a salt of thereof, wherein DDC, or the salt thereof, and copper, or the salt thereof, are adapted for combination. In some embodiments, the kit further includes a diluent for dissolving the DDC, or a salt thereof, and the copper, or the salt thereof. In some embodiments, the weight to weight ratio of the copper, or the salt thereof, to DDC, or the salt thereof, is between 1 :1 and 8:1. In some embodiments of the kit, the volume of the diluent is proportional to the weight of the DDC, or the salt thereof, to provide a solution having a concentration of DDC, or the salt thereof, of at least 0.125 pg/ml. In some embodiments of the kit, the volume of the diluent is proportional to the weight of copper, or the salt thereof, to provide a solution having a concentration of copper of at least 0.5 pg/ml.

[0046] In some embodiments, the kit further including an antimicrobial. In some embodiments, the antimicrobial agent is an antibiotic, an antiseptic or an antifungal.

[0047] The kit disclosed herein is useful, or is intended for use or limited for use in the methods disclosed herein

[0048] Further provided by the present disclosure is a method of reducing the viability of a microorganism, the method including, providing, or exposing the microorganism to, a composition or solution as disclosed herein. In some embodiments, the method further includes providing, or exposing the microorganism to, an antimicrobial agent. Brief Description of the Drawings

[0049] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description, taken in conjunction with the accompanying Figures which illustrate certain embodiments of the present invention.

[0050] Figure 1 shows the minimum concentration of sodium Diethyldithiocarbamate (DDC) trihydrate alone or gallium (III) chloride, iron (II) sulfate, zinc (II) sulfate, copper (II) sulfate alone or in combination with DDC which inhibits the growth of epidermidis 1 planktonic bacteria. The left column for each labelled metal solution on the x-axis represents the MIC for the metal alone, while the right column for each metal represents the MIC for the metal combined with DDC.

[0051] Figure 2 shows the MIC of sodium DDC trihydrate in combination with 16 pg/ml copper (II) sulfate, copper (II) chloride or copper (II) acetate in inhibiting the growth of S. epidermidis 1 planktonic bacteria. The left column for each labelled metal salt solution on the x-axis represents the MIC for the metal salt solution alone, while the right column for each metal represents the MIC for the metal combined with DDC.

[0052] Figures 3A to 3C shows the minimum concentration of sodium DDC trihydrate alone, copper sulfate alone or the combination of DDC and copper sulfate for inhibiting the growth of various planktonic Bacteria. Figures 3A and 3B illustrate the efficacy on gram-positive bacteria, and Figure 3C illustrates the efficacy on gram-negative bacteria. The bacteria strains shown in Figure 3A are Staphylococcus epidermidis 1 and 2 and Methicillin-resistant Staphylococcus aureus (MRSA) 1 to 6. The bacteria strains shown in Figure 3B are Enterococcus faecium 1 and 2, Enterococcus faecalis, Staphylococcus aureus and a small colony variant (SCV) of Staphylococcus aureus. The bacteria strains shown in Figure 3C are Acinetobacter baumannii 1 and 2, Escherichia coli 1 and 2, Klebsiella pneumonia, Moraxella catarrhalis and Pseudomonas aeruginosa 1 and 2. The left column for each labelled bacterium on the x-axis represents the MIC for DDC solution alone, the middle column represents the MIC for copper solution alone, while the right column for each bacterium represents the MIC for the solution of copper in combination with DDC.

[0053] Figure 4 shows the effect of the ratio of sodium DDC trihydrate to copper sulfate on the growth over time of planktonic MRSA in comparison to no treatment (max growth) or treatment with DDC alone or copper alone. Concentrations are expressed as pg/ml of sodium DDC trihydrate and pg/ml of copper sulfate. [0054] Figures 5A and 5B shows the effect of varying ratios of sodium DDC trihydrate to copper sulfate in killing biofilms formed by MRSA 2 (5A) and MRSA 3 (5B). Ratios are expressed as DDC:copper.

[0055] Figure 6 shows the effect of decreasing concentration of a 1 :4 sodium DDC trihydrate to copper sulfate ratio on the killing of MRSA 1 biofilms.

[0056] Figure 7 shows the effect of varying the ratio of sodium DDC trihydrate to copper sulfate on the killing of MRSA 2 biofilms. X-axis labels represent the concentration (ug/ml) of sodium DDC trihydrate plus copper sulfate (i.e. 256-32 represents 256 pg/ml of sodium DDC trihydrate plus 32 pg/ml or copper sulfate).

[0057] Figure 8 shows the effect of sodium DDC trihydrate alone, copper sulfate alone and a combination of sodium DDC trihydrate and copper sulfate on the killing of MRSA 1 , 2, 4 and 6 bacteria and S. epi 2 bacteria within biofilms. The left column for each labelled bacterium on the x-axis (not visible for MRSA 2, 4 and s. epi 2) represents the biofilm killing for DDC solution (4 pg/ml) alone, the middle column represents the biofilm killing for copper solution (32 pg/ml) alone, while the right column for each bacterium represents the biofilm killing for the solution of copper in combination with DDC (4 pg/ml sodium DDC trihydrate plus 32 pg/ml copper sulfate).

[0058] Figures 9A to D shows confocal microscopy of representative images of bacterial biofilms on glass slides treated with control media (Figure 9A), sodium DDC trihydrate alone (Figure 9B), copper sulfate alone (Figure 9C) and sodium DDC trihydrate and copper sulfate in combination (Figure 9D). In the images of Figure 9, the live cells fluoresce green, cells with compromised membranes fluoresce yellow, and dead cells fluoresce red.

[0059] Figure 10 shows microscopic images from the bioflux system of a time course over 24 hours of biofilm development in the presence of cell culture media alone (upper time course) or in the presence of cell culture media containing 64 pg/ml of sodium DDC trihydrate and 256 pg/ml of Copper sulfate.

[0060] Figure 11 shows real-time analysis of biofilm formation in cell culture wells in the presence of media alone (control), media supplemented with 8 pg/ml of sodium DDC trihydrate, media supplemented with 32 pg/ml of copper sulfate or media supplemented with 8 pg/ml of sodium DDC trihydrate and 32 pg/ml.

[0061] Figure 12 shows the number of cell forming units (CFU) from polyester (LFIS) and polypropylene (RFIS) surgical meshes which had been colonised by S. epi 1 or MRSA biofilms prior to treatment with cell culture media alone, cell culture media with 8 pg/ml sodium DDC trihydrate, cell culture media with 32 pg/ml of copper sulfate or media containing 8 pg/ml sodium DDC trihydrate combined with 32 pg/ml of copper sulfate. Colony forming units are expressed on a log 10 scale. The left-most column represents the maximum growth control (no treatment), the middle-left column represents 8 pg/ml of sodium DDC trihydrate alone, the right-middle column represents 32 pg/ml of copper sulfate and the right-most column represents a combination of 8 pg/ml of sodium DDC trihydrate and 32 pg/ml of copper sulfate.

[0062] Figures 13A and 13B shows confocal microscopy of strands of a polyester mesh which had been colonised by MRSA 3 biofilms prior to treatment with cell culture media alone, or media containing 8 pg/ml DDC combined with 32 pg/ml of copper sulfate. As per Figure 9, the live cells fluoresce green, cells with compromised membranes fluoresce yellow, and dead cells fluoresce red.

[0063] Figure 14 shows the fractional inhibitory concentration (FIC) index against MRSA 3 biofilms in a checkerboard assay. Developed biofilms were treated with varying concentrations of seven different antibiotics combined with varying concentrations of sodium DDC trihydrate and copper. A FIC below 0.5 indicates a synergistic effect, a FIC between 4 and 0.5 shows an additive interworking relationship, and a FIC above 4 indicates antagonistic effects between the components.

[0064] Figures 15A and 15B show the minimum concentration (MIC) of sodium DDC trihydrate alone, copper sulfate alone or sodium DDC trihydrate in combination with copper sulfate against six different species of Candida fungi, namely C. albicans, C. dubliniensis, C. kefyr, C. kruzei, C. auris, C. tropicalis and C. parapsilosis. The left column for each labelled fungus on the x-axis represents the MIC for DDC solution alone, the middle column represents the MIC for copper solution alone, while the right column for each bacterium represents the MIC for the solution of DDC in combination with copper.

[0065] Figures 16A and 16B show the efficacy of various antifungal compounds when combined with sodium DDC trihydrate alone, copper sulfate alone or sodium DDC trihydrate in combination with copper sulfate. Specifically, Figure 16A show the fraction inhibitory concentration (FIC) index, and Figure 16B shows the minimum inhibitory concentration (MIC) against C. auris of the combination of clotrimazole, fluconazole, itraconazole, miconazole and amphotericin B in combination with DDC and/or copper.

[0066] Figure 17 shows the viability of human endothelial cells after treatment of various concentrations of a 1 :4 ratio of sodium DDC trihydrate to copper sulfate (weight to weight) and one concentration of 1 :8 DDC to copper sulfate (weight to weight). The left column for each labelled concentration on the x-axis represents the cell viability for the relevant concentration of DDC solution alone, the middle column represents the cell viability for the relevant concentration of copper solution alone, while the right column for each concentration represents cell viability for the relevant concentration of the solution of DDC in combination with copper at the prescribed concentrations (i.e. 4+32 equals 4 pg/ml of sodium DDC trihydrate and 32 pg/ml copper sulfate).

[0067] Figures 18A to C show the chemical structure of DDC anions (Figure 18A) and complexes of DDC anions and divalent copper (Figure 18B) and DDC anions and monovalent copper (Figure 18C).

[0068] Figures 19A and 19B show the effect of the combination of DDC and copper (8 pg/ml of sodium DDC trihydrate and 32 pg/ml of copper sulfate) on biofilm formation in an artificial wound model when tested against MRSA 3 (Figure 19A) and S. epidermidis 35984 (Figure 19B) compared to DDC or copper alone.

Detailed Description

[0069] As discussed above, the present invention is predicated on the work of the Inventor who has determined that diethyldithiocarbamate (DDC) forms a working inter-relationship with copper in inhibiting the growth of, or killing, a broad spectrum of microbes. This includes bacterial biofilms which are typically resistant to antibiotic and antiseptic treatments. Further, the Inventor has established that DDC and copper, when combined, potentiate a range of antimicrobial treatments.

[0070] Accordingly, the present invention provides an antimicrobial composition including: diethyldithiocarbamate (DDC), or a salt thereof, and copper, or a salt thereof. As will be demonstrated in the Examples included herein, DDC in combination with copper can effectively treat a range of microbial infections, including infections resistant to other common antimicrobial treatments.

[0071] As will be disclosed, DDC and copper can synergistically combine to inhibit the growth of, or kill, a range of microorganisms.

[0072] While the mechanism of action that results in DDC and copper in combination acting as an antimicrobial is not established, it is hypothesised that DDC and copper form a stable complex that is transported intracellularly via copper uptake mechanisms within the microbe. The complex of DDC and copper may then bind to intracellular copper binding sites Flowever, as DDC strongly chelates copper the copper cannot be released from the complex of DDC and copper, which results in inhibition of vital cellular pathways and generation of toxic free radicals. In addition, DDC and copper is hypothesised to occupy efflux pumps that are essential for detoxification of excess copper, leading to accumulation of Cu inside bacteria and fungi which is ultimately lethal.

[0073] In mammalian cells DDC complexed with monovalent and divalent copper have been demonstrated to selectively inhibit cancer cells. Interestingly, the proposed mechanisms of action in the literature vary. A series of studies on the role of DDC and copper in cancer treatment propose that DDC in combination with copper act as a proteasome inhibitor (Han, J. et at, Toxicol Appl Pharmacol (2013), 273; 477-483; Chen, D. et at, Cancer Res. 2006. 66, 10425-10433; and Daniel, K. et at. Breast Cancer Res. (2005), 7:R897-R908). In contrast, Allensworth, J. et at. (Mol. Oncol. (2015), 9:1155-68) propose that DDC acts as a copper ionophore that leads to copper accumulation within the cancer cell which then acts to induce reactive oxygen species within the cell, leading to cell death. Consequently, there may be several mechanisms by which DDC in combination with copper induces cell death and the mechanism in microbes, such as those described herein, is likely significantly different to that for cancer cells and mammalian cells.

[0074] The present invention also provides an antimicrobial solution including: diethyldithiocarbamate (DDC) or a salt thereof, and copper or a salt thereof.

[0075] As discussed further herein, in solution, salts of DDC dissociate to provide DDC anions, and salts of copper dissociate to provide copper cations. Therefore, in some embodiments, the antimicrobial solution includes DDC anions and copper cations.

[0076] Diethyldithiocarbamate and Copper

[0077] Diethyldithiocarbamate (referred to herein as “DDC”) is the main metabolite of the chronic alcoholism drug disulfiram (trade name Antbuse), which is a member of the dithiocarbamate family. Diethyldithiocarbamate has the systematic names of: Carbamic acid, diethyldithio-; Carbamodithioic acid, diethyl-; Carbamodithioic acid, N,N-diethyl-; and Diethyldithiocarbamic acid, and is also known as Diethyl dithiocarbamate, Diethylcarbamodithioic acid, Diethyldithiocarbamic acid, Diethyldithione, Ditiocarb (NCBI MeSH entry), DETC, DDTC, DTC, Diecaimuthiol and Thiocarb.

[0078] DDC can be used therapeutically as a potent chelating agent to prevent acute poisoning from toxic metals such as nickel. However, it has been shown that it can function as an ionophore transporting metal ions such as Fe²⁺ across cellular membranes. [0079] Diethyldithiocarbamate exists as a monovalent anion in solution and has the molecular formula C5H10NS2 and the chemical of Formula I (set forth below and in Figure 18A):

Formula I

[0080] DDC is derived from its parent compound Diethyldithiocarbamic acid by removal of a proton from the dithiocarbamic acid moiety. In solution the molecular weight of the DDC anion is 148.3 g/mol.

[0081 ] DDC can be present in the form of the negatively charged anion, which can exist in the presence of a metal as a metal complex. As a starting compound in the process of the present invention, it may be used in the form of a neutral compound (C Fl ) NCFl (S)SFI or, preferably, in the form of a salt (C Fl ) NCFl (S)S]m-Catm+, where m is the valency of the cation.

[0082] Typically, DDC is provided in the form of a salt. Known salts include, but are not limited to, ammonium salts, diethylammonium salts, bismuth salts, sodium salts, lead salts, potassium salts, tin salts and zinc salts and hydrated forms thereof.

[0083] DDC salts are available commercially such as; ammonium diethyldithiocarbamate (Sigma-Aldrich, cat No. 359548), Lead diethyldithiocarbamate (Sigma-Aldrich, cat No. 15331), Silver diethyldithiocarbamate (Sigma-Aldrich, cat No. D3132), sodium diethyldithiocarbamate trihydrate (Sigma-Aldrich, cat No. D3506) and Zinc diethyldithiocarbamate (Sigma-Aldrich, cat No. 329703).

