Compounds and uses thereof

Abstract

Disclosed is the use of copper ionophores for overcoming or inhibiting the development of resistance of a gram-positive bacterium to an antibiotic. More particularly, this disclosure relates to the use of a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof for conferring antibiotic sensitivity to a resistant gram-positive bacterium, inhibiting the development of resistance of a gram-positive bacterium to an antibiotic, increasing the susceptibility of a gram-positive bacterium to an antibiotic and / or treating or inhibiting the development of a gram-positive bacterial infection in a subject. Also provided is the use of a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof as an antibiotic adjuvant or potentiator.

Description

TITLE OF THE INVENTIONCOMPOUNDS AND USES THEREOF'

[0001] This application claims priority to Australian Provisional Patent Application No. 2024901855 entitled "Compounds and uses thereof" filed 18 June 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] This invention relates generally to the use of copper ionophores for overcoming or inhibiting the development of resistance of a gram-positive bacterium to an antibiotic. More particularly, this invention relates to the use of a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof for conferring antibiotic sensitivity to a resistant gram-positive bacterium, inhibiting the development of resistance of a gram-positive bacterium to an antibiotic, increasing the susceptibility of a gram-positive bacterium to an antibiotic and / or treating or inhibiting the development of a gram-positive bacterial infection in a subject. Also provided is the use of a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof as an antibiotic adjuvant or potentiator.BACKGROUND OF THE INVENTION

[0003] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0004] Antibiotic-resistant bacterial pathogens represent an imminent global threat to human health. In 2019, antimicrobial resistance was associated with nearly five million deaths worldwide, with more than 2.8 million antimicrobial-resistant infections occurring each year in the United States alone. The range of antibiotics available to combat bacterial infections is diminishing due to the development of bacterial resistance to all classes of antibiotics.

[0005] In addition, the number of new classes of antibiotics being developed is decreasing. A 2017 World Health Organization (WHO) Report (Antibacterial agents in clinical development - an analysis of the antibacterial clinical development pipeline, including tuberculosis) found that most of the drugs being developed are only short-term solutions as they are modifications of existing classes of antibiotics, and that very few potential treatment options exist for antibiotic-resistant infections identified by the WHO as being the greatest risk to human health. The WHO lists a number of gram positive bacteria as being priority pathogens for new antibiotic development, includingEnterococcus faecium, Staphylococcus aureus, Streptococcus pneumoniae and, most importantly, Mycobacterium tuberculosis.

[0006] Antibiotic resistance is the ability of bacteria to resist antibiotic medication used to treat bacterial infections. Antibiotic resistance occurs when bacteria are intrinsically resistant to an antibiotic or when bacteria change in response to use of antibiotics allowing them to inactivate the antibiotic, resist the action of the antibiotic, or remove or exclude the antibiotic from the bacterial cell. The mechanism through which pathogenic bacteria develop resistance depends on several factors which can include the class and structure of antibiotic and the nature of the pathogenic bacteria. An increasing number of bacterial infections caused by gram-positive bacteria such as pneumonia and tuberculosis are becoming more difficult to treat as the antibiotics previously used successfully to treat them become less effective due to antibiotic resistance. Antibiotic resistance results in longer hospital stays, higher medical costs and increased mortality.

[0007] As such, there is an urgent need for the development of new therapies for combatting the development of bacterial resistance to antibiotics.SUMMARY OF THE INVENTION

[0008] The present invention is predicated in part on the identification of particular copper ionophores that can confer antibiotic sensitivity to a resistant grampositive bacterium. Without wishing to be bound by theory, the inventors have found that the copper ionophores transport copper across the bacterial cell membrane, thereby increasing the intracellular copper concentration within the bacterium. It is proposed that such copper intoxication of the bacterium promotes sensitivity to antibiotics through the dysregulation of metal ion homeostasis and disruption of essential cellular processes. Based on this activity, these ionophores are considered to be useful for conferring antibiotic sensitivity to a resistant gram-positive bacterium, inhibiting the development of resistance of a gram-positive bacterium to an antibiotic, increasing the susceptibility of a grampositive bacterium to an antibiotic and / or treating or inhibiting the development of a bacterial infection caused by a gram-positive bacterium in a subject. These ionophores are also considered to be useful for potentiating or enhancing the activity of an antibiotic.

[0009] Accordingly, in one aspect, there is provided a method of conferring antibiotic sensitivity to a resistant bacterium, comprising contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'1, / V'3-dimethyl- / V'1, / V'3-di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof, and the bacterium is a gram-positive bacterium.

[0010] In another aspect, there is provided a method of inhibiting the development of resistance of a bacterium to an antibiotic, comprising contacting thebacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof, and the bacterium is a gram-positive bacterium.

[0011] Also provided is a method of increasing the susceptibility of a bacterium to an antibiotic, comprising contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a gram-positive bacterium. In preferred embodiments, the bacterium is resistant to the antibiotic.

[0012] In some embodiments of any one of the aspects above, the method further comprises contacting the bacterium with an antibiotic, such as a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a glycopeptide, a lincosamide, a macrolide, a nitrofuran, an oxazolidinone, a penicillin, a polypeptide, a fluoroquinolone, a sulfonamide or a tetracycline. In particular embodiments, the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a penicillin, a macrolide or a tetracycline.

[0013] In some embodiments, the antibiotic is a macrolide or a tetracycline. In some embodiments, the antibiotic is a macrolide such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin or spiramycin. In other embodiments, the antibiotic is a tetracycline such as doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline and tigecycline.

[0014] In exemplary embodiments, the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.

[0015] In some embodiments of any one of the aspects above, the copper ionophore is a thiosemicarbazone selected from the group consisting of GTSM, di-2- pyridylketone 4-methyl-4-cyclohexyl-3-thiosemicarbazone (DpC) and pharmaceutically acceptable salts and solvates thereof; especially GTSM.

[0016] In other embodiments, the ionophore is nitroxoline. In still other embodiments, the ionophore is elesclomol.

[0017] In some embodiments of any one of the aspects above, the bacterium is selected from the group consisting of a Streptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species, such as Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile or Mycobacterium tuberculosis. In particular embodiments, the bacterium is S. pneumoniae, E. faecium, S. pyogenes or S. aureus.

[0018] In particular embodiments of any one of the aspects above, the copper ionophore is a selective copper ionophore.

[0019] In a further aspect, there is provided a method of treating or inhibiting the development of a bacterial infection in a subject, comprising administering a copper ionophore and an antibiotic to the subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterial infection is caused by a gram-positive bacterium.

[0020] In some embodiments, the infection is a lung, blood or brain infection, particularly a lung infection.

[0021] In particular embodiments, the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a glycopeptide, a lincosamide, a macrolide, a nitrofuran, an oxazolidinone, a penicillin, a polypeptide, a fluoroquinolone, a sulfonamide or a tetracycline. In specific embodiments, the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a penicillin, a macrolide or a tetracycline.

[0022] In some embodiments, the antibiotic is a macrolide or a tetracycline. In some embodiments, the antibiotic is a macrolide such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin or spiramycin. In other embodiments, the antibiotic is a tetracycline such as doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline and tigecycline.

[0023] In exemplary embodiments, the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.

[0024] Suitable embodiments of the copper ionophore are as discussed supra. For example, in some embodiments, the thiosemicarbazone is selected from the group consisting of GTSM, DpC and pharmaceutically acceptable salts and solvates thereof; especially GTSM. In alternative embodiments, the ionophore is nitroxoline. In other embodiments, the ionophore is elesclomol.

[0025] In some embodiments, the bacterial infection is caused by a bacterium selected from the group consisting of a Streptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species, such as Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile or Mycobacterium tuberculosis. In particular embodiments, the bacterial infection is caused by S. pneumoniae, E. faecium, S. pyogenes or S. aureus.

[0026] In preferred embodiments, the copper ionophore is a selective copper ionophore.

[0027] Also provided herein, in another aspect, is a pharmaceutical composition comprising a copper ionophore and an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0028] In particular embodiments, the composition further comprises a carrier or diluent.

[0029] In a further aspect, there is provided a use of a copper ionophore as an antibiotic adjuvant or antibiotic potentiator, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0030] In yet another aspect, there is provided a method of potentiating an activity of an antibiotic against a gram-positive bacterium, comprising contacting the bacterium with a copper ionophore concurrently with the antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0031] In particular embodiments, the wherein the bacterium is resistant to the antibiotic.

[0032] In some embodiments, the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a glycopeptide, a lincosamide, a macrolide, a nitrofuran, an oxazolidinone, a penicillin, a polypeptide, a fluoroquinolone, a sulfonamide or a tetracycline; including a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a penicillin, a macrolide or a tetracycline. In some embodiments, the antibiotic is a macrolide or a tetracycline. In some embodiments, the antibiotic is selected from the group consisting of azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin and spiramycin; or is selected from the group consisting of doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline and tigecycline. In particular embodiments, the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.

[0033] In some embodiments, the thiosemicarbazone is selected from the group consisting of glyoxal-bis(N(4)-methylthiosemicarbazone), di-2-pyridylketone 4-methyl-4- cyclohexyl-3-thiosemicarbazone and pharmaceutically acceptable salts and solvates thereof; especially glyoxal-bis(N(4)-methylthiosemicarbazone). In some embodiments, the ionophore is nitroxoline. In alternative embodiments, the ionophore is elesclomol.

[0034] In some embodiments, the bacterium is selected from the group consisting of a Streptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species. In particular embodiments, the bacterium is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile and Mycobacterium tuberculosis. In specific embodiments, the bacterium is S. pneumoniae, E. faecium, S. pyogenes or S. aureus.

[0035] In some embodiments, the copper ionophore is a selective copper ionophore.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a graph showing the growth of S. pneumoniae serotype 2 laboratory strain, D39 in the presence of increasing concentrations of Cu(II)GTSM.

[0037] Figure 2 is a graph showing the expression of the copper efflux pathway, copA, in S. pneumoniae serotype 2 laboratory strain, D39 in the absence (untreated) and presence of Cu(II)GTSM, and the presence of copper alone.

[0038] Figure 3 is a series of graphs showing the growth of S. pneumoniae serotype 1 multi-drug resistant clinical strain, 4496 (Sp4496), in the presence of increasing concentrations of Cu(II)GTSM (Figure 3A), elesclomol (Figure 3B), nitroxoline (Figure 3C) and ammonium pyrrolidinedithiocarbamate (PDTC) (Figure 3D). All media, cation adjusted Mueller Hinton broth, contained 8 pM CuS04.

[0039] Figure 4 is a series of graphs showing the cellular accumulation of copper (Figure 4A) and zinc (Figure 4B) in micrograms per gram of cells in S. pneumoniae serotype 1 multi-drug resistant clinical isolate, Sp4496, in the presence of Cu(II)GTSM, elesclomol, nitroxoline and PDTC. All media, cation adjusted Mueller Hinton broth, contained 8 pM CuS04.

[0040] Figure 5 is a series of graphs showing the intracellular accumulation of Cu (Figure 5A) and growth (Figure 5B) of S. pneumoniae 4496 treated at the respective concentrations of Cu with and without Cu(II)GTSM. Error bars represent standard error of the mean (SEM); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, p < 0.0001; n.s., not significant.

[0041] Figure 6 is a series of graphs showing the impact of Cu(II)GTSM on S. pneumoniae 4496 metal homeostasis. Figure 6A shows the impact on Mg homeostasis, Figure 6B shows the impact on Ca homeostasis, Figure 6C shows the impact on Mn homeostasis, Figure 6D shows the impact on Fe homeostasis, Figure 6E shows the impact on Zn homeostasis, Figure 6F shows the impact on Ni homeostasis, Figure 6G shows the impact on Co homeostasis and Figure 6H shows the impact on Cd homeostasis. Error barsrepresent standard error of the mean (SEM); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, p < 0.0001; n.s., not significant.

[0042] Figure 7 is a series of images showing the growth of S. pneumoniae treated with ampicillin (AMP, Figure 7A), tetracycline (TET, Figure 7B) and azithromycin (AZI, Figure 7C) and Cu(II)GTSM in 96-well plates. The strength of growth inhibition or confluence is indicated by the coloured scale to the right of each plate where a solid yellow represents confluent growth and blue represents less confluent growth.

[0043] Figure 8 is a series of graphs showing the impact on S. pneumoniae metal homeostasis by tetracycline (TET) and Cu(II)GTSM treatment. Figure 8A shows the impact on Ca homeostasis, Figure 8B shows the impact on Mg homeostasis, Figure 8C shows the impact on Mn homeostasis, Figure 8D shows the impact on Fe homeostasis, Figure 8E shows the impact on Zn homeostasis and Figure 8F shows the impact on Cu homeostasis. Error bars represent standard error of the mean (SEM); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, p < 0.0001; n.s., not significant.DETAILED DESCRIPTION OF THE INVENTION1. Definitions

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0045] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0046] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0047] When referred to herein, the term "adjuvant" refers to a pharmacological agent that alters or improves the efficacy of another pharmacologically active agent. An adjuvant is delivered in addition to the primary pharmacologically active agent to enhance its effectiveness. The term "antibiotic adjuvant" refers to an agent that alters or improves the efficacy of an antibiotic. When used herein, the term "potentiator" refers to an agent that enhances or increases the effect of an antibiotic, e.g. bactericidal activity. In some embodiments, the efficacy or effect or an antibiotic is increased, improved or enhancedcompared to the efficacy or effect in the absence of the agent (e.g. a copper ionophore of the invention).

[0048] The terms "administration concurrently," "administered concurrently" and the like refer to the administration of a single composition containing two or more agents, or the administration of each agent as separate compositions and / or delivered by separate routes contemporaneously, simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such agents are administered as a single composition. By "simultaneously" is meant that the agents are administered at substantially the same time, and desirably together in the same composition. By "contemporaneously" it is meant that the agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and suitably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term "same site" includes the exact location, but can be within about 0.5 to about 15 centimetres, preferably from within about 0.5 to about 5 centimetres. The term "separately" as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The agents may be administered in either order. The term "sequentially" as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the agents may be administered in a regular repeating cycle.

[0049] The term "agent" includes a compound that induces a desired pharmacological and / or physiological effect. The term also encompasses pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogues and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, solvates, esters, amides, prodrugs, metabolites, analogues, etc. The term "agent" is not to be construed narrowly but extends to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogues thereof as well as cellular agents.

[0050] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0051] The terms "antibiotic" and the like when used herein refer to a chemical substance used in medicine that is capable of destroying or weakening, inhibiting or reducing growth of certain microorganisms that cause infections or infectious diseases, especially pathogenic bacteria. Antibiotics may have activity against one or more classes of bacteria, for example, one or both of gram-positive and gram-negative pathogenic bacteria, and find application in the treatment of a wide range of bacterial infections. In particular embodiments, the antibiotic has activity against gram-positive pathogenic bacteria. In some embodiments, antibiotics of the present invention include, but are not limited to, those having obtained marketing authorisation or regulatory approval, and pharmaceutically acceptable salts, solvates or in vivo hydrolysable esters thereof.

[0052] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Thus, the use of the term "comprising" and the like indicates that the listed integers are required or mandatory, but that other integers are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0053] When used herein the term "copper ionophore" unless otherwise indicated refers to an ionophore that reversibly binds to a copper(II) ion. Preferably, a copper ionophore is an organic compound. Organic compounds that act as copper ionophores are well known in the art, and are commercially available or may be synthesised according to known routes. It will be appreciated that a copper ionophore may be capable of binding with other metal ions. In particular embodiments, the ionophore selectively binds Cu(II) over at least one other metal ion, such as a transition metal ion including Zn(II), Fe(III), Ni(II), and the like. In particular embodiments, the ionophore exhibits selectivity for binding to Cu(II) of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to binding to another metal ion, particularly another transition metal ion, such as Zn(II). In particular embodiments, the ionophore does not substantially bind to another metal ion. In specific embodiments, the ionophore does not substantially bind to Zn(II).

[0054] By "derivative" is meant a molecule, such as a small molecule, that has been derived from the basic molecule by modification, for example by conjugation or complexing with other chemical moieties or by standard medicinal chemistry techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to a parent molecule that provide for functionally equivalent molecules.

[0055] As used herein, the term "dosage unit form" refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle.

[0056] By "effective amount", in the context of treating or inhibiting the development of a condition is meant the administration of an amount of an agent or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and / or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. In the context of conferring antibiotic sensitivity to a resistant bacterium, inhibiting the development of resistance of a bacterium to an antibiotic and increasing the susceptibility of a bacterium to an antibiotic, an effective amount means an amount that is effective for the stated activity (e.g. conferring sensitivity to at least one antibiotic to a resistant bacterium and the like).

[0057] As used herein, the phrase "inhibit the development of" refers to a prophylactic treatment which increases the resistance of a subject to developing the disease, disorder or condition (e.g. a bacterial infection) or, in other words, decreases the likelihood that the subject will develop the disease, disorder or condition, as well as a treatment after the disease, disorder or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse (e.g. inhibit the infection from spreading). In the context of inhibiting the development of antibiotic resistance, this phrase refers to a prophylactic treatment which reduces the ability of the bacterium to develop resistance to one or more antibiotics. This treatment may, for example, stop the bacteria from developing resistance altogether or increase the duration of antibiotic treatment before resistance is developed.

[0058] When used herein the terms "pathogenic bacteria" or "pathogenic bacterium" refer to bacteria that can cause infection. In some embodiments the bacteria are human pathogenic bacteria and cause disease in humans.

[0059] When used herein the term "ionophore", unless otherwise stated, refers to a chemical moiety, for example an organic compound, that reversibly binds to ions such as metal ions and facilitates their transport across cell membranes. Examples of ionophores include lipid soluble moieties that transport ions, for example metal cations, across a cell membrane. Preferably, the ionophore is pharmaceutically acceptable.

[0060] By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, fillers, detergents, wetting or emulsifying agents, pH buffering agents, preservatives and the like as discussed in detail herein.

[0061] Similarly, a "pharmaceutically acceptable" salt, solvate, ester or prodrug of a compound as provided herein is a salt, solvate, ester or prodrug that is not biologically or otherwise undesirable.

[0062] The terms "reduce," "inhibit" and grammatical equivalents when used in reference to the level of a substance and / or phenomenon in a first sample relative to a second sample, mean that the quantity of substance and / or phenomenon in the first sample is lower than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis. When these terms are used to refer to the action of an agent (e.g. a copper ionophore), the first sample may be a sample in the presence of the agent and the second sample may be a comparative sample without the agent. In one embodiment, the reduction may be determined subjectively, for example when a subject refers to their subjective perception of disease symptoms (e.g. symptoms of a bacterial infection), such as pain, redness, inflammation etc. In another embodiment, the reduction may be determined objectively, for example when the number of bacteria in a sample from a patient is lower than in an earlier sample from the patient or lower than in a sample from an untreated patient. In another embodiment, the quantity of substance and / or phenomenon in the first sample is at least 10% lower than the quantity of the same substance and / or phenomenon in a second sample. In another embodiment, the quantity of the substance and / or phenomenon in the first sample is at least 25% lower than the quantity of the same substance and / or phenomenon in a second sample. In yet another embodiment, the quantity of the substance and / or phenomenon in the first sample is at least 50% lower than the quantity of the same substance and / or phenomenon in a second sample. In a further embodiment, the quantity of the substance and / or phenomenon inthe first sample is at least 75% lower than the quantity of the same substance and / or phenomenon in a second sample. In yet another embodiment, the quantity of the substance and / or phenomenon in the first sample is at least 90% lower than the quantity of the same substance and / or phenomenon in a second sample.

[0063] The term "resistant" and grammatical variants thereof is used herein to refer to the capacity of bacteria to withstand the effects of antibiotics that are intended to kill or control them.