[0084] Copper in its pure state forms a water insoluble metal, but can form a variety of compound, typically with an oxidative state of +1 or +2, referred to as cuprous and cupric compounds, respectively. Consequently, it is to be understood that a reference herein to copper will encompass the oxidative forms of copper, such as copper cations in solution as well as salts.

[0085] Soluble forms of copper typically take the form of copper salts. The most commonly used water soluble copper salts include: Copper(l) Chloride, Copper(ll) Fluoride, Copper(ll) Benzoate Dihydrate and Copper(ll) Sulfate Pentahydrate. It is within the knowledge of a person skilled in the art to select a copper compound, or salt, suitable for use in the antimicrobial compositions and solutions provided herein. The selection of the appropriate copper compound will be determined by many factors including (but not limited to) the desired solubility of the copper compound, the biproducts of dissolution of the copper compound, the toxicity to cells such as mammalian cells and to a subject, the stability of the complex, and the like.

[0086] However, as is known in the art a variety of copper salts exist with variable solubility in water (see Table 1). These include: Copper(l) acetate, Copper(l) bromide, Copper(l) butyrate, Copper(l) chlorate, Copper(l) chloride, Copper(l) chromate, Copper(l) chromite, Copper(l) citrate, Copper(l) cyanide, Copper(l) hydroxide, Copper(l) iodide, Copper(l) sulfide, Copper(l) thiocyanate, Copper(ll) bromide, Copper(ll) carbonate, Copper(ll) chlorate, Copper(ll) chloride, Copper(ll) chromate, Copper(ll) fluoride, Copper(ll) fluorosilicate, Copper(ll) formate, Copper(ll) gluconate, Copper(ll) hexafluorosilicate, Copper(ll) hydroxide, Copper(ll) iodate, Copper(ll) nitrate, Copper(ll) oleate, Copper(ll) oxalate, Copper(ll) oxide, Copper(ll) perchlorate, Copper(ll) phosphate, Copper(ll) selenate, Copper(ll) selenite, Copper(ll) stater, Copper(ll) sulfate, Copper(ll) sulfide, Copper(ll) tartrate and Copper(ll) tungstrate.

[0087] Table 1 : Solubility of common copper salts in water at one atmosphere of pressure.

[0088] In some embodiments of the invention, the copper is provided in the form of a salt having water solubility greater than 1 pg/ml (100 pg/100 ml at 20°C), or greater that 100 pg/ml (10 mg/100ml at 20°C), or greater that 1 mg/ml (100 mg/100 ml at 20°C).

[0089] In water, dissolved copper exists in the form of a cation. The simplest form of copper ion in solution is a hexaaquacopper(ll) ion ([Cu(H20)6]2+). Typically, monovalent copper cations in solution disproportionate to copper(ll) ions and produce a copper precipitate. However, stable monovalent forms of copper in combination with DDC have been formed.

[0090] Salts of copper are commercially available from a variety of providers including Sigma Aldrich and Fisher Scientific amongst others.

[0091] In some embodiments of the antimicrobial composition or solution of the invention the weight ratio of the salt of copper to the salt of DCC, or the weight ratio of copper to DDC, is greater than or equal to 1 :2, greater than or equal to 1 :1 , greater than or equal to 2:1 , greater than or equal to 3:1 , greater than or equal to 4:1 , greater than or equal to 5:1 , greater than or equal to 6:1 , or greater than or equal to 7:1 . In some preferred embodiments, the weight of the salt of copper is equal to or in excess of the salt of DCC.

[0092] In some embodiments of the antimicrobial composition or solution of the invention the weight ratio of the salt of copper to the salt of DCC, or the weight ratio of copper to DDC, is greater than or equal to about 1 :2, greater than or equal to about 1 :1 , greater than or equal to about 2:1 , greater than or equal to about 3:1 , greater than or equal to about 4:1 , greater than or equal to about 5:1 , greater than or equal to about 6:1 , or greater than or equal to about 7:1 . In some preferred embodiments, the weight of the salt of copper is equal to or in excess of the salt of DCC.

[0093] In some embodiments of the antimicrobial composition or solution of the invention the weight to weight ratio of the salt of copper to the salt of DCC is less than or equal to 2:1 , less than or equal to 3:1 , less than or equal to 4:1 , less than or equal to 5:1 , less than or equal to 6:1 , less than or equal to 7:1 , less than or equal to 8:1 , less than or equal to 9:1 , less than or equal to 10:1 , less than or equal to 11 :1 , less than or equal to 12:1 , less than or equal to 13:1 , less than or equal to 14:1 , less than or equal to 15:1 or less than or equal to 16:1.

[0094] In some embodiments of the antimicrobial composition or solution of the invention the weight to weight ratio of the salt of copper to the salt of DCC is less than or equal to about 2:1 , less than or equal to about 3:1 , less than or equal to about 4:1 , less than or equal to about 5:1 , less than or equal to about 6:1 , less than or equal to about 7:1 , less than or equal to about 8:1 , less than or equal to about 9:1 , less than or equal to about 10:1 , less than or equal to about 11 :1 , less than or equal to about 12:1 , less than or equal to about 13:1 , less than or equal to about 14:1 , less than or equal to about 15:1 , or less than or equal to about 16:1.

[0095] In some embodiments of the antimicrobial composition or solution of the invention the weight ratio of the salt of copper to the salt of DCC is from 1 :2 to 16:1 , or from 1 :1 to 8:1 .

[0096] In some embodiments of the antimicrobial composition or solution of the invention the weight ratio of the salt of copper to the salt of DCC is from about 1 :2 to about 16:1 , or from about 1 :1 to about 8:1.

[0097] As would be understood by a person skilled in the art, the molecular weight of the salt of copper and DDC in the antimicrobial composition or solution will affect the molar to molar ratio of copper to DDC when expressed as a weight to weight ratio. The molecular weight of common copper and DDC salts is provided in Table 2.

Table 2: Molecular Weight of Common DDC and Copper Compounds

[0098] In view of the above, it will be apparent that the ratio of copper to DDC can be expressed as a mole ratio which is applicable irrespective of the compounds used to provide the antimicrobial composition or solution of the present invention. Therefore, in some embodiments of the antimicrobial composition or solution of the invention, the mole ratio of the salt of copper to the salt of DCC is greater than or equal to 1 :2, greater than or equal to 1 :1 , greater than or equal to 2:1 , greater than or equal to 3:1 , greater than or equal to 4:1 , greater than or equal to 5:1 , greater than or equal to 6:1 , or greater than or equal to 7:1. In some preferred embodiments, the moles of the salt of copper is equal to or in excess of the moles of the salt of DCC. In some embodiments of the antimicrobial composition or solution of the invention, the mole ratio of the salt of copper to the salt of DCC is greater than or equal to about 1 :2, greater than or equal to about 1 :1 , greater than or equal to about 2:1 , greater than or equal to about 3:1 , greater than or equal to about 4:1 , greater than or equal to about 5:1 , greater than or equal to about 6:1 , or greater than or equal to about 7:1 .

[0099] In some embodiments of the antimicrobial composition or solution of the invention, the mole ratio of the salt of copper to the salt of DCC is greater than or equal to about 1 .4:2, greater than or equal to about 1 .4:1 , greater than or equal to about 2.8:1 , greater than or equal to about 4.2:1 , greater than or equal to about 5.6:1 , greater than or equal to about 7:1 , greater than or equal to about 8.4:1 or greater than or equal to about 9.8:1 .

[0100] In some embodiments of the antimicrobial composition or solution of the invention, the mole ratio of the salt of copper to the salt of DCC is less than or equal to about 1 .5:1 , less than or equal to 2:1 , less than or equal to 3:1 , less than or equal to 4:1 , less than or equal to 5:1 , less than or equal to 6:1 , less than or equal to 7:1 , less than or equal to 8:1. In some embodiments of the antimicrobial composition or solution of the invention the mole ratio of the salt of copper to the salt of DCC is less than or equal to about 2:1 , less than or equal to about 3:1 , less than or equal to about 4:1 , less than or equal to about 5:1 , less than or equal to about 6:1 , less than or equal to about 7:1 less than or equal to about 8:1 , less than or equal to about 9:1 , less than or equal to about 10:1 , less than or equal to about 11 :1 , less than or equal to about 12:1 , less than or equal to about 13:1 , less than or equal to about 14:1 , less than or equal to about 15:1 or less than or equal to about 16:1.

[0101] In some embodiments of the antimicrobial composition or solution of the invention, the mole ratio of the salt of copper to the salt of DCC is less than or equal to about 2.2:1 , less than or equal to about 2.8:1 , less than or equal to about 4.2:1 , less than or equal to about 5.6:1 , less than or equal to about 7:1 , less than or equal to about 8.4:1 , less than or equal to about 9.8:, less than or equal to about 11 .2:1 , less than or equal to about 12.6:1 , less than or equal to about 14:1 , less than or equal to about 15.4:1 , less than or equal to about 16.8:1 , less than or equal to about 18.2:1 , less than or equal to about 19.6:1 , less than or equal to about 21 :1 , or less than or equal to about 22.4:1 .

[0102] In some embodiments of the antimicrobial composition or solution of the invention, the mole ratio of the copper to the salt of DCC is from 1 :2 to 16:1 , or from 1.4:2 to 22.4:1 or from 1 :1 to 8:1 , or from 1 .4:1 to 11 .2:1 . In some embodiments of the antimicrobial composition or solution of the invention the mole ratio of the copper to the salt of DCC is from about 1 :2 to about 16:1 , or from about 1 .4:2 to about 22.4:1 or from about 1 :1 to 8:1 , or from about 1 .4:1 to 11.2:1.

[0103] DDC can complex with divalent copper, at a stoichiometric ratio of 2:1 DDC to copper, to form cupric diethyldithiocarbamate, and having the following Formula II (set forth below and in Figure 7B):

Formula II

[0104] Further, as disclosed in Han, J. et al (see above), DDC can complex with monovalent copper, at a stoichiometric ratio of 1 :1 DDC to copper, to form cuprous diethyldithiocarbamate having the following Formula III (set for below and in Figure 18C): +

Cu

Formula III

[0105] In some embodiments of the antimicrobial composition or solution of the present invention, the copper, or copper salt, includes divalent cations of copper. In some embodiments, the copper, or copper salt, includes monovalent cations. Accordingly, the ratio of copper to DDC in the antimicrobial compositions and solutions of the present invention may be proportionate to stoichiometric ratio between DDC anions and copper cations, with a commensurate change in moles of copper to DDC when monovalent copper is used compared to when divalent copper.

[0106] Further, as DDC and copper complex to form cupric diethyldithiocarbamate and cuprous diethyldithiocarbamate (Formula II and Formula III - see Figures 18B and C), in some embodiments, the composition or solution includes, or consist of, a complex of DDC and copper. In some embodiments, the composition or solution includes, or consist of, cupric diethyldithiocarbamate. In some embodiments, the composition or solution includes, or consist of, cuprous diethyldithiocarbamate.

[0107] Compositions and Formulations

[0108] The antimicrobial compositions and solutions of the present invention may be used for the treatment of a microbial infection in a subject. Therefore, in some embodiments, the compositions or solutions of the present invention is suitable for the administration, provision or application to a subject.

[0109] Therefore, in some embodiment the composition or solution is a pharmaceutical composition and includes a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients will be known in the art and include: additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the composition to be administered. Further, excipients include pharmaceutically acceptable carriers, which may be chosen based on various considerations including the route of administration, the antimicrobial agent being delivered and the time course of delivery of the composition. [0110] The preparation of such pharmaceutical compositions is known in the art, for example as described in Remington's Pharmaceutical Sciences, 18th ed., 1990, Mack Publishing Co., Easton, Pa. and U.S. Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton, Pa, which are incorporated herein by reference in their entirety.

[0111] Particularly envisaged uses of the compositions and solutions of the present invention include the treatment of topical infections. Therefore, in some embodiments, the composition or solution is for topical treatment, is in the form of a topical treatment, or is adapted or formulated for topical treatment.

[0112] Formulations for topical delivery are described in D Osborne and A Aman (eds),1990, Topical drug delivery formulations, CRC press Taylor & Francis and D Bhowmik et at. (2012) Recent Advances In Novel Topical Drug Delivery System, the Pharma innovation, 1 :9, 12-31. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Tarun Garg, Goutam Rath & Amit K. Goyal (2015) Comprehensive review on additives of topical dosage forms for drug delivery, Drug Delivery, 22:8, 969-987.

[0113] Such administrations may be carried out using the composition or solution of the present invention as described herein, in the form of a liquid (including drops or liquid sprays), a gel, a paste, a lotion, a cream, an ointment, a powder, a foam, a patch, a suspension, a solution or other suitable form.

[0114] Drops or liquid sprays may be formulated with DDC and copper of the invention in an aqueous, or non-aqueous, base also comprising one or more of: dispersing agents, solubilizing agents or suspending agents. Drops can be delivered via a simple eye dropper- capped bottle, via a plastic bottle adapted to deliver liquid contents drop-wise, or via a specially shaped closure. Liquid sprays can be pumped or are conveniently delivered from pressurized aerosols and can be delivered via a targeted spray opening such as a manipulable tube or can be delivered via a spray aperture which spreads the liquid over a uniform area.

[0115] A cream is a formulation that contains water and oil and is stabilized with an emulsifier. Lipophilic creams are called water-in-oil emulsions, and hydrophilic creams oil-in water emulsions. The cream base for water-in-oil emulsions are normally absorption bases such as vaseline, ceresin or lanolin. The bases for oil-in-water emulsions are mono-, di-, and tri-glycerides of fatty acids or fatty alcohols with soaps, alkyl sulphates or alkyl polyglycol ethers as emulsifiers. [0116] A lotion is an opaque, thin, non-greasy emulsion liquid dosage form for external application to the skin, which generally contains a water-based vehicle with greater than 50% of volatiles and sufficiently low viscosity that it may be delivered by pouring. Lotions are usually hydrophilic, and contain greater than 50% of volatiles as measured by LOD (loss on drying). A lotion tends to evaporate rapidly with a cooling sensation when rubbed onto the skin.

[0117] A paste is an opaque or translucent, viscous, greasy emulsion or suspension semisolid dosage form for external application to the skin, which generally contains greater than 50% of hydrocarbon-based or a polyethylene glycol-based vehicle and less than 20% of volatiles. A paste contains a large proportion (20-50%) of dispersed solids in a fatty or aqueous vehicle.

[0118] An ointment is an opaque or translucent, viscous, greasy emulsion or suspension semisolid dosage form for external application to the skin, which generally contains greater than 50% of hydrocarbon-based or a polyethylene glycol-based vehicle and less than 20% of volatiles. An ointment is usually lipophilic, and contains >50% of hydrocarbons or polyethylene glycols as the vehicle and <20% of volatiles as measured by LOD. An ointment tends not to evaporate or be absorbed when rubbed onto the skin.