[0064] As used herein, the terms "salts" and "prodrugs" include any pharmaceutically acceptable salt, ester, hydrate or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a compound of the invention (e.g. a copper ionophore as described herein), or an active metabolite or residue thereof. The term "pharmaceutically acceptable salts" refers without limitation to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g. by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate and valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salt can be synthesised from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.Lists of suitable salts are found in, for example, Remington: The Science and Practice of Pharmacy, Adeboye Adejare and Joseph Remington (Ed), Academic Press, London, 23rdEdition, 2021; Stahl and Wermuth (2002) Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; and Berge et al. (1977) Journal of Pharmaceutical Science, 66: 1-19, each of which is incorporated herein by reference in its entirety.

[0065] The term "selective" as used herein refers to the ability of an agent to affect one thing and not others. For example, a copper ionophore which is selective for binding Cu(II), binds to Cu(II) and does not substantially bind to at least one other metal ion, such as a transition metal ion including Zn(II), Fe(III), Ni(II), and the like.

[0066] The term "solvate" is a complex of variable stoichiometry formed by a solute (e.g. a copper ionophore) and a solvent. Such solvents should preferably not interfere with the biological activity of the solute. Solvents may be, by way of example, water, acetone, ethanol or acetic acid. Methods of solvation are generally known within the art. In some embodiments, a solvate is pharmaceutically acceptable. In some embodiments, a solvate is a hydrate, for example a mono-, di- or tri-hydrate.

[0067] The term "subject" as used herein refers to a vertebrate subject, particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable subjects include, but are not limited to, primates; avians (birds); livestock animals such as sheep, cows, horses, deer, donkeys and pigs; laboratory test animals such as rabbits, mice, rats, guinea pigs and hamsters; companion animals such as cats and dogs; and captive wild animals such as foxes, deer and dingoes. In particular embodiments, the subject is a primate, suitably a human. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0068] As used herein, the terms "treatment", "treating", and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease, disorder or condition and / or adverse effect attributable to the disease, disorder or condition. These terms also cover any treatment of a condition or disease in a subject, particularly in a human, and include: (a) inhibiting the disease or condition, i.e. arresting or slowing its development; or (b) relieving the disease or condition, i.e. causing regression of the disease or condition.

[0069] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.2. Abbreviations

[0070] The following abbreviations are used throughout the application:GTSM =glyoxal-bis(N(4)-methylthiosemicarbazone) elesclomol = / V'1, / V'3-dimethyl- / V'1, / V'3-di(phenylcarbonothioyl)malonodi hydrazideDpC = di-2-pyridylketone 4-methyl-4-cyclohexyl-3-thiosemicarbazoneATSM =diacetyl-bis(N(4)-methylthiosemicarbazone)COTI-2 = 4-(2-pyridinyl)-2-(6,7-dihydro-8(5H)-quinolinylidene)hydrazide-l- piperazinecarbothioic acidDp44mT =di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazonePDTC =ammonium pyrrolidinedithiocarbamateCA-MHB =cation-adjusted Mueller-Hinton brothFICI =fractional inhibitory concentration indexMIC =mean inhibitory concentrationMPC = mutant prevention concentrationMSW =mutant selection windowICP-MS = inductively-coupled plasma mass spectrometry MHB =Mueller Hinton Broth3. Methods of Use

[0071] The inventors have found that particular copper ionophores can confer antibiotic sensitivity to a resistant gram-positive bacterium. Based on this activity, these ionophores are considered to be useful for conferring antibiotic sensitivity to a resistant gram-positive bacterium, inhibiting the development of resistance of a gram-positive bacterium to an antibiotic, increasing the susceptibility of a gram-positive bacterium to an antibiotic and / or treating or inhibiting the development of a bacterial infection caused by a gram-positive bacterium in a subject.

[0072] Accordingly, in one aspect, there is provided a method of conferring antibiotic sensitivity to a resistant bacterium, comprising, consisting or consisting essentially of contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a gram-positive bacterium. Also provided is a copper ionophore for use in conferring antibiotic sensitivity to a resistant bacterium, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a grampositive bacterium; a use of a copper ionophore for conferring antibiotic sensitivity to a resistant bacterium, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a gram-positive bacterium; and a use of a copper ionophore in the manufacture of a medicament for conferring antibiotic sensitivity to a resistant bacterium, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a gram-positive bacterium.

[0073] The copper ionophore may be a thiosemicarbazone or a pharmaceutically acceptable salt or solvate thereof, elesclomol or a pharmaceutically acceptable salt or solvate thereof or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0074] In particular embodiments, the copper ionophore is a thiosemicarbazone or a pharmaceutically acceptable salt or solvate thereof. A thiosemicarbazone is a compound with the following general structure:

[0075] Thiosemicarbazones are well known in the art and a skilled person will be well aware of suitable thiosemicarbazones. For example, suitable thiosemicarbazones may include, but are not limited to, GTSM, DpC, diacetyl-bis(N(4)-methylthiosemicarbazone) (ATSM), N-methyl-isatin-p-thiosemicarbazone, triapine (3-aminopyridine-2- carboxaldehyde thiosemicarbazone), 4-(2-pyridinyl)-2-(6,7-dihydro-8(5H)- quinolinylidene)hydrazide-l-piperazinecarbothioic acid (COTI-2), di-2-pyridylketone 4,4- dimethyl-3-thiosemicarbazone (Dp44mT), di-2-pyridylketone 4-ethyl-4-methyl-3- thiosemicarbazone, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, 4- acetamidobenzaldehyde thiosemicarbazone, 2-benzoylpyridyl 4-methyl-3- thiosemicarbazone, 2-benzoylpyridyl 4,4-dimethyl-3-thiosemicarbazone, 2-benzoylpyridyl 4-ethyl-3-thiosemicarbazone, 2-benzoylpyridyl 4-allyl-3-thiosemicarbazone, 2- benzoylpyridyl 4-phenyl-3-thiosemicarbazone, 2-benzoylpyridyl 3-thiosemicarbazone, 2- benzoylpyridyl 4-methyl-4-ethyl-3-thiosemicarbazone, di-2-pyridylketone 3- thiosemicarbazone, di-2-pyridylketone 4-allyl-3-thiosemicarbazone, di-2-pyridylketone 4- phenyl-3-thiosemicarbazone, 3-amino-2-formylpyridine thiosemicarbazone, 5-amino-2- formylpyridine thiosemicarbazone, 3-amino-4-methyl-2-formylpyridine thiosemicarbazone, 5-amino-4-methyl-2-formylpyridine thiosemicarbazone, 5- hydroxyamino-4-methyl-2-formylpyridine thiosemicarbazone, pyruvaldehyde bis(N(4)- methylthiosemicarbazone), ethylglyoxal bis(thiosemicarbazone), alloxan-5- thiosemicarbazone, or a pharmaceutically acceptable salt or solvate thereof.

[0076] Suitable thiosemicarbazones are also disclosed in WO 2010000008 Al, WO 2007035489 A2, WO 2022086079 Al and US 5344842 A, the contents of which are incorporated by reference herein.

[0077] In some embodiments, the thiosemicarbazone is GTSM, DpC or a pharmaceutically acceptable salt or solvate thereof; especially GTSM or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the thiosemicarbazone is GTSM:or a pharmaceutically acceptable salt or solvate thereof.

[0078] In some embodiments, the thiosemicarbazone is DpC:or a pharmaceutically acceptable salt or solvate thereof.

[0079] In some embodiments, when the ionophore is GTSM or a pharmaceutically acceptable salt or solvate thereof and the bacterium is S. pyogenes, the antibiotic is other than a macrolide.

[0080] In some embodiments, the copper ionophore is other than ATSM or a pharmaceutically acceptable salt or solvate thereof.

[0081] In alternative embodiments, the ionophore is elesclomol:or a pharmaceutically acceptable salt or solvate thereof.

[0082] In further embodiments, the ionophore is nitroxoline:or a pharmaceutically acceptable salt or solvate thereof.

[0083] In some embodiments, the ionophore is GTSM, DpC, elesclomol, nitroxoline or a pharmaceutically acceptable salt or solvate of any of the foregoing.

[0084] The copper ionophore is preferably a selective copper ionophore [i.e. an ionophore which is selective for binding to Cu(II)]. In some embodiments, the ionophore selectively binds Cu(II) over at least one other metal ion, such as a transition metal ion including Zn(II), Fe(III), Ni(II), and the like. In particular embodiments, the ionophore exhibits selectivity for binding to Cu(II) of greater than about 2-fold, 5-fold, 10-fold, 20- fold, 50-fold or greater than about 100-fold with respect to binding to another metal ion, particularly another transition metal ion, such as Zn(II). In particular embodiments, the ionophore does not substantially bind to another metal ion. In specific embodiments, the ionophore does not substantially bind to Zn(II).

[0085] The ionophore may be used in an amount suitable to confer antibiotic sensitivity to a resistant bacterium, or to potentiate the activity of an antibiotic. In some embodiments, the ionophore is used in an amount which does not have bactericidal activity in the absence of an antibiotic. In other words, the ionophore may be used in a sub- inhibitory amount. The ionophore is, for example, used in an amount that does not inhibit the growth of or kill the bacterium in the absence of an antibiotic.

[0086] Antibiotics are generally classified according to their mode of action, chemical class and / or types of infections that they treat. Classes and examples of antibiotics useful for treating bacterial infections include:Aminoglycosides e.g. kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycins B, C, E and streptomycin;Carbacephems e.g. loracarbef;Carbapenems e.g. ertapenem, doripenem, imipenem and meropenem;Cephalosporins (first generation) e.g. cefadroxil, cephazolin, cefalotin and cephalexin;Cephalosporins (second generation) e.g. cefaclor, cefamandole, cefprozil and cefuroxime;Cephalosporins (third generation) e.g. cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime and ceftaroline fosamil;Cephalosporins (fourth generation) e.g. cefepime;Cephalosporins (fifth generation) e.g. ceftaroline fosamil, cefiderocol and ceftobipriole;Cephamycin e.g. cefoxitin, cefotetan and cefmetazole;Glycopeptides e.g. teicoplanin, vancomycin, telavancin, dalbavancin and oritavancin;Lincosamides e.g. clindamycin and lincomycin;Lipopeptides e.g. daptomycin;Macrolides e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin and spiramycin;Nitrofurans e.g. furazolidine and nitrofurantoin;Oxazolidinones e.g. linezolid, posizolid, radezolid and torezolid;Penicillins e.g. amoxicillin, ampicillin, azlocilin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), piperacillin, temocillin and ticarcillin;Polypeptides e.g. bacitracin;Quinolones / fluoroquinolones e.g. ciprofloxacin, enrofloxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid and norfloxacin;Sulfonamides e.g. mafenide, silfacetaminde, sulfadiazine, silver sulfadiazine, sulfadimethoxazine, sulfamethizole, sulfasalazine and sulfisoxazole;Tetracyclines e.g. chlortetracycline, demecolcycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, rolitetracycline, tetracycline, eravacycline, sarecycline, omadacycline and tigecycline; andOther antibiotics e.g. chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, thiamphenicol, tigecycline, tinidazole, trimethoprim, rifampicin, rifapentine, pyrazinamide, isoniazid, ethionamide, ethambutol, cycloserine, dapsone and clofazimine.

[0087] These antibiotics are named according to their international nonproprietary name (INN). It will be appreciated that each antibiotic mentioned above will also have an IUPAC chemical name in accordance with its chemical structure, and may also have one or more proprietary or brand names. An antibiotic may be in the form of a pharmaceutically acceptable derivative such as a salt, solvate (e.g. hydrate) or in vivo hydrolysable ester. In vivo hydrolysable esters are esters which hydrolyse after administration to the subject to provide the free carboxylate group, such as pivaloyloxymethyl ester. Suitable pharmaceutically acceptable derivatives of antibiotics are well known in the art.

[0088] In some embodiments, the antibiotic to which sensitivity is conferred is any one of the antibiotics listed above.

[0089] In some embodiments, the antibiotic, is a carbacephem (e.g. loracarbef), a carbapenem (e.g. ertapenem, doripenem, imipenem or meropenem), a cephalosporin (e.g. cefadroxil, cephazolin, cefalotin, cephalexin, cefaclor, cefamandole, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil orceftobipriole), a cephamycin (e.g. cefoxitin, cefotetan or cefmetazole), a glycopeptide (e.g. teicoplanin, vancomycin, telavancin, dalbavancin or oritavancin), a lincosamide (e.g. clindamycin or lincomycin), a macrolide (e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin or spiramycin), a nitrofuran (e.g. furazolidine or nitrofurantoin), an oxazolidinone (e.g. linezolid, posizolid, radezolid or torezolid), a penicillin (e.g. amoxicillin, ampicillin, azlocilin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin, penicillin G, penicillin V, piperacillin, temocillin or ticarcillin), a polypeptide (e.g. bacitracin), a quinolone or fluoroquinolone (e.g. ciprofloxacin, enrofloxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid or norfloxacin), a sulfonamide (e.g. mafenide, silfacetaminde, sulfadiazine, silver sulfadiazine, sulfadimethoxazine, sulfamethizole, sulfasalazine or sulfisoxazole) or a tetracycline (e.g. chlortetracycline, demecolcycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, rolitetracycline, tetracycline, eravacycline, sarecycline, omadacycline or tigecycline). In particular embodiments, the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a penicillin, a macrolide or a tetracycline; especially a macrolide or a tetracycline.

[0090] In some embodiments, the antibiotic is a macrolide such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin or spiramycin; especially erythromycin or azithromycin. In other embodiments, the antibiotic is a tetracycline such as doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline and tigecycline; especially tetracycline or doxycycline.

[0091] In particular embodiments, the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.

[0092] In some embodiments, the antibiotic is a penicillin, such as amoxicillin, ampicillin, azlocilin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin, penicillin G, penicillin V, piperacillin, temocillin or ticarcillin; particularly penicillin or ampicillin. In some embodiments, the antibiotic is a fluoroquinolone such as ciprofloxacin, enrofloxacin, gatifloxacin, levofloxacin, lomefloxacin or norfloxacin; particularly levofloxacin. In some embodiments, the antibiotic is a penicillin or a fluoroquinolone. In exemplary embodiments, the antibiotic is penicillin, ampicillin or levofloxacin.

[0093] In some embodiments, the antibiotic is a macrolide, tetracycline, penicillin or fluoroquinolone, such as tetracycline, doxycycline, erythromycin, azithromycin, penicillin, ampicillin or levofloxacin.

[0094] In some embodiments, the antibiotic is other than a g-lactam antibiotic.

[0095] The bacterium may be any gram-positive bacterium to which antibiotic sensitivity is desired. In particular embodiments, the bacterium is a pathogenic grampositive bacterium.

[0096] Suitable gram-positive bacteria include, but are not limited to, Actinomyces species (e.g. A. israelii, A. viscosus, A. naeslundii, A. turicensis, A. meyeri,A. odontolyticus, A. gerencseriae and A. radingae); Bacillus species (e.g. B. anthracis andB. cereus); Corynebacterium species (e.g. C. diptheriae and C. pseudotuberculosis); Clostridium species (e.g. C. difficile, C. tetani, C. botulinum, C. baratii, C. butyricum, C. sordellii and C. perfringens); Lactobacillus species (e.g. L. rhamnosus, L. easel, L. fermentum, L. gasseri, L. plantarum, L. acidophilus, and L. ultunensis); Listeria species (e.g. L. monocytogenes); Mycobacterium species (e.g. M. tuberculosis and M. leprae); Streptococcus species (e.g. S. pyogenes, S. pneumoniae, S. mitis, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis and S. mutans), Enterococcus species (e.g. E. faecalis and E. faecium), Streptomyces species (e.g. S. somaliensis and S. sudanesis) and Staphylococcus species (e.g. S. aureus and S. epidermis).

[0097] In some embodiments, the bacterium is selected from the group consisting of a Streptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species.

[0098] In some embodiments, the bacterium is a Streptococcus species. Suitable species include, but are not limited to, S. pyogenes, S. pneumoniae, S. mitis, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis and S. mutans; especially S. pneumoniae or S. pyogenes.

[0099] In some embodiments, the bacterium is a Staphylococcus species, representative examples of which include S. aureus and S. epidermis. In particular embodiments, the bacterium is S. aureus.

[0100] In particular embodiments, the bacterium is an Enterococcus species, such as E. faecalis and E. faecium; especially E. faecium.

[0101] In other embodiments, the bacterium is a Clostridium species. Suitable Clostridium species include, but are not limited to, C. difficile, C. tetani, C. botulinum, C. baratii, C. butyricum, C. sordellii and C. perfringens; especially C. difficile. In particular embodiments, the bacterium is S. pneumoniae, E. faecium, S. pyogenes or S. aureus.

[0102] In some embodiments: the antibiotic is a macrolide such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin or spiramycin;the bacterium is an Enterococcus species, such as E. faecalis or E. faecium; and / or the ionophore is a thiosemicarbazone.

[0103] In particular embodiments: the antibiotic is azithromycin or erythromycin; the bacterium is E. faecium; and / or the ionophore is GTSM or a pharmaceutically acceptable salt or solvate thereof.

[0104] In some embodiments: the antibiotic is a tetracycline such as doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline or tigecycline; the bacterium is a Streptococcus species, such as S. pyogenes, S. pneumoniae, S. mitis, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis or S. mutans; and / or the ionophore is a thiosemicarbazone.

[0105] In particular embodiments: the antibiotic is tetracycline; the bacterium is S. pyogenes; and / or the ionophore is GTSM or a pharmaceutically acceptable salt or solvate thereof.

[0106] In some embodiments: the antibiotic is a tetracycline such as doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline or tigecycline; or a macrolide such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin or spiramycin; the bacterium is a Streptococcus species, such as S. pyogenes, S. pneumoniae, S. mitis, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis or S. mutans; and / or the ionophore is a thiosemicarbazone.

[0107] In particular embodiments: the antibiotic is tetracycline, doxycycline or azithromycin; the bacterium is S. pneumoniae; and / orthe ionophore is GTSM or a pharmaceutically acceptable salt or solvate thereof.

[0108] In some embodiments: the antibiotic is a macrolide such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin or spiramycin; the bacterium is a Streptococcus species, such as S. pyogenes, S. pneumoniae, S. mitis, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis or S. mutans; or a Staphylococcus species, such as S. aureus or S. epidermis; and / or the ionophore is elesclomol, nitroxoline or a pharmaceutically acceptable salt or solvate of either of the foregoing.

[0109] In some embodiments: the antibiotic is azithromycin; the bacterium is S. pyogenes, S. pneumoniae or S. aureus; and / or the ionophore is elesclomol, nitroxoline or a pharmaceutically acceptable salt or solvate of either of the foregoing.

[0110] In some embodiments: the antibiotic is azithromycin; the bacterium is a S. pyogenes, S. pneumoniae or S. aureus; and / or the ionophore is elesclomol or a pharmaceutically acceptable salt or solvate thereof.

[0111] In some embodiments: the antibiotic is a macrolide such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin or spiramycin; the bacterium is a Streptococcus species, such as S. pyogenes, S. pneumoniae, S. mitis, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis or S. mutans; and / or the ionophore is nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0112] In some embodiments: the antibiotic is azithromycin; the bacterium is S. pneumoniae; and / or the ionophore is nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0113] In some embodiments:the antibiotic is a tetracycline such as doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline or tigecycline; the bacterium is a Streptococcus species, such as S. pyogenes, S. pneumoniae, S. mitis, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis or S. mutans; and / or the ionophore is elesclomol or a pharmaceutically acceptable salt or solvate thereof.

[0114] In some embodiments: the antibiotic is tetracycline; the bacterium is S. pyogenes or S. pneumoniae; and / or the ionophore is elesclomol or a pharmaceutically acceptable salt or solvate thereof.

[0115] In some embodiments: the antibiotic is a penicillin such as amoxicillin, ampicillin, azlocilin, ca rbenici II in, cioxacillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin, penicillin G, penicillin V, piperacillin, temocillin or tica rcil I in ; or a fluoroquinolone such as ciprofloxacin, enrofloxacin, gatifloxacin, levofloxacin, lomefloxacin or norfloxacin; the bacterium is a Streptococcus species, such as S. pyogenes, S. pneumoniae, S. mitis, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis or S. mutans; and / or the ionophore is a thiosemicarbazone.