[0119] A gel is usually a translucent, non-greasy emulsion or suspension semisolid dosage form for external application to the skin, which contains a gelling agent in quantities sufficient to impart a three-dimensional, cross-linked matrix. A gel is usually hydrophilic, and contains sufficient quantities of a gelling agent such as starch, cellulose derivatives, carbomers, magnesium-aluminum silicates, xanthan gum, colloidal silica, aluminium or zinc soaps.

[0120] The composition or solution of the present invention, when in a form for topical administration, may further include drying agents, anti-foaming agents, buffers, neutralizing agents, agents to adjust pH, colouring agents and decolouring agents, emollients, emulsifying agents, emulsion stabilizers and viscosity builders, humectants, odorants, preservatives, antioxidants, and chemical stabilizers, solvents, and thickening, stiffening, and suspending agents, and a balance of water or solvent.

[0121] Applications of the Composition or Solution

[0122] In some embodiments, the topical treatment is for the treatment of a wound, treatment of keratinised skin, treatment of a mucosal surface, treatment of the ear or treatment of the sinuses.

[0123] Wound Treatment [0124] A particularly envisaged topical treatment includes treatment of a wound. In some embodiments, the topical treatment is for the prevention or treatment of an infection in a wound.

[0125] In some embodiments, the composition of the present invention is for use in promoting wound healing or preventing the formation of a chronic wound. Further provided are methods for treating a wound including the administration or application of an effective amount of a composition of the present invention to a subject, preferably to the site of the wound.

[0126] Further provided herein is a method of preventing or treating a bacterial or fungal infection of a wound in a subject, the method including providing or administering an effective amount of an antimicrobial composition or solution in accordance with the present invention to the site of the wound. In some embodiments the method includes administering a topical composition in accordance with the present invention. Some embodiments of the method further include administering an antimicrobial to the subject, such as an antibiotic, an antiseptic or an antifungal.

[0127] In some embodiments, the wound is a chronic wound. In some embodiments, the wound is an acute wound. In some embodiments the infection is a biofilm.

[0128] Wounds can be categorised as acute or chronic. Chronic wounds are defined as wounds that have failed to repair in a timely and orderly process of repair for at least a period of 3 months. Chronic wounds can be identified by the presence of a raised, hyperproliferative but non-advancing wound margin. Fibroblasts derived from the wound bed of chronic wounds of various aetiologies represent a senescent, premature, or differentiated phenotype, which do not respond or respond inefficiently to normal stimulatory messages.

[0129] All wound types have the potential to become chronic. Chronic wounds are traditionally divided etiologically, which may inform the treatment of the chronic wound. Further, treatment of the chronic wounds may also be accompanied by treatment of the underlying condition which may have instigated, or contributed to the formation of, the wound such as venous insufficiency, arterial perfusion, diabetes, unrelieved pressure as well as systemic factors such as nutritional status (Type I orType II diabetes), immunosuppression and infection.

[0130] Common chronic wounds are venous ulcers, which usually occur in the legs and mostly affect the elderly, diabetic ulcers which are another major cause of chronic wounds, pressure ulcers, which usually occur in people with conditions such as paralysis that inhibit movement of body parts that are commonly subjected to pressure such as the heels, shoulder blades and sacrum, corneal ulcers, most commonly caused by an infection with bacteria, viruses, fungi or amoebae. Other types of chronic wounds may be due to causes such as ischemia and radiation poisoning.

[0131] As would be understood in the art, chronic wounds are particularly vulnerable to infection with bacteria or fungi, as well as biofilm formation. Therefore, in some embodiments, the composition or solution of the invention is for, or is adapted or formulated for, the treatment or prevention of an infection of a chronic wound.

[0132] Acute wounds can be categorised as any wound that does not meet the classification of a chronic wound. As such, acute wounds can be defined as wounds less than 3 months since the instigation of the wound site, often showing signs of wound healing within the first 4 weeks since instigation.

[0133] Acute wounds may be classified into different types, according to the object that caused the wound. For example, incisions or incised wounds, lacerations, abrasions and grazes, burns, puncture or penetration wounds. Acute wounds are less susceptible to infections that chronic wounds as the time of exposure of the dermal and subdermal layers is decreased. However, the occurrence of an infection in an acute wound is a risk factor for the development of a chronic wound. Therefore, in some embodiments, the composition or solution of the present invention is for, or is adapted or formulated for, the treatment or prevention of an infection of an acute wound.

[0134] As an infection of a wound often manifests as a decrease in the rate of wound healing and may lead to a chronic wound, measurement of wound growth can provide an indication of infection status. There are various techniques known in the art for measuring wound healing, including the following.

[0135] Ruler technique - The surface area of a wound can be approximated by multiplying the greatest length and perpendicular width measurements.

[0136] Acetate Tracing and Planimetry- An alternative method for estimating wound area is acetate tracings and contact planimetry. This technique involves placing a transparent sheet across the wound surface and then tracing its margins. The area of the wound is then determined manually by placing the tracing over a grid and counting the number of squares within the circumscribed area, or by using computer image analysis to accurately quantify area.

[0137] Three-dimensional estimation of wounds - More complicated three-dimensional topography measurement of wounds can be performed using structured light or laser light. These techniques, which are known in the art, use digital cameras and projected laser beams that distort with the curvature and depth of the wound surface.

[0138] Sinus Treatment

[0139] Further particularly envisaged topical treatments include treatment or the sinuses. Therefore, in some embodiments, the topical treatment is for prevention or treatment of an infection of the sinuses.

[0140] Further provided herein is a method of preventing or treating a bacterial or fungal infection of a sinus of a subject, the method including providing or administering an effective amount of an antimicrobial composition or solution in accordance with the present invention to a sinus. In some embodiments, the method includes administering a topical composition in accordance with the present invention. Some embodiments of the method further include administering an antimicrobial to the subject, such as an antibiotic, an antiseptic or an antifungal.

[0141 ] In some embodiments, the infection is a biofilm. In some embodiments, the infection is a bacterial infection. In some embodiments, the infection is a fungal infection.

[0142] Assessment of antimicrobial compositions of the present invention for treating infection of the nose or sinuses (such as microbial sinusitis and rhinosinusitis) can be performed using various sheep models. See for example, Fla KR et al., 2007, Am. J. Rhinol., 21 (3): 339- 345; Drilling A et al., 2014, International forum of allergy & rhinology, 4(3): 176-186; and Jardeleza C et al., 2015, Transl. Res., 166(6): 683-692. Exemplary formulations for treating sites of infection in the nose and sinuses include drops, emulsions, or other solutions which can be squirted into the sinuses (such as nebulizer or spray-type formulations) including sinus irrigation washes and dilute compositions for the same. Other formulations are contemplated and will be known to those skilled in the art.

[0143] Sinusitis may be classified by duration, namely: acute (less than four weeks), subacute (four to 12 weeks), or chronic (more than 12 weeks). Acute sinusitis may be further classified as acute bacterial sinusitis or viral sinusitis. Diagnostic criteria for different forms of sinusitis are provided below

[0144] Adult acute rhinosinusitis

[0145] Sudden onset of two or more symptoms, one of which should be either nasal blockage/congestion/obstruction or nasal discharge (anteriorly or posteriorly), plus facial pain/pressure and/or reduction or loss of smell. [0146] Adult chronic rhinosinusitis

[0147] Presence of two or more symptoms persisting for more than 12 weeks, one of which should be either nasal blockage/congestion/obstruction or nasal discharge (anteriorly or posteriorly), plus facial pain/pressure and/or reduction or loss of smell

[0148] Paediatric acute rhinosinusitis

[0149] Sudden onset of two or more of nasal blockage/congestion/obstruction, discoloured nasal discharge or cough (day and night time).

[0150] Paediatric chronic rhinosinusitis

[0151 ] Presence of two or more symptoms persisting for more than 12 weeks, one of which should be either nasal blockage/congestion/obstruction or nasal discharge (anteriorly or posteriorly), plus facial pain/pressure and/or cough.

[0152] Acute bacterial rhinosinusitis

[0153] At least three of: (i) discoloured, purulent nasal discharge. (2) severe, localised pain (3) fever >38°C, (4) elevated erythrocyte sedimentation rate/C- reactive protein, and (5) double sickening.

[0154] Therefore, the efficacy of the method for preventing or treating a bacterial or fungal infection of a sinus of a subject can be assessed against one or more of the criteria provided above.

[0155] Ear Infections

[0156] Microbial infections, such as fungal and bacterial infections, commonly infect the ear canal of a subject (otitis externa). Such infections can lead to inflammation, discomfort, pain, obstruction of the ear canal and in severe cases damage to the ear drum or infection of the middle and inner ear.

[0157] Acute otitis externa is caused primarily by bacterial infection, with Pseudomonas aeruginosa and Staphylococcus aureus the most common pathogens. Acute otitis externa presents with the rapid onset of ear canal inflammation, resulting in otalgia, itching, canal edema, canal erythema, and otorrhea, and often occurs following swimming or minor trauma from inappropriate cleaning. Tenderness with movement of the external ear is typically indicative. [0158] In some embodiments, the composition or solution of the present invention is for treatment of an infection of the ear, in particular the ear canal.

[0159] Therefore, a particularly envisaged topical treatment includes the treatment of the ear canal. Further provided herein is a method of preventing or treating a bacterial or fungal infection of an ear canal of a subject, the method including providing or administering an effective amount of an antimicrobial composition or solution in accordance with the present invention to the ear canal. In some embodiments, the method includes administering a topical composition in accordance with the present invention. Some embodiments of the method further include administering an antimicrobial to the subject, such as an antibiotic, an antiseptic or an antifungal.

[0160] Compositions for administration to the ear canal are known in the art and include drops and ointments. Furthers, such treatments can also include additional therapeutic agents such as anti-inflammatories (for example corticosteroids). Therefore, in some embodiments, the composition or solution for treatment of the ear may include additional therapeutic agents, such as immune regulators and antimicrobials.

[0161 ] Diagnosis of Otitis Externa may be indicated based on the following criteria:

[0162] Onset of symptoms within 48 hours in the past three weeks and: (1a) symptoms of ear canal inflammation, ear pain, itching, or fullness (1 b) with or without hearing loss or jaw pain; and (2a) signs of ear canal inflammation, tenderness of tragus/pinna or ear canal edema/erythema (2b) with or without otorrhea, tympanic membrane erythema, cellulitis of the pinna, or local lymphadenitis

[0163] Therefore, the efficacy of the method for preventing or treating a bacterial or fungal infection of the ear canal of a subject can be assessed against one or more of the criteria provided above.

[0164] Medical Devices and Implants

[0165] The composition or solution of the present invention may also be used for the treatment of a surgical device or implant. Therefore, in some embodiments, the composition or solution of the invention is for the treatment of a surgical device or implant.

[0166] In some embodiments there is provided a method of inhibiting the growth of a bacterium or a fungus, or killing a bacterium or a fungus, or preventing the formation of a bacterial or fungal biofilm, or treating a bacterial or fungal biofilm, on a surgical device or implant the method comprising providing, or exposing the bacterium or fungus or biofilm to, an effective amount of the antimicrobial composition or solution of the present invention. Further, the method may also include providing, or exposing the bacterium or fungus or biofilm to, an antimicrobial.

[0167] Such treatments can be to prevent or treat microbial growth on the device or implant. In some embodiments, the treatment results in the surgical implant being impregnated with a composition or solution of the present invention.

[0168] Further, in some embodiments the present invention provides a surgical device or implant including an effective amount of DDC, or a salt thereof, and copper, or a salt thereof. In some embodiments, the device or implant is impregnated with an effective amount of DDC, or a salt thereof, and copper or, a salt of thereof, or the implant is coated with an effective amount of DDC, or a salt thereof, and copper, or a salt of thereof. The coating may be attached to the device or implant, or may be applied as a solution, such as a gel or cream or the like.

[0169] Suitable surgical devices and implants will be known in the art but include, surgical meshes, sutures, stents, stent-less support structures, dental implants, pins, rods, screws, prosthesis, subcutaneous delivery devices, catheters, cannulas and needles. Further, the surgical device or implant may include externally used devices and implants includes patches, gauzes, wound fillers, cavity fillers, poultices, bandages, adhesive polymers and films.

[0170] Materials for medical devices and implants include: titanium and titanium alloys, gold and gold alloys, cobalt-chromium alloys, iron-chromium-nickel alloys, stainless steel, ceramics, niobium, tantalum, zirconia, aluminium oxides, zirconium oxide, hydroxyapatite, tricalcium phosphate, bio glass, silicon and polymers.

[0171] In some embodiments, the surgical device includes, or is formed of, a polymer. In some embodiments, the polymer is nylon, polyamide, polyester, polyethylene, polyvinylidene fluoride (PVDF), polypropylene, ultra-high molecular weight polyethylene (UHMWPE) polydimethylsiloxane, parylenepolytetrafluoroethylene, polymethylmethacrylate, polyimide, and polyurethane. In some embodiments, the polymer is polypropylene or polyester.

[0172] Further provided herein are dressings or patches which include a composition or solution in accordance with the present invention. In some embodiments, these patches or dressings are for dressing, or sealing, wounds. In some embodiments, the dressing or patches are adhesive dressings or patches.

[0173] Bacterial Infections and Biofilms [0174] As is understood in the art, bacteria can exist in a planktonic form or in a biofilm. In nature almost all bacteria grow in biofilms. Once adhered to a tissue, or the surface of a substrate, planktonic bacteria immediate begin to form a colony and secrete a polymer matrix, ultimately forming a biofilm. After forming, biofilms can release individual free-form bacterium which are motile and can colonise new locations forming remote biofilms.

[0175] Once formed, bacteria within a biofilm are extremely resistant to a host’s immune defence mechanisms. Further, they are resistant to antimicrobials such as antiseptics, disinfectants and antibiotics. By contrast the mobile planktonic bacteria are more susceptible to the immune system and antimicrobial treatments.

[0176] There are distinct differences between the external appearance and functionality of bacteria in a biofilm and that of planktonic bacteria. Bacteria in biofilms are visually distinct due to their location in close proximity to other bacteria, their adhesive nature and the surrounding self-produced matrix containing exopolysaccharides, proteins, nucleic acids and other bacterial detritus. In addition, studies indicate that biofilms exhibit low metabolic activity together with an upregulation of genes needed for anaerobic growth, all under the strict control of quorum sensing. Therefore, biofilms are easily identified by a skilled technician.

[0177] In some embodiments of the present invention, the antimicrobial composition or solution is for inhibiting the growth of, or killing, a planktonic bacterium. In some embodiments of the present invention, the antimicrobial composition or solution is for inhibiting the growth of, or killing, a bacterium in a biofilm. In some embodiments, the antimicrobial composition or solution is for preventing the formation of a biofilm.

[0178] Bacteria are often categorised based on their cell wall types and the relative abundance of peptidoglycan in their cell walls. Gram staining utilises crystal violet (triarylmethane) dye which integrates with the peptidoglycan in the cell wall of the bacteria, which is then variably retained when washed out with ethanol. Gram-positive bacteria include more peptidoglycan and therefore retain more of the crystal violet dye when washed and appear purple/blue under microscopy. In contrast gram-negative bacteria retain little of the of the dye and are typically coloured pink/red by a counter stain to permit visualisation.