[0116] In particular embodiments: the antibiotic is penicillin, ampicillin or levofloxacin; the bacterium is S. pneumoniae; and / or the ionophore is GTSM or a pharmaceutically acceptable salt or solvate thereof.

[0117] In some embodiments, the method and uses further comprise contacting the bacterium with an antibiotic. Suitable antibiotics are as described supra. The bacterium may be contacted simultaneously, separately or sequentially with the ionophore and antibiotic.

[0118] The ionophores are also useful for inhibiting the development of antibiotic resistance in gram-positive bacteria. Accordingly, in another aspect, there is provided a method of inhibiting the development of resistance of a bacterium to an antibiotic, comprising, consisting or consisting essentially of contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxolineor a pharmaceutically acceptable salt or solvate thereof, and the bacterium is a grampositive bacterium. Also provided is a use of a copper ionophore for inhibiting the development of resistance of a bacterium to an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof, and the bacterium is a gram-positive bacterium; a copper ionophore for use in inhibiting the development of resistance of a bacterium to an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof, and the bacterium is a gram-positive bacterium; and a use of a copper ionophore in the manufacture of a medicament for inhibiting the development of resistance of a bacterium to an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof, and the bacterium is a gram-positive bacterium.

[0119] Suitable embodiments of the ionophore, bacterium and antibiotic are as discussed above.

[0120] In some embodiments, the method and uses further comprise contacting the bacterium with an antibiotic. Suitable antibiotics are as described supra. The bacterium may be contacted simultaneously, separately or sequentially with the ionophore and antibiotic.

[0121] In particular embodiments, the antibiotic is one which is one to which the bacterium is known to develop resistance.

[0122] The invention also provides the use of the copper ionophore for increasing the susceptibility of a gram-positive bacterium to an antibiotic. Accordingly, also provided, in a further aspect, is a method of increasing the susceptibility of a bacterium to an antibiotic, comprising, consisting or consisting essentially of contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a gram-positive bacterium; a use of a copper ionophore for increasing the susceptibility of a bacterium to an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a gram-positive bacterium; a copper ionophore for use in increasing the susceptibility of a bacterium to an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a gram-positive bacterium; and a use of a copper ionophore in the manufacture of a medicament for increasing the susceptibility of a bacterium to an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterium is a gram-positive bacterium.

[0123] In preferred embodiments, the bacterium is substantially resistant to the antibiotic or is developing resistance to the antibiotic.

[0124] Suitable embodiments of the ionophore, bacterium and antibiotic are as discussed above.

[0125] The methods and uses may further comprise contacting the bacterium with the antibiotic. The bacterium may be contacted simultaneously, separately or sequentially with the ionophore and antibiotic.

[0126] Due to their role in conferring sensitivity to antibiotics and for inhibiting the development of antibiotic resistance, the copper ionophores are also useful for treating or inhibiting the development of a gram-positive bacterial infection in a subject, when administered concurrently with an antibiotic. Accordingly, in another aspect of the invention, there is provided a method of treating or inhibiting the development of a bacterial infection in a subject, comprising, consisting or consisting essentially of administering a copper ionophore and an antibiotic to the subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterial infection is caused by a gram-positive bacterium; a use of a copper ionophore and an antibiotic for treating or inhibiting the development of a bacterial infection in a subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterial infection is caused by a gram-positive bacterium; a copper ionophore and an antibiotic for use in treating or inhibiting the development of a bacterial infection in a subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof and the bacterial infection is caused by a gram-positive bacterium; and a use of a copper ionophore in the manufacture of a medicament for treating or inhibiting the development of a bacterial infection in a subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof, the bacterial infection is caused by a gram-positive bacterium, and the copper ionophore is to be administered with an antibiotic.

[0127] In particular embodiments, the copper ionophore and antibiotic are administered concurrently, for example, simultaneously.

[0128] Suitable embodiments of the ionophore, bacterium causing the infection and antibiotic are as discussed above.

[0129] In some embodiments, the ionophore is administered in an amount which does not have bactericidal (or antibacterial) activity in the absence of the antibiotic. In other words, the ionophore may be administered in a sub-inhibitory amount. In someembodiments, the ionophore is administered in an amount that does not inhibit the growth or kill the bacterium in the absence of the antibiotic.

[0130] The antibiotic may be administered in an amount which is known to be active against a non-resistant bacterium.

[0131] The infection may be caused by a gram-positive bacterium that is resistant to one or more antibiotics or is known to develop antibiotic resistance. In some embodiments, the bacterium is one which is substantially resistant to the antibiotic. In some embodiments, the bacterium is one which is known to develop resistance to the antibiotic.

[0132] Examples of antibiotic resistant gram-positive bacteria include, but are not limited to, Group A Streptococcus (GAS) e.g. S. pyogenes (e.g. which is resistant to a macrolide such as erythromycin and / or azithromycin, and / or a tetracycline such as tetracycline), drug resistant S. pneumoniae (e.g. which is resistant to a penicillin such as penicillin, a macrolide such as azithromycin, a cephalosporin such as ceftazidime and / or ceftriaxone, and / or a tetracycline such as doxycycline and / or tetracycline); vancomycin- resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA).

[0133] The infection may be an infection of any tissue of the subject. For example, the infection may be a lung, blood, brain, skin, throat, urinary tract (e.g. bladder or urethra), vaginal, penile, gastrointestinal tract (e.g. peritoneum or bowel), meninges, ear, sinus, eye, cardiac, intra-abdominal, liver joint, pelvic, bone, kidney, or spinal infection; especially a lung, blood or brain infection. In some embodiments, the infection is a lung infection.

[0134] In some embodiments, the subject has a disease, disorder or condition associated with a bacterial infection, such as sepsis, pneumonia, meningitis, bronchiolitis, bronchitis, endocarditis, osteomyelitis, peritonitis, pyelonephritis, cystitis or urethritis. The subject may have, for example, a sexually transmitted infection or a urinary tract infection.

[0135] In particular embodiments, the subject is a human subject.

[0136] The copper ionophore and / or antibiotic may be administered by any route, including, but not limited to, oral, rectal, topical, intranasal, inhalation, intraocular, transmucosal, intestinal, enteral, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intracerebral, intravaginal, intravesical, intravenous, otic, urethral or intraperitoneal administration. In particular embodiments, the copper ionophore and / or antibiotic are administered via oral or intravenous administration. The copper ionophore and / or antibiotic may be administered in the form of a composition, as discussed elsewhere herein.

[0137] In a further aspect, there is provided a use of a copper ionophore as an antibiotic adjuvant or antibiotic potentiator, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0138] Suitable embodiments of the ionophore and antibiotic are as discussed supra. In particular embodiments, the copper ionophore is used in combination with an antibiotic (e.g. administered concurrently), especially used simultaneously with an antibiotic.

[0139] In some embodiments, the copper ionophore is the sole antibiotic adjuvant or antibiotic potentiator present.

[0140] The antibiotic adjuvant or antibiotic potentiator may be administered concurrently with an antibiotic for treating or inhibiting the development of a gram-positive bacterial infection, especially an infection caused by a bacterium which is resistant to one or more antibiotics. In particular embodiments, the bacterium is resistant to the antibiotic being potentiated.

[0141] Also provided herein is a method of potentiating an activity of an antibiotic against a gram-positive bacterium, comprising contacting the bacterium with a copper ionophore concurrently with the antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; a copper ionophore for use in potentiating an activity of an antibiotic against a gram-positive bacterium, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'1, / V'3-dimethyl- / V'1, / V'3-di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and a use of a copper ionophore for potentiating an activity of an antibiotic against a gram-positive bacterium, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl- / V'^ / V^-di phenylcarbonothioyljmalonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0142] Suitable embodiments of the ionophore, antibiotic and bacterium are as discussed supra. In particular embodiments, the bacterium is resistant to the antibiotic being potentiated.

[0143] In some embodiments, the activity of an antibiotic is antibacterial activity. In particular embodiments, the activity of the antibiotic is increased compared to the activity of the antibiotic in the absence of the copper ionophore, such as by at least about 2-fold to 1000-fold (and all integers therebetween). In some embodiments, the activity isincreased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or 1000-fold.

[0144] The invention also contemplates a method of potentiating an activity of an antibiotic against a gram-positive bacterium in a subject, comprising administering a copper ionophore and the antibiotic to the subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; a copper ionophore for use in potentiating an activity of an antibiotic against a gram-positive bacterium in a subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and a use of a copper ionophore for potentiating an activity of an antibiotic against a gram-positive bacterium in a subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0145] Suitable embodiments of the ionophore, antibiotic and bacterium are as discussed supra. In particular embodiments, the bacterium is resistant to the antibiotic being potentiated.

[0146] In some embodiments, the activity of an antibiotic is antibacterial activity. In particular embodiments, the activity of the antibiotic is increased compared to the activity of the antibiotic in the absence of the copper ionophore, such as by at least about 2-fold to 1000-fold (and all integers therebetween). In some embodiments, the activity is increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or 1000-fold.

[0147] In some embodiments of any of the methods and uses described herein, the copper ionophore increases the intracellular copper concentration within the bacterium.

[0148] Any one of the methods and uses described herein may involve administration or use of an effective amount of the copper ionophore and, where applicable, antibiotic.

[0149] Suitable unit dosages and maximum daily dosages of antibiotic used in combination with a copper ionophore of the invention may be determined in accordance with the unit doses and maximum daily doses used conventionally for a given antibiotic. Accordingly, an antibiotic may be administered to a patient at a daily dosage of, for example, from about 250 mg to about 750 mg intravenously or orally every 6 hours toabout 500 mg to about 1 g intravenously or orally every 6 to 8 hours, with a maximum dose of about 50 mg / kg / day or about 4 g / day.