[0179] In addition to gram-positive and gram-negative bacteria there are additional categories of gram-variable and gram-indeterminate groups. Gram-variable bacteria typically exhibit mixed staining showing a mixture of blue/purple and red/pink staining. Further, gram- intermediate bacteria do not respond predictably to Gram stain and therefore cannot be considered as gram-positive or gram negative. A classic example of gram-intermediate bacteria are Mycobacterium species which are considered as acid-fast and therefore do not readily stain with Gram stain.

[0180] In some embodiments of the present invention, the composition, solution and methods are not for inhibiting the growth of, or killing, mycobacterium. In some embodiments, the composition, solution and methods are not for inhibiting the growth of, or killing, acid-fast, gram-variable and/or gram-indeterminate bacteria.

[0181 ] As will be demonstrated in the Examples, the antibacterial composition and solution of the present invention shows efficacy against a broad range of gram-positive bacteria. Therefore, in some embodiments of the present invention the antimicrobial composition, solution or method is for inhibiting the growth of, or killing, gram-positive bacteria.

[0182] Common gram-positive genera include (but are not limited to): Bacillus, Clostridia, Corynebacterium, Listeria, Staphylococcus, Streptococcus and Enterococcus. Therefore, in some embodiments to the present invention, the composition, solution or methods prevent, kill or treat a gram-positive bacterium selected from the genus: Bacillus, Clostridia, Corynebacterium, Listeria, Staphylococcus, Streptococcus or Enterococcus. In some embodiments, the gram-positive bacterium is selected from the genus: Enterococcus or Staphylococcus.

[0183] Common gram-positive bacteria species include (but are not limited to): Bacillus anthracis, Bacillus cereus, Clostridia tetani, Clostridia botulinum, Clostridia perfringens, Clostridia difficile, Corynebacterium diphtheria, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus aqui, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, Bacillus subtilis, Nocardia asteroides, Actinomyces israelii, Propionibacterium acnes and Enterococcus species. Other types of bacteria are contemplated.

[0184] Further the solutions and compositions of the present invention may be efficacious against some strains of gram-negative bacteria. Specifically, as shown in the examples herein, DDC in combination with copper is effective at killing bacteria in the genus Moraxella, specifically Moraxella catarrhalis.

[0185] Efficacy of the combination of DDC and copper has been demonstrated against bacterium selected from the species: Enterococcus faecium, Enterococcis faecalisor, Staphylococcus aureus or Moraxella catarrhalis. [0186] While the examples provided herein demonstrate efficacy against the above specified species, the compositions, solutions and methods of the present invention may be effective against other species of bacteria. For example, the compositions, solutions and methods of the present invention may be effective against: Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Pseudomonas aeruginosa, Vibrio cholerae, Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurella multocida, Legionella pneumophila, Salmonella typhi, Brucella abortus, Chlamydi trachomatis, Chlamydia psittaci, Coxiella bumetti, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, Yersinia pestis, Yersinia enterolitica, Escherichia hirae, Burkhoideria cepacia, Burkhoideria pseudomallei, Francisella tularensis, Bacteroides fragilis, Fusobascterium nucleatum, and Cowdria ruminantium. Other types of bacteria are contemplated.

[0187] Methods for screening the inhibition or killing of bacteria are provided in the examples herein and are known in the art. For examples O’Neill, A. and Chopra, I., Expert Opin. Investig. Drugs (2004), 13(8); 1045-63. Therefore, and in view of the teachings of the present disclosure, a person skilled in the art can screen the compositions and solutions of the present invention against a range of bacteria which are not exemplified in the examples.

[0188] For example, the activity of the composition or solution may be reflective of the measured minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and/or minimum biofilm inhibitory concentration (MBIC) of the antibiotic or antiseptic, or of the short-kill assay times with respect to an in vitro analysis. For example, the combination of DDC and copper may decrease the MIC, MBC, and/or MBIC of the DDC or copper alone, or reduce the short-kill time for bacteria which are resistant to the DDC or copper alone. The activity may also be observed in the form of an improvement of the condition of the subject, for example, as determined by a clinician.

[0189] As the use of antibiotics have increased in medicine and in agriculture, the number of bacteria resistant to common antibiotics has increased. These bacteria present a public health problem as the transmission of these bacteria within healthcare environments and the community results in a considerable risk of morbidity for patients who may be compromised or vulnerable as well as increased costs from prolonged hospital stays and readmissions. The present invention has shown high efficacy against antibiotic-resistant bacteria, in particular methicillin-resistant Staphylococcus aureus (MRSA).

[0190] Methicillin-resistant Staphylococcus aureus are a group of genetically distinct gram positive bacteria that are related to other strains of Staphylococcus aureus. Methicillin-resistant Staphylococcus aureus demonstrate resistance to killing by a range of beta-lactam antibiotics including penams, cephems and cephalosporins. Diagnosis to MRSA often relies on bacterial cultures of collected sample such as sputum, blood, urine or other body fluids in the presence of various antibiotics. More rapid, but less conclusive, test exist which include quantitative polymerase chain reaction and rapid latex agglutination.

[0191 ] The present invention has demonstrated efficacy in killing and inhibiting the growth of antibiotic-resistant bacteria and has also shown the ability to potentiate the efficacy of antibiotics against such bacteria. Therefore, in some embodiments, the composition, solution and methods of the present invention are for inhibiting the growth of, or killing, antibiotic resistant bacteria. In some embodiments, the antibiotic-resistant bacteria are MRSA.

[0192] Fungal Infections

[0193] In addition to the anti-bacterial properties discussed above, the combination of DDC and copper is effective against fungi. Accordingly, in some embodiments, the composition, solution and methods of the present invention is for inhibiting the growth of, or killing, a fungus.

[0194] Examples of fungal infections are described herein and include infections associated with a fungal species such as Aspergillus, Alternaria, Aureobasidium, Candida, Cladosporium, Cryptococcus, Curvularia, Coniophora, Diplodia, Epidermophyton, Engodontium, Fusarium, Gliocladium, Gloeophylium, Humicola, Histoplasma, Lecythophora, Lentinus, Malassezia, Memnionella, Mucor, Oligoporus, Paecilomyces, Penicillium, Petriella, Paracoccidioides, Phanerochaete, Phoma, Pneumocystis, Poria, Pythium, Rhodotorula, Rhizopus, Schizophyllum, Sclerophoma, Scopulariopsis, Serpula, Sporobolomyces, Stachybotrys, Stemphylium, Trichosporon, Trichtophyton, Trichurus, or Ulocladium. Other types of fungi are contemplated.

[0195] In some embodiments, the infection may be due to a fungal skin or mucosal infection.

[0196] In some embodiments, the fungus is a member of the Candida genus. In some embodiments, the fungus is a stain of Candida albicans, Candida auris, Candida dubliniensis, Candida kruzei or Candida parapsilosis.

[0197] Methods, Uses and Kits

[0198] As demonstrated herein, the composition and solution of the present invention can be used to inhibit, or kill, a range of microorganisms including bacteria and fungi. Therefore, the present invention also provides a method of inhibiting the growth of a bacterium or a fungus, or killing a bacterium or a fungus, or preventing the formation of a bacterial biofilm, or treating a bacterial biofilm, the method comprising providing, or exposing the bacterium or fungus or biofilm to, an effective amount of the antimicrobial composition or solution as described herein.

[0199] The method can be performed on any suitable subject, surface or object. These include a subject in need of treatment, a surgical device or implant (such as those discussed herein), a substrate or a surface.

[0200] Accordingly, in some embodiments there is provided a method of inhibiting the growth of a bacterium or a fungus, or killing a bacterium or a fungus, or preventing the formation of a bacterial or fungal biofilm, or treating a bacterial or fungal biofilm, on a medical device or implant the method comprising providing, or exposing the bacterium or fungus or biofilm to, an effective amount of the antimicrobial composition or solution as described herein.

[0201 ] Further, in some embodiments, there is provided a method of preventing or treating an infection in a subject, the method comprising providing to the site of infection an effective amount of a composition or solution of the present invention or exposing the infection to an effective amount of the antimicrobial composition or solution as described herein.

[0202] Exposing microorganisms to compositions and solution of the present invention may not directly result in the killing or inhibition of the microorganism but rather may reduce the viability of the microorganism, which may result in the microorganism being vulnerable to other antimicrobial treatments or immunological attack. Therefore, in some embodiments, there is provided a method of reducing the viability of a microorganism, the method including providing, or exposing the microorganism to, a composition or solution of the present invention.

[0203] The methods of the present invention may further include the additional step of providing, or exposing the microorganism to, an antimicrobial agent. In the context of treating a patient or object the antimicrobial agent may be administered as part of the composition or solution of the present invention or may be administered sequentially to the composition or solution of the present invention.

[0204] Further, and in relation to treating a subject, the antimicrobial agent may be administered systemically, while the compositions and solutions of the present invention may be administered locally. Such treatments are particularly envisaged when oral or intravenous antibiotics and antifungals are used as a first line treatment. Suitable antimicrobial agents are described below. [0205] In some embodiments of the method, the antimicrobial composition or antimicrobial solution, when administered or provided in combination with an antimicrobial agent, potentiates the antimicrobial agent. In some embodiments, the administration or provision of the antimicrobial agent potentiates the antimicrobial composition or solution of the present invention.

[0206] Antimicrobial agents for use with the methods of the present invention are provided below. Further, microorganisms to be treated, inhibited, killed or have their viability reduced are disclosed and discussed herein.

[0207] Further provided herein is the use of DDC, or a salt thereof, and copper, or a salt thereof in the preparation of a medicament for the treatment or prevention of a microorganism infection in a subject. In some embodiments, the medicament is a topical medicament.

[0208] Further provided is a kit including diethyldithiocarbamate (DDC), or a salt thereof, and copper, or a salt thereof. In forms of the kit the DDC or copper, or the salts thereof, are adapted for combination or simultaneous or sequential administration such that the DDC and copper (or salts thereof) interact. Suitably adapted forms include: the combination of DDC and copper (or salts thereof) into a composition; co-packaging of DDC with copper (or salts thereof) in a device adapted for coadministration or sequential administration; or co-packaging of DDC with copper (or salts thereof) in a manner intended for combination, co-administration or sequential administration, this may include directions or instructions for combination, co administration or sequential administration.

[0209] In some embodiments of the kit, the weight to weight ratio the salt of copper to DDC, or the salt thereof, is between 1 :1 and 8:1 . In some embodiments of the kit, the mole to mole ratio the salt of copper to DDC, or the salt thereof, is between 1 :1 and 8:1. In some embodiments, the ratio is any appropriate ratio of the composition disclosed herein.

[0210] In some embodiments, the kit includes a diluent for dissolving the DDC and copper, or salts thereof. The diluent will be chosen based on the intended use of the resultant antimicrobial solution and may include liquid diluents such as sterile water, 0.9% sodium chloride injection, USP (normal saline), 5% dextrose solution, USP (D5W), Ringer's injection, USP and Lactated Ringer's injection or USP, amongst others.

[0211] When the kit includes a diluent, the volume of the diluent may be proportional to the weight of the DDC, or a salt thereof, to provide a solution having a concentration of DDC of at least 0.125 pg/ml. Moreover, the volume of the diluent may be proportional to the weight of the copper or salt thereof, to provide a solution having a concentration of copper of at least 0.5 pg/ml.

[0212] In some embodiments, the kit may further include an antimicrobial, such as those disclosed herein.

[0213] It is intended that in some embodiments, the kit is for use in methods such as those disclosed herein.

[0214] Antimicrobial Agents

[0215] The compositions and solutions described above include DCC and copper (or salts thereof), however it is demonstrated in the Examples that the combination of DDC with copper can potentiate the efficacy of antimicrobials or interact synergistically with antimicrobials to inhibit or kill microbes. Accordingly, in some embodiments the antimicrobial compositions and solutions of the present invention further include an antimicrobial agent. Moreover, in some embodiments the methods of the invention include the additional step of administering or providing an antimicrobial agent.

[0216] In some embodiments, of the composition, solution and methods of the present invention the antimicrobial agent is an antibiotic, an antiseptic or an antifungal.

[0217] The means by which the antimicrobial agent is administered or provided will be understood by a person skilled in the art and will be suitable for the treated infection or purpose with regard to the site of treatment, the subject, the tissue or the material to be treated and the antimicrobial agent used. Specific embodiments include: oral administration of antifungals and antibiotics, topical administration of antifungals, antibiotics and antiseptics and the application of antifungals, antibiotics, antiseptics or disinfectants to the surface of an object.

[0218] Antibiotics

[0219] In some embodiments, of the composition, solution and methods of the present invention the antimicrobial agent is an antibiotic.

[0220] Antibiotics for use in the methods, composition or solution of the present invention may be selected from the group consisting of one or more of: a protein synthesis inhibitor, a cell wall synthesis inhibitor, a beta-lactam antibiotic, a beta-lactamase inhibitor, a lipopeptide, a peptidoglycan synthesis inhibitor, a DNA synthesis inhibitor, a RNA synthesis inhibitor, a mycolic acid synthesis inhibitor, a mechanosensitive channel of large conductance (MscL), and a folic acid synthesis inhibitor, or a combination of the aforementioned antibiotics or a combination of the aforementioned antibiotics. Antibiotics for use in the present invention can be purchased from relevant commercial suppliers such as Sigma-Aldrich (Castle Hill, NSW, Australia), and methods for their use are known in the art, for example as described in “Therapeutic Guidelines - Antibiotic”, Version 15, 2014, published by eTG complete.

[0221] Examples of cell wall synthesis inhibitors include, but are not limited to, carbapenems (such as ertapenem, doripenem, imipenem and meropenem), penicillins (such as amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, fluloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin and ticarcillin), cephalosporins (such as cefadroxil, cefazolin, cefalotin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil and ceftobiprole), monobactams (such as aztreonam), fosfomycin, polymyxin B, bacitracin, colistin, glycopeptides (such as teicoplanin, vancomycin, telavancin, dalbavancin and oritavancin), and beta-lactamase inhibitors (such as clavulanic acid, sulbactam, tazobactam, tebipenem, avibactam and relebactam).

[0222] Examples of DNA synthesis inhibitors include, but are not limited to: quinolones or fluoroquinolones (such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin,grepafloxacin, sparfloxacin and temafloxacin) or metronizadole.

[0223] Examples of RNA synthesis inhibitors include, but are not limited to, rifamycins such as rifampin and rifapentine.

[0224] Mechanosensitive channels of large conductance (MscL) consists of pore-forming membrane proteins that are responsible for translating physical forces applied to cell membranes into electrophysiological activities. MscL have a relatively large conductance, 3 nS, making them permeable to ions, water, and small proteins when opened. Examples of MscL can be found at http://www.tcdb.org/search/result.php?tc=1.A.22.3. One specific example is Ramizol, which belongs to the styrylbenzene class of antibiotics.

[0225] Examples of folic acid synthesis inhibitors include, but are not limited to: sulfonamides (such as mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole and sulfonamidochrysoidine) or pyrimethamine.

[0226] In some embodiments of the present invention the antibiotic is selected from the group consisting of: a quinolone antibiotic, a tetracycline antibiotic, a glycopeptide antibiotic, an aminoglycoside antibiotic, a penicillin antibiotic, a cephalosporin antibiotic, a cephamycin antibiotic or a macrolide antibiotic. In some embodiments, the antibiotic is ciprofloxacin, doxycycline, vancomycin, gentamycin, amoxicillin, ceftazidime or erythromycin.

[0227] Antiseptics

[0228] In some embodiments of the composition, solution and methods of the present invention the antimicrobial agent is an antiseptic.

[0229] Antiseptics encompass a range of antimicrobial substances and solution intended to be applied to living tissue such as the skin or sinuses. Unlike antibiotics, antiseptics are intended for external use (including use on internal membranes such as the sinuses) rather than systemic use. Antiseptics include antibacterials, antimycotics and fungicides.

[0230] Antiseptics include, but are not limited to the group consisting of: an alcohol (primarily ethanol and isopropanol), benzethonium chloride, chlorhexidine, chloroxylenol, hexachlorophene, hydrogen peroxide, iodine, octenidine dihydrochloride, oxychlorosene sodium, polyhexanide, povidone iodine, sodium hypochlorite or triclosan.

[0231] In some embodiments, the antiseptic is for use on a mammalian subject, such as a human, and therefore it is suitable for administration to a mammalian subject or is approved for use, or human use.

[0232] Antifungals

[0233] In some embodiments of the antimicrobial composition, solution and methods of the present invention the antimicrobial agent is an antifungal. In some embodiments the antifungal is selected from the group consisting of: a polyene, an azole, an allylamine or an echinocandin.

[0234] A polyene is a molecule with multiple conjugated double bonds. A polyene antifungal is a macrocyclic polyene with a heavily hydroxylated region on the ring opposite the conjugated system. This makes polyene antifungals amphiphilic. Polyene antimycotics bind with sterols in the fungal cell membrane, principally ergosterol. This changes the transition temperature of the cell membrane, thereby placing the membrane in a less fluid, more crystalline state. As a result, the contents of the fungal cell leak and result in cell death.

[0235] In some embodiments, the polyene antifungal is selected from one or more of: amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin or rimocidin. [0236] An azole antifungal can inhibit the enzyme lanosterol 14 a-demethylase, which is necessary to convert lanosterol to ergosterol. Depletion of ergosterol in fungal membrane disrupts the structure and many functions of the membrane ultimately leading to inhibition of fungal growth.

[0237] In some embodiments, the azole antifungal is selected from an imidazole, a triazole, and/or a thiazole. For example, the imidazole may be selected from bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole and tioconazole. The triazole may be selected from albaconazole, efinaconazole, epoxyconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole or voriconazole. The thiazole may include abafungin

[0238] An allylamine can inhibit squalene epoxidase, which is another enzyme required for ergosterol synthesis in the fungal membrane. In some embodiments, the allylamine antifungal may be selected from: amorolfin, butenafine, naftifine or terbinafine.

[0239] An echinocandin inhibits the synthesis of glucan in the cell wall via the enzyme 1 ,3- Beta-glucan synthase. In some embodiments, the echinocandin antifungal may be selected from anidulafungin, caspofungin and micafungin.

[0240] The antifungal for use in the composition of the present invention may also be selected from the group consisting of: an aurone, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, tolnaflate, undecylenic acid, crystal violet or Balsam of Peru.

[0241] In some embodiments of the present invention the antifungal is selected from the group consisting of: amphotericin B, anidulafungin, candicidin, caspofungin, clotrimazole, filipin, fluconazole, flucytosine, griseofulvin, hamycin, isavuconazonium, itraconazole, ketoconazole natamycin, micafungin, miconazole, nystatin, posaconazole, rimocidin, terbinafine or voriconazole.

[0242] In some embodiments, the antifungal is suitable for administration to a mammalian subject, preferably a human.

[0243] Examples

[0244] The efficacy of DDC in combination with copper has been tested against various microorganisms in accordance with the following Examples which are included to exemplify particular embodiments of the invention and should not be considered to limit the invention as defined herein. [0245] Through the Examples the following bacterial strains have been used: Staphylococcus epidermidis ATCC 35984 (S. epi 1 ), Staphylococcus epidermidis ATCC 14990 (S. epi 2), methicillin-resistant Staphylococcus aureus (MRSA) clinical isolate 1 (MRSA 1), MRSA clinical isolate 2 (MRSA 2), MRSA clinical isolate 3 (MRSA 3), MRSA clinical isolate 4 (MRSA 4), MRSA clinical isolate 5 (MRSA 5), MRSA clinical isolate 6 (MRSA 6), Enterococcus faecium ATCC 19434 (E. faecium 1), Enterococcus faecium clinical isolate (E. faecium 2), Enterococcus faecalis clinical isolate (E. faecalis), Staphylococcus aureus ATCC 25923 (S. aureus), Staphylococcus aureus small colony variant/clinical isolate (S. aureus SCV), Acinetobacter baumanii ATCC 19606 (A. baum 1), Acinetobacter baumanii clinical isolate (A. baum 2), Escherichia coli clinical isolate 1 (E. coli 1 ), Escherichia coli clinical isolate 2 (E. coli 2), Klebsiella pneumonia ATCC 13883 (K. pneum), Moraxella catarrhalis ATCC 49143 (M. catarr), Pseudomonas aeruginosa PA01 (P. aerug 1 ), Pseudomonas aeruginosa clinical isolate (P. aerug 2).

[0246] Further, and unless specified otherwise, a reference to DDC in the following methods refers to sodium DDC trihydrate and a reference to copper refers to copper II sulfate. Both of these are readily available from commercial chemical suppliers such as Sigma Aldrich.

[0247] Example 1 - Assessment of Minimal Inhibitory Concentration of DDC and/or Metal Cations on Planktonic Bacteria

[0248] The ability of DDC, various metal cations and combinations of DDC and the various metal cations to inhibit the growth of S. epidermidis 1 planktonic bacteria was assess in accordance with the following protocol.

[0249] The minimum inhibitory concentration (MIC) values of various metal cations (gallium (III), iron (II), zinc (II) or copper (II)), DDC and combinations of DDC with the metal cation were assessed against planktonic (free floating/non-adherent) bacteria using standard methods (i.e. the colony suspension and broth microdilution method as described in Wiegand I. eta\. Nat Protoc. (2008); 3(2): pp.163-75).

[0250] Briefly, S. epidermidis 1 bacteria were suspended in 0.9% saline and adjusted to 0.5 McFarland units (approx. 1.5x10⁸ colony forming units (CFU)/ml). After a 1 in 100 dilution in Mueller-Hinton-Broth, the bacterial suspension was added to a 96-well microtiter plate with 100 mI per well. One hundred mI of treatment was added to the first well of column 1 and then serially diluted up to column 10. Column 11 served as maximum growth control and column 12 as negative control (i.e. pure broth without bacterial growth). Treatments were incubated for 24 hours at 37^eC and the MIC was determined visually as the lowest concentration of a treatment to inhibit bacterial growth. [0251 ] The MIC is determined as the lowest concentration of a drug to inhibit the bacterial growth. Lower MIC values indicate an increased activity of the compound in solution.

[0252] Individual treatment concentrations ranged from 0.0625 to 512 pg/ml of DDC (sodium DDC trihydrate, purchased from Sigma Aldrich) and 1 to 512 pg/ml of the following metal salts: gallium (III) chloride, iron (II) sulfate, zinc (II) sulfate, copper (II) sulfate in media.

[0253] For treatment combinations of metal salts with DDC, the DDC concentration was varied from 0.0625 pg/ml to 512 pg/ml, while the metal salt concentration was maintained at 16 pg/ml. The MIC was the lowest concentration of DDC combined with 16 pg/ml of the cationic salt to inhibit cell growth.

[0254] As can be seen in Figure 1 , the MIC for DDC was 64 pg/ml while all of the metals had a MIC of 256pg/ml indicating that concentrations below these would not inhibit the growth of the planktonic bacteria. Further, the combination of gallium with DDC did not result in a lower MIC compared to DDC alone. Flowever, all of iron, zinc and copper potentiated the efficacy of DDC with the MIC of DDC decreasing by 50% when combined with iron (MIC DDC:Fe²⁺ = 32 pg/ml), 75% when combined with zinc (MIC DDC:Zn²⁺ = 16 pg/ml) and over 96% when combined with copper (MIC DDC:Cu²⁺ = 2 pg/ml).

[0255] Example 2 - Varying Copper(ll) Salts are Effective in Inhibiting Planktonic Bacteria Growth.

[0256] Flaving established that DDC in combination with copper sulfate effectively kills planktonic bacteria, different divalent cationic salts of copper were assessed. The protocol outlined above was performed with 0.125 pg/ml to 64 pg/ml of DDC in combination with 16 pg/ml of either copper sulfate, copper chloride or copper acetate.

[0257] As can be seen in Figure 2 each of these copper salts provided a comparable MIC when combined with DDC.

[0258] Example 3 - DDC and Copper Demonstrate a Synergistic Decrease in the Minimum Inhibitory Concentration for a Broad Spectrum of Gram-Positive Bacteria.

[0259] Flaving established that copper when combined with DDC results in a significant improvement in antimicrobial activity against S. epidermidis 1 strain (Figure 1) the efficacy of DDC and copper against a range of gram positive and gram-negative bacteria was assessed using the protocol describe in Example 1 , with the exception of variations in the strains of bacteria. [0260] Gram-positive bacteria tested were two strains of Staphylococcus epidermidis (S. epi 1 and 2), six strains of Methicillin-resistant Staphylococcus aureus (MRSA 1 to 6), two strains of Enterococcus faecium (E. faecium 1 and 2), one strain of Enterococcus faecalis, one strain of Staphylococcus aureus and one strain of small colony variants of Staphylococcus aureus (S. aureus SCV).

[0261] Gram-negative bacteria tested were two strains of Acinetobacter baumannii (A. baum 1 and 2), two strains of Escherichia coli (E. coli 1 and 2), Klebsiella pneumoniae (K. pneum), Moraxella catarrhalis (M. catarr) and two strains of Pseudomonas aeruginosa (P. aerug 1 and 2).

[0262] As can be seen in Figures 3A and 3B the combination of DDC and copper sulfate resulted in significantly improved antimicrobial effects compared to DDC or copper alone, as evidenced by a lower MIC, for a broad range of gram-positive bacteria. These included both strains of S. epi, all six strains of MRSA, both strains of E. faecium and E. faecalis.

[0263] However, as illustrated in Figure 3C, DDC and copper only decreased the MIC for one gram-negative species, Moraxella catarrhalis, while combining DDC and copper either had no effect, or had an inhibitory effect, on the antimicrobial effect of DDC for the other 7 stains of gram-negative bacteria.

[0264] Example 4 - DDC and Copper Inhibit the Growth of MRSA in a Ratio Dependent Manner.

[0265] The efficacy of DDC in combination with copper at various ratios in preventing the growth of a strain of gram-positive bacteria was assessed using the following protocol.

[0266] Bacteria were suspended in 0.9% saline and adjusted to an OD600 of 0.1 (approx. 1 x10^s CFU/ml). After a 1 in 10 dilution in Mueller-Hinton-Broth, the bacterial suspension was added to a 96-well microtiter plate with 100 mI per well. One hundred mI of treatment containing various concentrations of DDC (as set out below) in combination with a fixed concentration of copper was added to each well and incubated with Staphylococcus epidermidis over a 24 hr period at 37^eC. The optical density (OD 600) was measured at specific time points (i.e. 0, 1 , 2, 3, 4, 5, 6, 7, 8, 22, 23, 24 hours) supplemented with either: 16 pg/ml of copper sulfate alone, no supplementation (max growth - control) or one of the following combinations of DDC and copper: 128 pg/ml of DDC and 16 pg/ml of copper sulfate (8:1 ratio); 64 pg/ml of DDC and 16 pg/ml of copper sulfate (4:1 ratio); 32 pg/ml of DDC and 16 pg/ml of copper sulfate (2:1 ratio); 16 pg/ml of DDC and 16 pg/ml of copper sulfate (1 :1 ratio); 8 pg/ml of DDC and 16 pg/ml of copper sulfate (1 :2 ratio); 4 pg/ml of DDC and 16 pg/ml of copper sulfate (1 :4 ratio); 2 pg/ml of DDC and 16 pg/ml of copper sulfate (1 :8 ratio); 1 pg/ml of DDC and 16 pg/ml of copper sulfate (1 :16 ratio); or 0.5 pg/ml of DDC and 16 pg/ml of copper sulfate (1 :32 ratio).

[0267] The optical density (OD) of the cells in culture was measured at 600nm to assess growth, with higher optical density indicating higher growth of the bacteria.

[0268] Figure 4 illustrates that copper and DDC optimally prevent the growth of MRSA 3 at ratios from about 1 :2 (8 pg of DDC and 16 pg/ml of copper sulfate) and 1 :16 (1 pg/ml of DDC and 16 pg/ml of copper sulfate). Further, while not as effective, a 1 :1 ratio of copper to DDC (to 16 pg/ml DDC and 16 pg/ml copper sulfate) significantly decreased bacterial growth.

[0269] Surprisingly, a higher concentration of DDC which results in a lower copper to DDC ratio (i.e. 1 :2 to 1 :8 copper to DDC), was not as effective at preventing the growth of MRSA, despite the concentration of copper being fixed. Consequently, it appears that the amount of copper sulfate needs to be in excess on a weight to weight basis. As set out in table 2, the molar mass of copper sulfate is 159.6 g/mol and the molar mass of Sodium DDC trihydrate is 225.3 g/mol. As such a 1 :1 ratio of copper sulfate to Sodium DDC trihydrate by mass concentration approximates a 1.4:1 molar ratio of copper to DDC ions in solution.

[0270] Example 5 - DDC and Copper Inhibit Bacterial Biofilms.

[0271 ] As established above, the combination of DDC with copper results in effective killing of multiple strains of planktonic bacteria. Flowever, bacterial biofilms are known to be significantly resistant (up to 1000 times) to traditional antibacterial treatments such as antibiotics and antiseptics (see for example Gebreyohannes, G. et al. Heliyon 019 Aug; 5(8): e02192). Therefore, the efficacy of DDC and copper in treating bacterial biofilms was assessed using the AlamarBlue viability assay as briefly set out below.