[0150] The amount of copper ionophore administered will vary and can be determined according to the circumstances and the route of administration. The amount of copper ionophore administered should be non-toxic to the subject. In some embodiments the amount of ionophore administered is about 2 to about 100 mg / kg per day (and all integer mg therebetween), including about 2.5 to about 60 mg / kg per day, 2.5 to about 50 mg / kg per day, about 2.5 to about 30 mg / kg per day, about 2.5 to about 25 mg / kg per day, or about 2.5 to about 10 mg / kg per day intravenously or orally. In some embodiments the amount of copper ionophore administered does not exceed about 60 mg / kg per day, for example about 20 mg / kg per day or about 10 mg / kg per day intravenously or orally. The ionophore may be administered as a single dose or, alternatively, as a divided dose, such as up to 30 mg / kg twice daily.

[0151] A copper ionophore, as hereinbefore described, may be the sole active ingredient administered to the subject. However in preferred embodiments the ionophore is administered with another therapeutic agent, especially an antibiotic. For example, the copper ionophore may be administered with one or more therapeutic agents in combination. The combination may allow for separate, sequential or simultaneous administration of the compound as hereinbefore described with the other active ingredient(s). The combination may be provided in the form of a pharmaceutical composition as discussed elsewhere herein. Administration with one or more other active ingredients is within the scope of the invention, such as other inhibitors of bacterial resistance, antibiotic potentiators, antibiotics or antibiotic adjuvants, including p-lactamase inhibitors such as clavulanic acid, or other antibiotic adjuvants such as cilastatin, tazobactam, sulbactam, avibactam, relebactam, vaborbactam, ETX2514, aspergillomarasmine A, phosphonoformate, phosphonoacetate, acetylphosphonate, a- hydroxytropolone 3e, a-hydroxytropolone 3o, CKI-7, quercetin, damnacanthal, wortmannin, pyrazolopyrimidines 2a, pyrrolidine-3-ol, eisinhibitor, aranorosin or pterostilbene (trans-3,5-dimethoxy-4'-hydroxystilbene).

[0152] In some embodiments of any one of the uses and methods described herein, the copper ionophore is in the form of a complex with a metal (e.g. a coordination complex), such as a complex with copper (e.g. Cu(II)GTSM). In particular embodiments of any one of the uses and methods described herein, the copper ionophore is administered concurrently with a metal salt, e.g. a zinc or copper salt. In some embodiments of any one of the uses and methods described herein, the copper ionophore is administered concurrently with a copper salt.

[0153] In particular embodiments of any one of the uses and methods described herein, the copper ionophore is not in the form of a complex with a metal (e.g. a coordination complex). In particular embodiments of any one of the uses and methods described herein, the copper ionophore is not administered concurrently with a metal salt, e.g. a zinc or copper salt.4. Compositions

[0154] The invention also contemplates pharmaceutical compositions comprising a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof as hereinbefore described, and the use of such compositions in the methods and uses described in Section 3 supra. The composition may further comprise an antibiotic and / or a carrier or diluent. The use of such compositions for therapy is contemplated.

[0155] Accordingly, in another aspect, there is provided a pharmaceutical composition comprising, consisting or consisting essentially of a copper ionophore and an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

[0156] Suitable embodiments of the antibiotic and copper ionophore are as discussed in Section 3 supra.

[0157] In particular embodiments, the composition further comprises a carrier or diluent.

[0158] As will be appreciated by those skilled in the art, the choice of pharmaceutically acceptable carrier or diluent will be dependent on the route of administration and on the nature of the condition and subject to be treated. The particular carrier or delivery system and route of administration may be readily determined by a person skilled in the art. The carrier or delivery system and route of administration should be carefully selected to ensure that the activity of the copper ionophore and / or antibiotic is not depleted during preparation of the formulation and the copper ionophore and / or antibiotic is able to reach the site of action intact. The pharmaceutical compositions of the invention may be administered through a variety of routes including, but not limited to, oral, rectal, topical, intranasal, inhalation, intraocular, transmucosal, intestinal, enteral, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intracerebral, intravaginal, intravesical, intravenous, otic, urethral or intraperitoneal administration. In particular embodiments, the copper ionophore and / or antibiotic are administered via oral or intravenous administration.

[0159] The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions and sterile powders for the preparation of sterileinjectable solutions. Such forms should be stable under the conditions of manufacture and storage and may be preserved against reduction, oxidation and microbial contamination.

[0160] A person skilled in the art will readily be able to determine appropriate formulations for the copper ionophores using conventional approaches. Techniques for formulation and administration may be found in, for example, Remington: The Science and Practice of Pharmacy, Adeboye Adejare and Joseph Remington (Ed), Academic Press, London, 23rdEdition, 2021.

[0161] Identification of preferred pH ranges and suitable excipients, such as antioxidants, is routine in the art, for example, as described in Katdare and Chaubel (2006) Excipient Development for Pharmaceutical, Biotechnology and Drug Delivery Systems (CRC Press). Buffer systems are routinely used to provide pH values of a desired range and may include, but are not limited to, carboxylic acid buffers, such as acetate, citrate, lactate, tartrate and succinate; glycine; histidine; phosphate; tris(hydroxymethyl)aminomethane (Tris); arginine; sodium hydroxide; glutamate; and carbonate buffers. Suitable antioxidants may include, but are not limited to, phenolic compounds such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole; vitamin E; ascorbic acid; reducing agents such as methionine or sulfite; metal chelators such as ethylene diamine tetraacetic acid (EDTA); cysteine hydrochloride; sodium bisulfite; sodium metabisulfite; sodium sulfite; ascorbyl palmitate; lecithin; propyl gallate; and alpha-tocopherol.

[0162] For injection, the copper ionophore may be formulated in an aqueous solution, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, dextrose solution or physiological saline buffer, such as phosphate buffered saline (PBS). For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0163] The compositions of the present invention may be formulated for administration in the form of liquids, containing acceptable diluents (such as saline and sterile water), or may be in the form of lotions, creams or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity and appearance. Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, emulsifying agents such as non-ionic organic and inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives and hydrophilic beeswax derivatives.

[0164] Alternatively, the copper ionophore can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable the copper ionophore to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject. These carriers may be selected from sugars, chitosan, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and pyrogen-free water.

[0165] Pharmaceutical formulations for parenteral administration include aqueous solutions of the composition in water-soluble form. Additionally, suspensions of the copper ionophore may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilisers or agents that increase the solubility of the copper ionophore to allow for the preparation of highly concentrated solutions.

[0166] Sterile solutions may be prepared by combining the copper ionophore in the required amount in the appropriate solvent with other excipients as described above as required, followed by sterilisation, such as filtration. Generally, dispersions are prepared by incorporating the various sterilised active agents into a sterile vehicle which contains the basic dispersion medium and the required excipients as described above. Sterile dry powders may be prepared by vacuum- or freeze-drying a sterile solution comprising the active agents and other required excipients as described above.

[0167] Pharmaceutical preparations for oral use can be obtained by combining the copper ionophore with solid excipients and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilising processes.

[0168] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, PEG, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of particle doses.

[0169] Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilisers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilisers may be added.

[0170] Preparations for intranasal administration may include solutions, aerosols, dry powders, suspensions, gels or emulsions; especially a solution or suspension. Intranasal compositions may include, for example, a carrier (e.g. saline or water), cosolvent (e.g. ethanol, polypropylene glycol or a PEG such as PEG400), preservative (e.g. benzyl alcohol, benzalkonium chloride, chlorobutanol, methylparaben, phenylethyl alcohol or propylparaben), surfactant (e.g. a PEG such as PEG3500, polyoxyl 400 stearate, polysorbate 20 or polysorbate 80), pH adjuster (e.g. hydrochloric acid, sodium hydroxide or sulfuric acid), antioxidant (e.g. butylated hydroxyanisole or EDTA), stabiliser (e.g. cellulose microcrystalline), buffering agent or salt (e.g. trisodium citrate, sodium citrate, citric acid, sodium phosphate or sodium chloride), polymer (e.g. cellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone) and / or sugar or sugar alcohol (e.g. sorbitol, sucrose, glycerol or glucose / dextrose).

[0171] The copper ionophore may be administered in a local or systemic manner. Preferably the ionophore is systemically administered.

[0172] It is advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. The determination of the novel dosage unit forms of the present invention is dictated by and directly dependent on the unique characteristics of the active material, the particular therapeutic effect to be achieved and the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects as herein described.

[0173] While the copper ionophore as hereinbefore described may be the sole active ingredient administered to the subject, the administration of other active ingredients concurrently with said copper ionophore is within the scope of the invention, particularly an antibiotic. For example, in some embodiments, the copper ionophore may beadministered or used concurrently with one or more antibiotics, other inhibitor of bacterial resistance, antibiotic potentiator and / or antibiotic adjuvant. The copper ionophore may be therapeutically used after the other active ingredient or may be therapeutically used together with the other active ingredient. The copper ionophore may be administered separately, simultaneously or sequentially with the other active ingredient.

[0174] Accordingly, in another aspect of the invention, there is provided a composition comprising, consisting or consisting essentially of a copper ionophore and an antibiotic, other inhibitor of bacterial resistance, antibiotic potentiator and / or antibiotic adjuvant. Suitable antibiotics, inhibitor of bacterial resistance, antibiotic potentiator and / or antibiotic adjuvant are as discussed in Section 3 supra.

[0175] The present invention also contemplates the use of a composition of the invention as a coating on surgical instruments, needles, cannulae, sutures, staples, catheters, stents, artificial joint replacements, and the like as a prophylactic to mitigate against contracting bacterial infection during surgical procedures, intravenous injections, catheterisation etc. In some embodiments, there is provided a use of a composition of the invention for coating a catheter.

[0176] As mentioned above the principal active ingredient may be compounded for convenient and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.25 pg to about 200 mg. Expressed in proportions, the active compound may be present in from about 0.25 pg to about 200 mg / mL of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

[0177] The copper ionophores of the invention are commercially available from a number of sources or may be prepared by synthetic routes well known in the art.

[0178] For example, GTSM may be prepared by the procedure outlined in Beraldo et al. (1998) Transition. Met. Chem. 23: 67-71. In brief, GTSM may be prepared by stirring 4-methyl-3-thiosemicarbazide (e.g. 2.019 g, 19.2 mmol) in ethanol (e.g. 40 mL), followed by the addition of a glyoxal solution (e.g. 1.090 mL, 40% in H2O, 9.5 mmol). The mixture is then stirred at ambient temperature for about 3 h, and the resulting suspension is filtered. The solid is washed with hot ethanol followed by hot acetone, then dried under vacuum to give GTSM (H2GTSM). Cu(II)GTSM may be prepared by refluxing a suspension of H2GTSM (2.14 g, 9.22 mmol) and copper acetate monohydrate (1.84 g, 9.24 mmol) in ethanol (100 mL) for 3 hours. The brown suspension may be allowed to cool to ambient temperature and then filtered. The brown solid may then be washed with ethanol and dried under vacuum to give Cu(II)GTSM as a brown solid (2.08 g, 77%).

[0179] DpC and triapine are commercially available from, for example, Sigma- Aldrich Co. LLC (Merck KGaA, Darmstadt, Germany). DpC and Dp44mT may be synthesised using the procedure described in, for example, Lovejoy et al. (2012) J. Med. Chem., 55(16): 7230-7244; and / or Richardson et al. (2006) J. Med. Chem., 49(22): 6510- 6521, the entire contents of which are encompassed herein by reference. Other methods for preparing thiosemicarbazones may be found in Gingras et al. (1961) Can J Chem, 39: 973-985, Gingras et al. (1962) Can J Chem, 40: 1053-1059, WO 2010000008 Al, WO 2007035489 A2, WO 2022086079 Al and US 5344842 A, the entire contents of which are encompassed herein by reference.

[0180] Elesclomol is commercially available from, for example, Sigma-Aldrich Co. LLC (Merck KGaA, Darmstadt, Germany) and MedChemExpress (Monmouth Junction, New Jersey, USA). Elesclomol may be synthesised using the procedure described in, for example, Vo et al. (2014) J Inorg Biochem, 130: 69-73, the entire content of which is encompassed herein by reference.

[0181] COTI-2 is commercially available from, for example, MedChemExpress (Monmouth Junction, New Jersey, USA). COTI-II may be synthesised using the procedure described in, for example, Nunes et al. (2020) J. Med. Chem., 63: 13719-13732, the entire content of which is encompassed herein by reference.

[0182] Nitroxoline is commercially available from, for example, Sigma-Aldrich Co. LLC (Merck KGaA, Darmstadt, Germany). Nitroxoline may be synthesised using the procedure detailed in, for example, Voronin et al. (1976) Technology of Drug Manufacture, 10: 1215-1217; and / or Isaev et al. (2005) Chemistry of Heterocyclic Compounds, 41: 1027-1030, the entire contents of which are encompassed herein by reference.

[0183] The copper ionophore of the invention may be in crystalline form, and may exist as polymorphic forms. The copper ionophore of the invention may exist in an amorphous form. In some embodiments, the copper ionophore is in the form of a solvate (e.g. hydrate). Methods of solvation are generally known within the art. The copper ionophore may be in the form of a salt as discussed elsewhere herein.

[0184] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.EXAMPLES

[0185] All starting materials, reagents, media, bacterial strains and equipment used are commercially available (e.g. from Sigma-Aldrich, Burlington, Massachusetts, USA; Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA; Merck KGaA,Darmstadt, Germany; and the like) and were obtained from commercial sources unless otherwise indicated.EXAMPLE 1 - ACTIVITY OF CU(II)GTSM AGAINST STREPTOCOCCUS PNEUMONIAEMaterials and Methods

[0186] Glyoxal-bis(N(4)-methylthiosemicarbazonato)-copper(II) (Cu(II)GTSM) was prepared by first synthesising the starting compound, H2GTSM, as outlined in Beraldo et al. (1998) Transition. Met. Chem. 23: 67-71. Briefly, H2GTSM was prepared by stirring 4-methyl-3-thiosemicarbazide (e.g. 2.019 g, 19.2 mmol) in ethanol (e.g. 40 mL), followed by the addition of a glyoxal solution (e.g. 1.090 mL, 40% in H2O, 9.5 mmol). The mixture was then stirred at ambient temperature for about 3 h, and the resulting suspension was filtered. The solid was washed with hot ethanol followed by hot acetone, then dried under vacuum to give H2GTSM. Cu(II)GTSM was prepared by refluxing a suspension of H2GTSM (2.14 g, 9.22 mmol) and copper acetate monohydrate (1.84 g, 9.24 mmol) in ethanol (100 mL) for 3 hours. The brown suspension was allowed to cool to ambient temperature and then filtered. The brown solid was washed with ethanol and dried under vacuum to give Cu(II)GTSM as a brown solid (2.08 g, 77%).

[0187] Activity of Cu(II)GTSM was tested against S. pneumoniae serotype 2 laboratory strain, D39. Briefly, S. pneumoniae was grown on Columbia agar plates supplemented with 5% (v / v) horse blood for 18 h at 37°C + 5% CO2. Overnight biomass was then suspended in cation-defined media (C+Y) and grown to optical density 600 nm (ODsoo) of 0.3. Cultures were diluted back to ODsoo 0.01 in fresh C+Y supplemented with 8 pM CuSO4 and added to a 96 well plate with increasing concentrations of Cu(II)GTSM. Bacteria were incubated at 37°C + 5% CO2 and growth was monitored for 10 h with readings taken every 30 mins.

[0188] To determine if growth in the presence of Cu(II)GTSM was eliciting copper accumulation within the bacteria, transcriptional analyses were conducted to monitor expression of the sole copper efflux pathway, copA. Cultures of S. pneumoniae were prepared as above and grown in C+Y supplemented with a sub-inhibitory concentration of Cu(II)GTSM (39 nM). 400 pL of culture was then used for RNA extraction and purified using RNeasy Protect Bacterial Mini kit (Qiagen, Hilden, Germany) after enzymatic lysis using lysozyme and mutanolysin, all according to manufacturer's instructions. DNase treatment was performed on-column during RNA extraction using RNase-free DNase (Qiagen , Hilden, Germany). Quantitative reverse transcription PCR was conducted in a 96-well plate with 10 ng total RNA per well using the Superscript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, Waltham, USA) on a QuantStudio 7 Real-time PCR system (Applied Biosystems, Waltham, USA) as per manufacturer's instructions. The transcription levels of genes analysed were normalised to those obtained for 16s.Results

[0189] The antimicrobial activity of Cu(II)GTSM against S. pneumoniae serotype 2 laboratory strain, D39 is presented in Figure 1. These data show that Cu(II)GTSM is independently antimicrobial and exerts a dose-dependent perturbation of growth with increasing concentrations.

[0190] Expression of the copper efflux pathway, copA, is presented in Figure 2. These data show that addition of copper alone (8 pM) causes ~40-fold up-regulation of the copper efflux pathway compared to untreated. When Cu(II)GTSM is also added, expression of copA increases ~150-fold compared to untreated. These data show that Cu(II)GTSM is highly efficient at transporting copper into the bacterial cell and eliciting a copper intoxication response in S. pneumoniae D39.

[0191] GTSM was used in the form of Cu(II)GTSM throughout the examples to ensure that the effects, shown in Figures 1 and 2, were due to GTSM mobilisation of copper in the growth medium and not the presence of a potentially adventitious metal, which could occur as a contaminant during GTSM synthesis and purification. It is expected that GTSM alone would result in similar effects to the complex in vitro and in vivo. For subsequent experiments, the concentration of copper in the growth medium was experimentally determined to optimally be 8 pM to observe the activity of the added GTSM complex in CA- MHB and mimic the role of copper present in in vivo niches infected by bacteria.EXAMPLE 2 - SYNERGY BETWEEN GTSM AND ANTIBIOTICS AGAINST STREPTOCOCCUS PNEUMONIAEMaterials and Methods

[0192] To investigate the antimicrobial synergy between Cu(II)GTSM and current frontline antibiotics, minimal inhibitory concentration (MICs) were conducted. The antibiotics azithromycin, doxycycline, tetracycline, penicillin, ampicillin and levofloxacin were tested against antibiotic resistant clinical isolates of S. pneumoniae in the presence of sub-inhibitory concentrations of Cu(II)GTSM. MIC assays were conducted according to Clinical and Laboratory Standards Institute (CLSI) guidelines, modified for multiple agents. Briefly, S. pneumoniae was grown on Columbia agar plates supplemented with 5% (v / v) horse blood for 18 h at 37°C + 5% CO2. Bacteria were collected from blood agar plates, suspended in cation-adjusted Mueller Hinton Broth (CA-MHB), and adjusted to a concentration of 1 x 106CFU.mL-1in CA-MHB supplemented 1: 10 with lysed horse blood (LHB), with or without Cu(II)GTSM. The cell suspension was then added to a 96-well flat bottom plate containing 2-fold serial dilutions of antibiotic (azithromycin, tetracycline, doxycycline, penicillin, ampicillin or levofloxacin) in CA-MHB+LHB at a final concentration of 5 x 105CFU.mL'1. The plates were sealed with gas-permeable seals and incubated at37°C for 20 h. The MIC was recorded as the antibiotic concentration of the well displaying no visible growth.Results

[0193] The results are presented in Table 1 and Table 2. Data show that Cu(II)GTSM treatment was able to potentiate the antimicrobial efficacy of the antibiotics by reducing the MIC, in some instances, breaking antibiotic resistance in the S. pneumoniae (Sp) clinical isolates (strains 19, 27, 304 and 4496).TABLE 1MINIMUM INHIBITORY CONCENTRATIONS (MIC; PG / ML) OF THE ANTIBIOTICS AZITHROMYCIN, DOXYCYCLINE AND TETRACYCLINE FOR S. PNEUMONIAE CLINICAL ISOLATES IN THE ABSENCE AND PRESENCE OF SUB-INHIBITORY CONCENTRATIONS (0.25XMIC) OF CU(II)GTSMTABLE 2MINIMUM INHIBITORY CONCENTRATIONS (MIC; PG / ML) OF THE ANTIBIOTICS PENICILLIN, AMPICILLIN AND LEVOFLOXACIN FOR S. PNEUMONIAE CLINICAL ISOLATES IN THE ABSENCE AND PRESENCE OF SUB-INHIBITORY CONCENTRATIONS (0.5XMIC) OF CU(II)GTSMEXAMPLE 3 - SYNERGY BETWEEN GTSM AND ANTIBIOTICS AGAINST GRAM POSITIVEBACTERIAMaterials and Methods