[0272] A bacterial suspension was prepared by immersing single colonies of two strains of MRSA (MRSA 2 and MRSA 3) in 0.9% saline, adjusted to 1.0 McFarland units (3x10⁸ CFU/ml), and diluted 1 :15 in nutrient broth media. One hundred pi of the prepared bacterial suspension was added to each well of a 96-well black microtiter plate and incubated at 37°C for 48 hours on a rotating platform at 70 rpm to allow biofilm formation. The formed biofilms were washed with 0.9% saline to remove planktonic cells, followed by exposure to 100 mI DDC (ranging from 0.5 pg/ml to 32 pg/ml), 100 mI Copper sulfate (16 pg/ml) or varying ratios of DDC and copper sulfate dissolved in media. Specifically, DDC and copper were tested at ratios of 2:1 (32 pg of DDC and 16 pg of copper), 1 :1 (16 pg of DDC and 16 pg of copper), 1 :2 (8 pg of DDC and 16 pg of copper), 1 :4 (4 pg of DDC and 16 pg of copper), 1 :8 (2 pg of DDC and 16 pg of copper), 1 :16 (1 pg of DDC and 16 pg of copper) and 1 :32 (0.5 pg of DDC and 16 pg of copper).

[0273] After 24 hours incubation at 37°C on a rotating platform, the supernatants were aspired and biofilms were washed in saline. To assess viability of cells within the biofilm 100 pi of a freshly prepared 10% AlamarBlue dilution in media was added to each well. Viable bacteria metabolise the AlamarBlue dye (resazurin) to a pink dye (resorufin), permitting quantification of cell viability by measuring fluorescence intensity. Following the addition of AlmarBlue dye, plates were incubated in the dark at 37°C on a rotating platform for up to 5 hours. The fluorescence was measured hourly using 530 nm light for excitation and reading emissions at 590 nm. Maximum intensities were typically reached after 4 hours incubation and were used for quantification according to the equation:

[0274] Bacterial viability was determined as the percentage of biofilm killing (%BK), where 1C represents the fluorescence intensity of the controls (i.e. 100% bacterial growth) and IT indicates the maximum intensity of each treatment. Both IC and IT were corrected by the intensity of background (i.e. 0% bacterial growth). The results illustrated in Figure 5A (MRSA 2) and Figure 5B (MRSA 3) indicate the percentage of biofilm killing after treatment exposure.

[0275] As can be seen in Figures 5A (MRSA 2) and 5B (MRSA 3) killing of bacteria within a biofilm by DDC alone was negligible, while copper was only moderately effective at killing bacteria within a biofilm (approximately 20% to 35% killing). Flowever, DDC when combined with copper sulfate resulted in greater than 85% killing (approximately 90% killing) at DDC to copper sulfate ratios of 1 :1 to 1 :8 (wt:wt). Interestingly, a higher ratio of DDC to copper (2:1 ) was less effective than copper sulfate alone, while once the concentration of copper sulfate was 32 times that of DDC the efficacy of the treatment also decreased.

[0276] These data show that DDC and copper synergistically combine to effectively kill bacterial within a biofilm. This is particularly significant in view of the difficulty in treating biofilm infections.

[0277] In order to assess the effect of concentration of DDC and copper on killing bacterial biofilms a 1 :4 weight to weight ratio of DDC to copper sulfate was prepared at varying concentrations and tested against MRSA 1 bacteria. Specifically, serial dilutions of DDC and copper were prepared at the concentrations of: 32 pg/ml DDC + 128 pg/ml copper sulfate, 16 pg/ml DDC + 64 pg/ml copper sulfate, 8 pg/ml DDC + 32 pg/ml copper sulfate, 4 pg/ml DDC + 16 pg/ml copper sulfate, 2 pg/ml DDC + 8 pg/ml copper sulfate, 1 pg/ml DDC + 4 pg/ml copper sulfate and 0.5 pg/ml DDC + 2 pg/ml copper sulfate.

[0278] MRSA 1 bacterial biofilms were prepared as described above for MRSA 2 and 3 before being incubated at 37°C for 24 hours with the varying concentrations of DDC and copper sulfate listed above.

[0279] As shown in Figure 6, comparable killing of MRSA 1 biofilm forming bacteria was seen at concentrations of DDC to copper sulfate of 32 pg/ml and 128 pg/ml through to 4 pg/ml and 16pg/ml demonstrating a large therapeutic range.

[0280] Having identified above that 32 pg of copper sulfate was effective at treating MRSA biofilms, when combined with 8 pg/ml of DDC, varying concentrations of DDC were combined with 32 pg/ml of copper sulfate. Specifically, DDC and copper sulfate were prepared at the concentrations of: 256 pg/ml of DDC + 32 pg/ml of copper sulfate (8:1 ratio), 128 pg/ml of DDC + 32 pg/ml of copper sulfate (4:1 ratio), 64 pg/ml of DDC + 32 pg/ml of copper sulfate (2:1 ratio), 32 pg/ml of DDC + 32 pg/ml of copper sulfate (1 :1 ratio), 16 pg/ml of DDC + 32 pg/ml of copper sulfate (1 :2 ratio), 8 pg/ml of DDC + 32 pg/ml of copper sulfate (1 :4 ratio), 4 pg/ml of DDC + 32 pg/ml of copper sulfate (1 :16 ratio), 2 pg/ml of DDC + 32 pg/ml of copper sulfate (1 :32 ratio) and 1 pg/ml of DDC + 32 pg/ml of copper sulfate (1 :64 ratio).

[0281] MRSA 2 bacterial biofilms were prepared as described above before being incubated at 37°C for 24 hours with the varying concentrations of DDC and copper sulfate listed above.

[0282] As can be seen in Figure 7, weight to weight ratios of DDC to copper sulfate of 1 :1 through to 1 :16 showed comparable killing above approximately 90%. Consistent with the results for planktonic bacteria, higher concentrations of DDC, resulting in a higher DDC to copper sulfate ratio, had surprisingly lower rates of efficacy as demonstrated by less effective killing, despite the concentration of copper sulfate being consistent. Further, once the concentration of DDC dropped to 2 pg/ml (1 :16 ratio to copper sulfate) then efficacy of killing MRSA 2 in biofilms decreased.

[0283] Example 6 - DDC and Copper Demonstrate Synergistic Killing for a Broad Spectrum of Biofilm Formino Bacteria.

[0284] Biofilms of four strains of MRSA (MRSA 1 , 2, 4 and 6) and one strain of Staphylococcus epidermidis (S. epi 2) were prepared as described above, before being incubated with 4 pg/ml of DDC, 32 pg/ml of copper sulfate or a combination of 4 pg/ml and 32 pg/ml of copper sulfate.

[0285] The AlamarBlue assay was performed as described above to assess the ability of DDC and copper to kill a range of biofilm forming bacterial strains.

[0286] As can be seen in Figure 8, DDC alone had negligible efficacy and copper sulfate alone had minimal efficacy against any of the five selected bacterial stains. However, DDC when combined with copper showed greater than 80% killing for all strains tested, with over 90% for all MRSA strains tested. Consequently, DDC when combined with copper is an effective treatment for a range of biofilm forming bacterial strains.

[0287] Example 7 - Microscopic Analysis of DDC and Copper in Bacterial Biofilm.

[0288] The effect of the combination of DDC and copper in killing bacterial biofilms was visualised using microscopy as described below.

[0289] A bacterial suspension was prepared by immersing single colonies of MRSA 3 in 0.9% saline, adjusted to 0.5 McFarland units (1 x10⁸ CFU/ml), and diluted 1 :100 in nutrient broth media. Three hundred mI of the prepared bacterial suspension was added to each chamber of an 8-chamber glass slides suitable for microscopy. MRSA biofilms were grown for 24 hrs at 37^eC on a rotating platform at 70 rpm. Subsequently, biofilms were exposed for 24 hrs to one of the following four treatments (performed in duplicate): (1) nutrient broth media alone (maximum growth control), (2) 8 pg/ml of DDC in media, (3) 32 pg/ml copper sulfate in media or (4) 8 pg/ml of DDC and 32 pg/ml copper sulfate in media. After the treatment period, the viability of bacteria was quantified by Live/Dead BacLight staining (i.e. 1 mI SYTO 9 and 2 mI propidium iodide in 1 ml media, staining for 30 min in the dark at room temperature), with viable cells fluorescing green, membrane compromised bacteria fluorescing yellow and dead cells fluorescing red. The biofilm viability was visualised using confocal laser scanning microscopy with a 60x objective.

[0290] Example images are provided in Figures 9A (maximum growth control), 9B (sodium DDC trihydrate alone), 9C (copper sulfate alone) and 9D (DDC and copper sulfate). As can be seen, almost all cells in Figures 9A and 9B are green indicating that the biofilms have a high rate of viability. Figure 9C has predominantly live, green, cells with a small number of yellow cell with compromised membranes indicating a minimal effect of copper on biofilm viability. However, by comparison, the majority of cells treated with DDC and copper sulfate (Figure 9D) were red with only a limited number of yellow and green cells. [0291 ] These data are consistent with the previous data, which shows a potent anti-biofilm effect of DDC when combined with copper.

[0292] Example 8 - DDC and Copper Kill Three-Dimensional Biofilms.

[0293] Biofilms can grow in two-dimensions across a surface and later develop into three- dimensional structures. As three-dimensional biofilm structures form, the surface area to cell ratio decreases thereby reducing the exposure of the bacteria within the biofilm to treatments. Consequently, the effect of DDC and copper on killing three dimensional biofilms was assessed using the Bioflux system and the xCELLigence system, as set out below.

[0294] The Bioflux system (Fluxion Biosciences) is a live cell microscopy assay whereby continual media flow can be provided to wells of a cell culture plate which can produce sheer force across the cultured cells. This allows for modelling and microscopic visualisation of a 3D biofilm model.

[0295] Briefly, MRSA 3 bacteria were dispersed in media (i.e. half strength tryptone soy broth containing 0.2% glucose) and adjusted to an OD600 of 0.2. Then, 75 mI of the bacteria were seeded for 30 min at 37^eC in wells of a Bioflux plate under a light microscope. After bacterial attachment to the surface of the wells, bacteria were exposed to a consistent flow of either culture media, media containing 64 pg/ml of DDC and 256 pg/ml of Copper sulfate under steady flow (0.5 Dn) for 24 hours.

[0296] As can be seen in the upper time series of Figure 10 expansive three-dimensional biofilms grew under steady nutrient flow when exposed to control media alone. Flowever, as seen in the lower time series of Figure 10, the combination of DDC and copper was effective at preventing three-dimensional biofilm formation.

[0297] Comparable Bioflux results were also generated for MRSA 6, S.epi 1 and S.epi 2 (data not shown).

[0298] Flaving microscopically analysed the effect of DDC and copper on three- dimensional biofilm formation, the xCELLigence system was used to study biofilm formation in real time.

[0299] The xCELLigence system (Acea Biosciences, Inc.) measures cell growth, morphology and cell viability in real time by measuring impedance of electrical flow between two opposing electrodes in the cell culture well. Greater biofilm formation results in greater electrical impedance and a higher cell “index”. When a treatment is added, the change in impedance is assessed which is correlated with the cell index. Consequently, a treatment with a strong ability to inhibit biofilm formation will have a cell index near zero and may be indicated as a slightly positive or negative plot over time, while a treatment with no or little efficacy will have a strongly positive signal over time.

[0300] To analyse the growth of three-dimensional biofilms over time, three different strains of gram-positive bacteria, namely S. epi 1 , MRSA 3 and MRSA 6, were dispersed in nutrient broth and adjusted to an OD600 of 0.4. Following a baseline measurement with 100 mI media per well in an xCELLigence plate, 50 mI of the bacterial suspension was added to the baseline volume in each well. The seeded cells were then treated with nutrient media (maximum growth control), media supplemented with 8 pg/ml of DDC, media with 32 pg/ml of copper sulfate or media with 8 pg/ml of DDC and 32 pg/ml of copper sulfate. To assess biofilm formation the impedance of the cells was continuously and automatically measured every 15 minutes for 48 hours while incubated at 37°C.

[0301] As can be seen in Figure 11 , biofilm growth in all three strains was inhibited when DDC and copper were combined, while treatment with DDC or copper alone resulted in biofilm growth comparable to that for the maximum growth control.

[0302] These data demonstrate that DDC and copper combine for a synergistic effect in inhibiting biofilm growth.

[0303] Example 9 - DDC and Copper Inhibit Bacterial Cell Growth on Surgical Implants.

[0304] To assess if DDC and copper would synergistically inhibit growth of bacteria on surgical implants, surgical meshes woven of two different polymers were tested as set out below.

[0305] Polypropylene and polyester surgical meshes were incubated with 2x10⁸ CFU/ml of S. epi 1 or MRSA 3 dispersed in tryptone soy broth and incubated at 37°C for 72 hours on a rotating platform at 70 rpm to allow extensive biofilm formation. Nutrient broth media was changed after 24 hours. Subsequently, the biofilm containing meshes were incubated for 24 hrs with one of the following four treatments: (1) Nutrient broth media alone (maximum growth control), (2) 8 pg/ml of sodium DDC trihydrate in media, (3) 32 pg/ml copper sulfate in media or (4) 8 pg/ml of sodium DDC trihydrate and 32 pg/ml copper sulfate in media.

[0306] Following treatment, the meshes were collected in 0.9% saline to extract bacteria by a series of vortexing (5 min) and sonication (15 min), prior to serial dilutions and plating on tryptone soy agar. Treatment efficacy was quantified by CFU counting. [0307] Figure 12 illustrates the number of CFUs for each treatment group, with the CFUs on the y axis expressed on a logarithm scale with the base of 10. As can be seen, DDC and copper synergistically combined to reduce the number of S. epi 1 on the polyester mesh by a log 10 reduction of 2.63 (equating to 99.69 % reduction in biofilm bacterial numbers) and a log 10 reduction of 3.45 in the number of viable S. epi 1 bacteria on polypropylene mesh (correlating to a 99.96 % reduction in biofilm bacterial numbers). By comparison DDC alone and copper sulfate alone showed negligible reduction or a minor increase in CFUs. Further, DDC synergistically combined with copper sulfate to kill MRSA 3 on polyester and polypropylene meshes with a log 10 reduction of 1.23 for the polyester mesh (correlating to 92.64 % biofilm reduction) and a Iog10 reduction of 1.88 for the polypropylene mesh (correlating to 96.53 % biofilm reduction). While, DDC alone and copper sulfate alone showed a negligible reduction in the number of viable cells compared to maximum growth controls.

[0308] In addition to quantifying the number of viable cells on the implantable meshes following treatment, the biofilm present on the polyester mesh was visualised via microscopy, as set out below.

[0309] Biofilms were stained as set out in Example 7 before being visualised via confocal microscopy at 20x magnification. As discussed in Example 7, viable cells fluoresce green, cells having compromised membranes fluoresce yellow and dead cells fluoresce red.

[0310] Figure 13A is a confocal microscope image of a biofilm on the polyester mesh following exposure to control media alone. As can be seen, the majority cells in the biofilm are green indicating they are viable, with only a small portion of yellow (compromised cells) and red (dead) cells. By comparison Figure 13B is a confocal microscope image of the biofilm on the polyester mesh after the treatment with sodium DDC trihydrate (8 pg/ml) and coper sulfate (32 pg/ml). Notably, the majority of cells are red, with only small areas of green cells, indicating that the majority of bacteria in the biofilm have been killed by the treatment.

[0311] These data demonstrate that DDC and copper can kill biofilms on common forms of implantable surgical devices.

[0312] Example 10 - DDC and Copper Synergistically Kill Bacterial Biofilms When Combined with Antibiotics.

[0313] The checkerboard synergy assay was used (as described below) to determine synergistic, additive or antagonistic interrelationship of DDC and copper sulfate when combined with antibiotics. Seven different antibiotics classes were tested alone and in combination with DDC and copper sulfate against MRSA 3 biofilms. Further the combination of DDC and copper was also tested as a control.

[0314] One representative antibiotic was chosen for each antibiotic class, i.e. ciprofloxacin (fluoroquinolone), doxycycline (tetracycline), vancomycin (glycopeptide), gentamycin (aminoglycoside), amoxicillin (penicillin), ceftazidime (cephalosporin) and erythromycin (macrolide).

[0315] Briefly, bacteria were suspended in 0.9% saline and adjusted to 0.5 McFarland units (approx. 1.5x10⁸ CFU/ml). After a 1 in 100 dilution in nutrient broth, the bacterial suspension was added to a 96-well microtiter plate with 100 mI per well. Following biofilm formation over 24 hours at 37^eC, media was aspired and biofilms were washed in 0.9% saline to remove planktonic bacteria. Then, 100 mI of treatment (i.e. 50 mI DDC and Cu and 50 mI antibiotic dissolved in media) was added to each well and incubated for 24 hours at 37^eC, before determining the antibiofilm activity via the AlamarBlue assay as described herein. Nine concentrations of each antibiotic tested (ranging from 128-0.5 pg/ml) were combined with 7 concentrations of DDC+Cu (i.e. 32+128, 16+64, 8+32, 4+16, 2+8, 1+4, 0.5+2 pg/ml) resulting in different combinations of antibiotics combined with DDC and copper in each well. The antibacterial activity of DDC and Cu in combination with antibiotics was compared to the antibacterial activity of the individual antibiotic alone and DDC and Cu (without antibiotics).

[0316] Synergy was calculated as the fractional inhibitory concentration (FIC) index according to the following equation:

MIC of DDC+Cu MIC of DDC+Cu

FIC index = MIC of DDC + MIC of Cu

[0317] An FIC index <0.5 indicates synergistic effects in this particular assay; an FIC index 0.5-4.0 indicates additive or indifferent effects; and an FIC index >4.0 indicates antagonistic effects

[0318] As can be seen in Figure 14, DDC and Cu when combined with antibiotics showed synergistic effects (FIC index < 0.5) with fluoroquinolone, tetracycline and glycopeptide antibiotics, near synergy with aminoglycoside, penicillin and cephalosporin, and additive effects with macrolide antibiotics.

[0319] Critically, these data show that DDC and copper synergistically combine with antibiotics to kill bacterial biofilms.

[0320] Example 11 - DDC Synergistically Combines with Copper to Kill Fungi. [0321] Having established that DCC and copper synergistically combine to kill planktonic bacteria and bacteria in a biofilm, the activity of DDC and copper against fungi was assessed.

[0322] Using the MIC assay described in Examples 1 to 3, the antifungal activity of DDC alone, copper sulfate alone and DDC in combination with copper sulfate was assessed against six species of the genus Candida. Specifically, the efficacy of DDC in combination with copper was assessed against C. albicans, C. dubliniensis, C. kefyr, C. kruzei, C. tropicalis and C. parapsilosis.

[0323] Briefly, Candida strains were immersed in media (i.e. yeast nitrogen base containing 0.5% glucose, adjusted to pH 7) and adjusted to an OD600 of 0.1 (1 x10⁶ CFU/ml). After a 1 in 1000 dilution in media (1 x10³ CFU/ml), the fungal suspension was added to a 96- well microtiter plate with 100 mI per well. One hundred mI of treatment dissolved in media was added and incubated for 48 hours at 30^eC. Controls included maximum growth control (i.e. fungi incubated with media) and negative control (i.e. pure media without fungi). Measurements of the OD600 were taken at 24 hours and 48 hours.

[0324] The DDC concentrations tested ranged between 0.0625 to 32 pg/ml in 2x concentration increments, and the concentration of copper sulfate ranged between 0.25 pg/ml and 128 pg/ml. When DDC and copper sulfate were combined a 1 :4 ratio was maintained for MIC testing.

[0325] As can be seen in Figure 15, copper when combined with DDC potentiated the efficacy of DDC for 5 of the 6 fungi strains, namely C. albicans, C. dubliniensis, C. kruzei, and C. parapsilosis.

[0326] To further assess the antifungal effects of DDC and copper, a second inhibition assay was performed using a different growth media, different strains of C. albicans, C. dubliniensis, C. kefyr, C. kruzei and C. parapsilosis, as well as assessing activity against C. auris.

[0327] Candida auris is identified by the US Centers for Disease Control and Prevention (the CDC) as an emerging fungus which presents a serious global health threat due to commonly being multi-drug resistant, hard to identify using standard microbiology protocols and its tendency to cause outbreaks in healthcare settings.

[0328] Candida species (C. albicans 2, C. auris, C. dubliniensis 2, C. kefyr 2, C. kruzei 2, C. tropicalis 2 and C. parapsilosis 2) were suspended in media (Sabouraud Dextrose Broth) and adjusted to an OD600 of 0.1 (approximately 1 x10⁶ CFU/ml). The fungal suspension was diluted 1 in 1000 dilution in media to a concentration of 1 x10³ CFU/ml, before 100mI of the fungal suspension was added to each well in a 96-well microtiter plate. One hundred mI of treatment (DDC alone, copper anions alone (Cu), or DDC + Cu) dissolved in media was then added to each well followed by incubation for 24 hours at 37^eC. Controls included maximum growth control (i.e. fungi incubated with media) and negative control (i.e. pure media without fungi). Growth was quantified by OD600 measurements taken after 24 hours of incubation.

[0329] The sodium DDC trihydrate concentrations tested ranged between 0.25pg/ml and 128 pg/ml in 2x concentration increments, and the concentration of copper sulfate ranged between 0.25 pg/ml and 128 pg/ml. When DDC and copper sulfate were combined a 1 :4 (DDC:copper, wt:wt) ratio was maintained for MIC testing.

[0330] As can be seen in Figure 15b, the combination of copper and DDC had an antifungal efficacy greater than DDC and copper alone for all Candida species tested with the greatest efficacy against Candida auris, Candida kefyr and Candida kruzei.

[0331] These data show that the combination of DDC and copper effectively kills fungi including multi-drug resistant species.

[0332] Example 12 - The Combination of DDC and Copper Potentiates the Efficacy of Antifungal Agents.

[0333] Flaving established the antifungal efficacy of DDC in combination with copper against C. auris, the combined efficacy of DDC and copper with common antifungal agents was tested.

[0334] A checkerboard assay, such as that described in Example 10, was performed as briefly described below.

[0335] C. auris was suspended in 0.9% saline and adjusted to 0.5 McFarland units (approx. 1.5x10⁸ CFU/ml). After a 1 in 100 dilution in Sabouraud Dextrose Broth, 50 mI of the fungal suspension was added to each well of a 96-well microtiter plate followed by 50 mI of treatment (i.e. 12.5 mI DDC, 12.5 mI Cu and 25 mI each antifungal dissolved in media, with Sabouraud Dextrose Broth containing 1% DMSO used for Amphotericin). The fungal cultures were incubated for 24 hours at 37^eC, before determining the antifungal activity by measuring the optical density.

[0336] Nine concentrations of DDC in combination with Copper (i.e. 8+32, 4+16, 2+8, 1 +4, 0.5+2, 0.25+1 , 1/8+1/2, 1/16+1/4, 1/32+1/8 pg/ml) were combined with 7 concentrations of each antifungal tested (ranging from 8 to 0.125 pg/ml for clotrimazole, 32 to 0.5 pg/ml for fluconazole, 1 to 1/64 pg/ml for itraconazole, 16 to 0.25 pg/ml for miconazole and 2 to 1/32 pg/ml for amphotericin B) thereby providing each well with a unique combinations of antifungal, DDC and copper concentrations in each well. The antifungal activity of DDC and copper in combination when combined with antifungal agents was compared to the antifungal activity of the individual antifungal drugs alone and the combination of DDC and copper in the absence of the antifungal agents.

[0337] Figure 16A shows the fractional inhibitory concentration (FIC) index against C. auris of the checkerboard assay. As can be seen, DDC and copper potentiated the efficacy of clotrimazole, itraconazole, miconazole and amphotericin B against C. auris which is known to be multi-drug resistant. While DDC and copper had an indifferent effect when combined with fluconazole.

[0338] Figure 16B shows the reduction in minimum inhibitory concentration (MIC) of the assayed antifungal agents in combination with DDC and copper and confirms the potentiation of the efficacy of clotrimazole, itraconazole, miconazole and amphotericin B against C. auris.

[0339] Example 13 - Therapeutic Ratios and Concentrations of DDC and Copper Do Not Kill Mammalian Cells.

[0340] Fluman fibroblast cells (GM00038) were cultured at 37°C and 5% CO2 in Eagle's Minimum Essential Medium with Earle's salts and non-essential amino acids supplemented with 15% fetal bovine serum and 2.2 g/l sodium bicarbonate. The cytotoxicity of DDC and Cu was determined using the CellTiter-Glo Luminescent Viability Assay. Briefly, cells were seeded at 5x10³ in 100 mI culture medium per well in 96-well plates and incubated for 24 hours (37°C, 5% CO2) to allow attachment. Then, cells were exposed for 18 hours to media containing varying concentrations of sodium DDC trihydrate alone (4, 8, 16, 32, 64 pg/ml), varying concentrations of copper sulfate alone (16, 32, 64, 128, 256 pg/ml) or varying concentrations of sodium DDC trihydrate combined with copper sulfate. The specific concentrations tested were 4 pg/ml of DDC and 16 pg/ml of copper, 4 pg/ml of DDC and 32 pg/ml of copper, 8 pg/ml of DDC and 32 pg/ml of copper, 16 pg/ml of DDC and 64 pg/ml of copper, 32 pg/ml of DDC and 128 pg/ml of copper and 64 pg/ml of DDC and 256 pg/ml of copper. Negative controls included untreated cells.

[0341] The Cell Titer-Glo Luminescent Cell Viability Assay (Promega) was performed as per manufacturer’s instruction to determine cell viability of human endothelial cells after exposure to DDC, copper sulfate or the combination of DDC and copper sulfate. Cell viability above 80% indicated non-harmful effects of a treatment. [0342] As demonstrated in Figure 17, treatment of human fibroblasts with media containing DDC alone or copper sulfate alone resulted in cell viabilities above 80% for all concentrations tested indicating that these treatments had no adverse effect on the endothelial cells.

[0343] Further, therapeutic concentrations of DDC and copper sulfate of up to 8 pg/ml of DDC and 32 pg/ml of copper sulfate (ratio 1 :4) and DDC 4 pg/ml + Cu 32 pg/ml (ratio 1 :8) showed no harmful effect on the endothelial cells.

[0344] Example 14 - DDC and Copper Decrease Biofilm in a Wound Model.

[0345] To assess the ability of DDC and copper to reduce biofilm formation in a wound, and therefore improve wound healing, an artificial wound model was used. The artificial wound model is disclosed in Richter, Katharina, et al. "A topical hydrogel with deferiprone and gallium- protoporphyrin targets bacterial iron metabolism and has antibiofilm activity." Antimicrobial agents and chemotherapy 61.6 (2017): e00481 -17 and Brackman G et al. “ Dressings loaded with cyclodextrin-hamamelitannin complexes increase Staphylococcus aureus susceptibility toward antibiotics both in single as well as in mixed biofilm communities". Macromol Biosci (2016): 16:859-869. https://doi.org/10.1002/mabi.201500437, and described in brief below. The disclosure of these documents is incorporated herein.

[0346] An artificial dermis of hyaluronic acid (1 .20 to 1 .80 MDa; Lifecore Biomedical, MN, USA) and collagen (Corning, NY, USA) was prepared. A mixture of lyophilized bovine plasma (Sigma), 19 ml of Bolton broth, 1 ml of horse blood and 10 IU of heparin was added to the artificial dermis. The dermis was infected with 10 mI of an overnight culture of either MRSA 3 or S. epidermidis 35984 adjusted to 1 x 10⁶ CFU/ml. After 24 hrs of biofilm formation at 37°C, biofilms were exposed to 200 mI of media (maximum growth control in Tryptic Soy Broth), sodium DDC trihydrate 8 pg/ml alone, copper sulfate 32 pg/ml alone or a combination of sodium DDC trihydrate 8 pg/ml and copper sulfate 32 pg/ml for 24 hrs at 37°C. The dermis was washed and placed in 10 ml of 0.9% saline. Biofilms were extracted by 3 alternating cycles of 30 seconds of vortexing and 30 seconds of sonication, before being diluted and plated for CFU counting. The percentage of Biofilm reduction was calculated relative to maximum growth control (Figures 19A and B).

[0347] As demonstrated in Figures 19A, DDC alone or copper alone had a negligible effect on bacterial biofilms for MRSA 3. Flowever, the combination of DDC with copper showed an 82.4% reduction in MRSA 3 biofilms. Figure 19B shows that DDC alone and copper alone had resulted in some reduction in biofilm formation for S. epidermidis 35984 and a 70.9% reduction when DDC and copper were combined. [0348] These data show that DDC and copper, when combined, significantly reduce the biofilm formation in an artificial wound model and suggest that DDC and copper in combination could reduce biofilm formation during in vivo wound healing and thereby improve the rate of wound healing or prevent the formation of, or treat, chronic wounds.

Definitions and Qualifications

[0349] Certain disclosed embodiments provide compositions, solutions, methods, products, uses and the like that have one or more described advantages. It is to be understood that such advantages may not be achieved by all of the disclosed embodiments of the invention and therefore should not be considered as the promise or object of the invention in totality.

[0350] It is to be understood that any mechanism of action proposed, or discussed, herein is not intended to limit the scope of the specification or claims and it is not intended to form part of the invention as claimed, unless explicitly specified. Further, the mechanisms of action proposed herein may not reflect the mechanisms by which the compositions, solutions and methods of the invention work, or work in each and every microorganism within the scope of the claims. As such, the mechanisms proposed herein are proposed based on the data provided in combination with that known in the art and are subject to change.

[0351] “Synergism” as used herein means that the efficacy, or potency, of DDC in combination with copper is greater than the sum of the individual effects of each drug. Methods are known in the art for assessing synergy between antimicrobial assay (see for example Doern, C., J. Clin. Micobiol. (2014), 52(12): pp4124-28. On such methods is the FIC index and checkerboard assay as disclosed herein. Although this assay has limitations as set forth in Odds, F., J. Antimicrob. (2003) 52(1 ):1.

[0352] The term "topical composition" or “composition for topical use” refers to a composition that is formulated for topical administration, being the application primarily to keratinous tissue, primarily the skin, but may include hair and nails. Topical generally relates to delivery to the skin, but can also mean delivery to lumen spaces lined with epithelial cells, for example mucosal tissue such as the lips, mouth etc.

[0353] The term “wound” as used herein refers to any wound to the tissue of the body. In a preferred form the term “wound” relates to dermal wounds where the skin is disrupted forming a tear, cut, puncture, incision, laceration, abrasion, rip, slash, scratch, slit, burn, rupture or ulceration in the skin of an animal. [0354] The terms “effective amount” or “pharmaceutically effective amount” as used herein is the quantity of the antimicrobial composition or solution, or components thereof, which will result in the stasis or killing of bacteria (planktonic or in a biofilm), or fungus over any period. When used in reference to a subject or patient the terms are used to specify the quantity of the composition or solution, or components thereof, which, when administered to a subject, improves the prognosis and/or health state of the subject with respect to their infection status. The amount of antimicrobial composition, solution or components thereof to be administered to a subject will depend on the particular characteristics of one or more of the level or amount of resistance to the antimicrobial agent in the subject, the type of infection being inhibited, prevented or treated, the mode of administration of the antimicrobial compositions or solutions, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors. The effective amount of the antimicrobial composition or solution to be used in the various embodiments of the present invention is not particularly limited.

[0355] The term "pharmaceutically acceptable carrier" refers to a substantially inert solid, semi-solid or liquid filler, diluent, excipient, encapsulating material or formulation auxiliary of any type. An example of a pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known in the art. Some examples of materials which can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN 80; buffering agents such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as perfuming agents, preservatives and antioxidants.

[0356] The term “treatment” and related terms as used herein refer to obtaining a desired pharmacologic and/or physiologic effect that is therapeutic in nature. For example, the effect may be therapeutic in terms of improving the condition of the subject, ameliorating, relieving and/or slowing the progression of one or more symptoms in the subject, a partial or complete stabilization of the subject, or a cure in the subject. [0357] In some embodiments, the combination of DDC, or a salt thereof, and copper, or a salt therof, inhibits the growth of bacteria or fungi by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99% over a specified period of time. In some embodiments the specified period of time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours or 48 hours.

[0358] The term “preventing”, and related terms, as used herein refer to obtaining a desired pharmacologic and/or physiologic effect that is prophylactic in nature. For example, the effect may be a complete or partial prevention of a disease, condition, state or symptom in the subject, or the complete or partial prevention of the progression or occurrence of symptoms or pathology in the subject.

[0359] An “antimicrobial agent” is to have the ordinary meaning in the art. However, in the case of doubt an antimicrobial agent is to be considered to be any agent that inhibits the growth of or kills a microbe, in particular bacteria or fungi. These include microbicidal agents and biostatic agents. Specific antimicrobial agents include disinfectants, antiseptics, antibiotics and antifungals.

[0360] An “antibiotic” as used herein means a compound or solution, suitable for ingestion, that can inhibit the growth of, or kill, a bacterium.

[0361 ] An “antiseptic” as used herein means a compound, including a solution, which when applied topically can inhibit the growth of, or kill, a bacterium or fungus. Typically, antiseptics are tolerated by living organisms and are intended for use, or restricted to use, on living tissue.

[0362] A “disinfectant” as used herein means a compound, including a solution, which when applied to a surface can inhibit the growth of, or kill, a bacterium or fungus. Typically, disinfectants may incorporate antiseptics but are not required to be tolerated by living organisms and therefore are not restricted to use on living tissue and are rather used on inanimate objects.

[0363] An “antifungal” as used herein means a compound, including a solution, that can inhibit the growth of, or kill, a fungus or a fungal spore.

[0364] In some embodiments, the antimicrobial in combination with DDC and copper inhibits the growth of bacteria or fungi by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99% over a specified period of time. In some embodiments the specified period of time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours or 48 hours.

[0365] In some embodiments, the activity of the antimicrobial may be enhanced or increased in the presence of DDC and copper by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater, or by 1 -fold, 2-fold, 3-fold, 4-fold, 5-fold, 6.0-fold, 7- fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 125-fold, 150-fold, 175-fold, 200-fold, 225-fold, 250- fold, 275-fold, 300-fold, 400-fold, 500-fold, or greater, when compared to the activity of the antimicrobial when used in the absence of DDC and copper.

[0366] Where exact numerical ranges and ratios are specified in respect of embodiments disclosed herein it is to be understood that these are intended to provide support for deviations from these ranges of 10%.

[0367] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters or referenced documents, acts, materials, devices, articles and the like formed part of the base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0368] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers

[0369] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.

[0370] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as", “i.e.”) provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[0371] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0372] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0373] The invention also includes all of the steps, features, components and compounds referred to, or disclosed in this specification, individually or collectively. Further the invention also includes any and all combinations of any two or more of the steps, components or features. Unless specifically indicated, the steps of a method do not need to be performed in the listed order, or the order of any numbering.

[0374] Also, it is to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

[0375] As used throughout this specification in reference to numeric ranges the term “between” is to be understood to be inclusive of the bounding number. For example, between 1 and 10 refers to the range of 1 to 10, inclusive. Likewise, a reference “from” and “to” bounding numbers is to be understood as inclusive of the bounding numbers.

[0376] Future patent applications may be filed in Australia or overseas on the basis of or claiming priority through the present application, for example by claiming divisional status or continuation status. It is to be understood that the following claims are provided to define particular embodiments of the invention of the present application only, and are not intended to limit the scope of what may be claimed in any such future application(s). Features may be added to, or omitted from, the claims at a later date so as to further define or re-define the invention or inventions.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An antimicrobial composition including: a) copper, or a salt thereof, and b) diethyldithiocarbamate (DDC), or a salt thereof.

2. The antimicrobial composition of claim 1 , wherein the weight to weight ratio of copper, or the salt thereof, to DCC, or the salt thereof, is greater than or equal to about 1 :1 and/or is less than or equal to about 8:1.

3. The antimicrobial composition of claim 1 , wherein the molar to molar ratio of copper, or the salt thereof, to DCC, or the salt thereof, is greater than or equal to about 1 :1 and/or is less than or equal to about 8:1 .

4. The antimicrobial composition of any one of claims 1 to 3, wherein the antimicrobial composition is for inhibiting the growth of, or killing, a bacterium.

5. The antimicrobial composition of any one of claims 1 to 4, wherein the antimicrobial composition is for inhibiting the growth of, or killing, a bacterium in a biofilm.

6. The antimicrobial composition of any one of claims 1 to 5, wherein the antimicrobial composition is for inhibiting the growth of, or killing, a gram-positive bacterium.

7. The antimicrobial composition of claim 4 or claim 5, wherein the bacterium is selected from the genus: Enterococcus or Staphylococcus.

8. The antimicrobial composition of claim 4 or claim 5, wherein the bacterium is a strain selected from the species: Enterococcus faecium, Enterococcis faecalisor, Staphylococcus aureus or Moraxella catarrhalis.

9. The antimicrobial composition of claim 4 or claim 5, wherein the bacterium is Methicillin-resistant Staphylococcus aureus.

10. The antimicrobial composition of any one of claims 1 to 3, wherein the antimicrobial composition is for inhibiting the growth of, or killing, a fungus.

11 . The antimicrobial composition of claim 10, wherein the fungus is a member of the Candida genus.

12. The antimicrobial composition of claim 10, wherein the fungus is a strain of Candida albicans, Candida auris, Candida dubliniensis, Candida kruzei or Candida parapsilosis.

13. The antimicrobial composition of any one of claims 1 to 12, wherein the composition is for topical treatment.

14. The antimicrobial composition of claim 13, wherein the topical treatment is treatment of a wound, treatment of keratinised skin, treatment of a mucosal surface, treatment of an ear or treatment of the sinuses.

15. The antimicrobial composition of any one of claims 1 to 12, wherein the composition is for treatment of a surgical device or implant.

16. The antimicrobial composition of any one of claims 1 to 15, wherein the antimicrobial composition further includes an antimicrobial agent.

17. The antimicrobial composition of claim 16, wherein the antimicrobial agent is an antibiotic, an antiseptic or an antifungal.

18. The antimicrobial composition of claim 17, wherein the antibiotic is selected from the group consisting of a protein synthesis inhibitor, a cell wall synthesis inhibitor, a beta- lactam antibiotic, a beta- lactamase inhibitor, a lipopeptide, a peptidoglycan synthesis inhibitor, a DNA synthesis inhibitor, a RNA synthesis inhibitor, a mycolic acid synthesis inhibitor, a mechanosensitive channel of large conductance (MscL), a folic acid synthesis inhibitor, a quinolone antibiotic, a tetracycline antibiotic, a glycopeptide antibiotic, an aminoglycoside antibiotic, a penicillin antibiotic, a cephalosporin antibiotic, a cephamycin antibiotic, a macrolide antibiotic, ciprofloxacin, doxycycline, vancomycin, gentamycin, amoxicillin, ceftazidime or erythromycin, or a combination of the aforementioned.

19. The antimicrobial composition of claim 17, wherein the antiseptic is selected from the group consisting of: an alcohol, ethanol, isopropanol, benzethonium chloride, chlorhexidine, hexachlorophene, hydrogen peroxide, iodine, octenidine dihydrochloride, oxychlorosene sodium, polyhexanide, povidone iodine, sodium hypochlorite or triclosan.

20. The antimicrobial composition of claim 17, wherein the antifungal is selected from the group consisting of: azole antifungals, an allylamine, echinocandins, polyenes, amphotericin B, anidulafungin, candicidin, caspofungin, clotrimazole, filipin, fluconazole, flucytosine, griseofulvin, hamycin, isavuconazonium, itraconazole, ketoconazole, micafungin, miconazole, natamycin, nystatin, posaconazole, rimocidin, terbinafine or voriconazole.

21 . The antimicrobial composition of any one of claims 1 to 20, wherein the composition is a solution including DDC anions and copper cations.

22. The antimicrobial solution of claim 21 , wherein the copper cations in solution are divalent.

23. The antimicrobial solution of claim 21 or claim 22, wherein the molar to molar ratio of copper cations to DCC anions in solution is greater than or equal to about 1 :1 and/or is less than or equal to about 8:1 .

24. The antimicrobial solution of any one of claims 21 to 23, wherein the concentration of DDC in solution is at least about 0.25 pg/ml.

25. The antimicrobial solution of any one of claims 21 to 24, wherein the concentration of copper, or a salt thereof, in solution is at least about 0.25 pg/ml.

26. A surgical implant or device including, impregnated with or coated with an effective amount of an antimicrobial composition according to any one of claims 1 to 20 or the solution of any one of claims 21 to 25.

27. The surgical implant or device of claim 26, wherein the surgical device or surgical implant includes polyester, or polypropylene.

28. The surgical implant or device of claim 26 or claim 27, wherein the surgical implant is an implantable mesh.

29. A method of inhibiting the growth of a bacterium or a fungus, or killing a bacterium or a fungus, or preventing the formation of a bacterial biofilm by a bacterium, or treating a bacterial biofilm, the method comprising exposing the bacterium or fungus or biofilm to an effective amount of the antimicrobial composition of any one of claims 1 to 20 or the antimicrobial solution of any one of claims 21 to 25.

30. The method of claim 29, wherein the bacterium, is a gram-positive bacterium.

31 . The method of claim 29, wherein the bacterium is a strain selected from the species: Enterococcus faecium, Enterococcis faecalisor, Staphylococcus aureus or Moraxella catarrhalis.

32. The method of claim 29, wherein the fungus is a member of the candita genus.

33. The method of claim 29, wherein the fungus is a strain of Candida albicans, Candida auris, Candida dubliniensis, Candida kruzei or Candida parapsilosis.

34. The method of any one of claims 29 to 33, wherein the method is performed on a surgical device or surgical implant.

35. The method of claim 34, wherein the surgical device or surgical implant includes polyester, or polypropylene.

36. The method of claim 34 or claim 35, wherein the surgical implant is an implantable mesh.

37. A method of preventing or treating an infection in a subject, the method comprising providing to the site of infection an effective amount of an antimicrobial composition according to any one of claims 1 to 20, or an antimicrobial solution according to any one of claims 21 to 25, or performing the method of any one of claims 29 to 33.

38. The method of any one of claims 29 to 37, wherein the method further includes providing an antimicrobial agent.

39. The method of claim 38, wherein the antimicrobial composition or antimicrobial solution is potentiated by the antimicrobial agent.

40. The method of claim 38 or claim 39, wherein the antimicrobial agent is an antibiotic, an antiseptic or an antifungal.

41 . The method of claim 40, wherein the antibiotic is selected from the group consisting of a protein synthesis inhibitor, a cell wall synthesis inhibitor, a beta-lactam antibiotic, a beta-lactamase inhibitor, a lipopeptide, a peptidoglycan synthesis inhibitor, a DNA synthesis inhibitor, a RNA synthesis inhibitor, a mycolic acid synthesis inhibitor, a mechanosensitive channel of large conductance (MscL), a folic acid synthesis inhibitor, a quinolone antibiotic, a tetracycline antibiotic, a glycopeptide antibiotic, an aminoglycoside antibiotic, a penicillin antibiotic, a cephalosporin antibiotic, a cephamycin antibiotic, a macrolide antibiotic, ciprofloxacin, doxycycline, vancomycin, gentamycin, amoxicillin, ceftazidime or erythromycin, or a combination of the aforementioned.

42. The method of claim 40, wherein the antiseptic is selected from the group consisting of: an alcohol, ethanol, isopropanol, benzethonium chloride, chlorhexidine, hexachlorophene, hydrogen peroxide, iodine, octenidine dihydrochloride, oxychlorosene sodium, polyhexanide, povidone iodine, sodium hypochlorite or triclosan.

43. The method of claim 40, wherein the antifungal is selected from the group consisting of: azole antifungals, echinocandins, polyenes, amphotericin B, anidulafungin, candicidin, caspofungin, clotrimazole, filipin, fluconazole, flucytosine, griseofulvin, hamycin, isavuconazonium, itraconazole, ketoconazole, micafungin, miconazole, natamycin, nystatin, posaconazole, rimocidin, terbinafine or voriconazole.

44. A kit including diethyldithiocarbamate (DDC), or a salt thereof, and copper, or a salt thereof, wherein DDC, or the salt thereof, and copper, or the salt thereof, are adapted for combination.

45. The kit of claim 44, wherein the weight to weight ratio of copper, or the salt thereof, to DDC, or the salt thereof, is from about 1 :1 to about 8:1.

46. The kit of claim 44 or claim 45, further including a diluent for dissolving the DDC, or the salt thereof, and the copper, or the salt thereof.

47. The kit of claim 46, wherein the volume of the diluent is proportional to the weight of DDC, or the salt thereof, to provide a solution having a concentration of DDC, or the salt thereof, of equal to or greater than 0.125 pg/ml, or equal to or greater than 0.5 pg/ml.

48. The kit of any one of claims 44 to 47, further including an antimicrobial.

49. The kit of claim 48, wherein the antimicrobial agent is an antibiotic, an antiseptic or an antifungal.