[0194] To investigate the antimicrobial synergy between Cu(II)GTSM and current frontline antibiotics in a broader range of Gram-positive bacterial pathogens, further MICanalyses were conducted with methicillin-resistant Staphylococcus aureus (USA300; MRSA), Streptococcus pyogenes (HKU16), and vancomycin-resistant Enterococcus faecium (AUSMDU48083; VRE). S. aureus was cultured overnight on Mueller Hinton agar at 37°C, followed by inoculation of a single colony into MHB and grown overnight at 37°C with shaking at 200 rpm. Bacterial cells were then diluted into fresh MHB, with or without sub- inhibitory Cu(II)GTSM supplementation, to a concentration of 1 x 106CFU.mL-1. The cell suspension was then added to a 96-well flat bottom plate containing 2-fold serial dilutions of antibiotic (azithromycin, tetracycline or erythromycin) in CA-MHB at a final concentration of 5 x 105CFU.mL'1. The plates were sealed with gas-permeable seals and incubated at 37°C for 20 h. The MIC was recorded as the antibiotic concentration of the well displaying no visible growth. S. pyogenes and E. faecium were cultured overnight on Columbia agar supplemented with 5% (v / v) horse blood for 18 h at 37°C, with and without 5% CO2, respectively. Bacteria were collected from blood agar plates, suspended in CA-MHB, and diluted to a concentration of 1 x 106CFU.mL-1in CA-MHB supplemented 1: 10 with LHB, with or without Cu(II)GTSM. The cell suspension was then added to a 96-well flat bottom plate containing 2-fold serial dilutions of antibiotic (azithromycin, tetracycline or erythromycin) in CA-MHB+LHB at a final concentration of 5 x 105CFU.mL-1. The plates were sealed with gas-permeable seals and incubated at 37°C for 20 h. The MIC was recorded as the antibiotic concentration of the well displaying no visible growth.Results

[0195] The results are presented in Table 3. Data show that Cu(II)GTSM was able to potentiate the antimicrobial activity of selected antibiotics against multi-drug resistant isolates of S. aureus, S. pyogenes and E. faecium. In some, cases the reduction of MIC restored the bacteria to clinical sensitivity, breaking antimicrobial resistance.TABLE 3MINIMUM INHIBITORY CONCENTRATIONS (MIC; MG / ML) OF THE ANTIBIOTICS AZITHROMYCIN, ERYTHROMYCIN AND TETRACYCLINE FOR S. AUREUS, S. PYOGENES AND E. FAECIUM IN THE ABSENCE AND PRESENCE OF SUB-INHIBITORY CONCENTRATIONS (0.25XMIC) OF CU(II)GTSMEXAMPLE 4 - ACTIVITY OF CUdDGTSM, ELESCLOMOL, NITROXOLINE AND PDTC AGAINST STREPTOCOCCUS PNEUMONIAEMaterials and Methods

[0196] To determine if alternative classes of copper ionophores could also potentiate current antibiotics, the ionophores elesclomol, nitroxoline, and ammonium pyrrol id inedith iocarbamate [PDTC] (dithiocarbonate) were purchased commercially. Ionophores were solubilised as per manufacturer's instructions. To assess the innate antimicrobial activity of these compounds, the multi-drug resistant S. pneumoniae strain, Sp4496, was grown in CA-MHB supplemented with 8 pM CuS04 (baseline concentration) until mid-log phase of growth (ODsoo ~0.3). These cultures were then diluted back to ODsoo 0.01 in fresh CA-MHB supplemented with increasing concentrations of elesclomol, nitroxoline or PDTC. Growth of the treated cultures was assessed in a 96 well plate, with ODsoo readings taken every 30 minutes for 18 h. The growth perturbation (i.e. antimicrobial activity) was then compared to Cu(II)GTSM.Results

[0197] The results are provided in Figures 3A to 3D. These data show that both elesclomol and nitroxoline exert a concentration-dependent inhibition of growth at concentrations comparable to Cu(II)GTSM. This suggests that these copper ionophores are also efficacious in Gram-positive bacterial pathogens and are therefore candidates for breakage of antibiotic resistance. PDTC also displayed antimicrobial activity, but was required at much higher concentrations.EXAMPLE 5 - CELLULAR COPPER AND ZINC ACCUMULATION IN STREPTOCOCCUS PNEUMONIAE IN THE PRESENCE OF CU(II)GTSM, ELESCLOMOL, NITROXOLINE AND PDTCMaterials and Methods

[0198] To ensure that the alternative copper ionophores were also eliciting accumulation of copper specifically within the bacteria, metal accumulation assays were conducted. The multi-drug resistant Sp4496 strain was grown overnight on Columbia agar supplemented with 5% (v / v) horse blood and overnight biomass was inoculated into CA- MHB supplemented with a sub-inhibitory concentration of each Cu(II)GTSM, elesclomol, nitroxoline and PDTC. Bacterial cultures were grown until a mid-log phase of growth (ODsoo ~0.3) and cells were harvested via centrifugation at 7000 xg for 7 mins at 4°C. Cell pellets were then washed twice in 20 mL PBS, 5 mM EDTA and twice in 20 mL PBS alone. Cell pellets were then left at 96°C overnight to desiccate. Desiccated cell pellets were weighed, and digested in 65% [v / v] HNO3 at 96°C for 30 mins. Samples were then diluted with MilliQ H2O and cellular copper and zinc content was analysed using an Agilent 8900QQQ ICP-MS (Agilent Technologies, Santa Clara, USA). Data are representative of at least 3 biological replicates.Results

[0199] The results are provided in Figure 4A and 4B. These data demonstrate that all copper ionophores tested are capable of significantly enhancing copper accumulation within the bacteria, compared to supplementation with 8 pM copper alone (untreated). Data show that copper accumulation increases by approximately 50-fold (Cu(II)GTSM), ~250-fold (elesclomol), ~150-fold (nitroxoline) and ~100-fold (PDTC) compared to untreated. These copper ionophores are able to shuttle copper ions across the S. pneumoniae membrane and effectively accumulate intracellularly, thereby overcoming the bacterial copper homeostatic mechanisms.

[0200] The zinc accumulation data confirms that, in the presence of equimolar concentrations of copper and zinc, the ionophores are not causing the accumulation of zinc within the bacteria, illustrating specificity for copper binding and transport.EXAMPLE 6 - SYNERGY BETWEEN ELESCLOMOL OR NITROXOLINE AND ANTIBIOTICS AGAINST GRAM POSITIVE BACTERIAMaterials and Methods

[0201] To test the ability of alternative copper ionophores to break azithromycin and tetracycline resistance, MIC assays were conducted with S. aureus (USA300), S. pyogenes (HKU16) and S. pneumoniae (Sp4496). Briefly, S. aureus was cultured overnight on Mueller Hinton agar at 37°C, followed by inoculation of a single colony into MHB andgrown overnight at 37°C with shaking at 200 rpm. Cultures were then diluted into fresh MHB, with or without sub-inhibitory elesclomol supplementation, to a concentration of 1 x 106CFU.mL1. The cell suspension was then added to a 96-well flat bottom plate containing 2-fold serial dilutions of azithromycin in CA-MHB at a final concentration of 5 x 105CFU.mL T The plates were sealed with gas-permeable seals and incubated at 37°C for 20 h. The MIC was recorded as the antibiotic concentration of the well displaying no visible growth. S. pyogenes and S. pneumoniae were cultured overnight on Columbia agar supplemented with 5% (v / v) horse blood for 18 h at 37°C, with and without 5% CO2, respectively. Bacteria were collected from blood agar plates, suspended in CA-MHB, and diluted to a concentration of 1 x 106CFU.mL1in CA-MHB supplemented 1: 10 with LHB, with or without elesclomol, nitroxoline or PDTC. The cell suspension was then added to a 96-well flat bottom plate containing 2-fold serial dilutions of azithromycin or tetracycline in CA- MHB+LHB at a final concentration of 5 x 105CFU.mL1. The plates were sealed with gas- permeable seals and incubated at 37°C for 20 h. The MIC was recorded as the antibiotic concentration of the well displaying no visible growth.Results

[0202] Results are presented in Table 4. The data show that elesclomol and nitroxoline were able to potentiate azithromycin activity against all bacterial species tested. PDTC had no effect on the MIC of azithromycin against species tested. Tetracycline activity was also potentiated by both elesclomol and nitroxoline against all species tested. PDTC did not alter the MIC of tetracycline against the species tested. Collectively, these data show that both elesclomol and nitroxoline are able to potentiate the antimicrobial activity of frontline antibiotics against multiple, multi-drug resistant bacterial speciesTABLE 4MINIMUM INHIBITORY CONCENTRATIONS (MIC; MG / ML) OF AZITHROMYCIN AND TETRACYCLINE FOR S. AUREUS, S. PYOGENES AND S. PNEUMONIAE IN THE ABSENCE AND PRESENCE OF SUB-INHIBITORY CONCENTRATIONS OF ELESCLOMOL, NITROXOLINE AND PDTCEXAMPLE 7 - SUSCEPTIBILITY OF MULTI-DRUG RESISTANT S. PNEUMONIAE TO CU AND CU(II)GTSMMaterials and Methods

[0203] The elemental composition of cation-adjusted Mueller-Hinton broth (CA-MHB) (Becton Dickinson), which is the standard media for antimicrobial susceptibility testing, was first established using inductively-coupled plasma mass spectrometry (ICP- MS). This was done to determine the baseline values of metal ions in the growth medium, including Cu which could contribute to ionophore activity.

[0204] Whole-cell Cu accumulation was measured using ICP-MS and growth was monitored by optical density (OD) at 600 nm of 1 mL culture at 3 h post-treatment. Statistical significance was determined by one-way ANOVA with Tukey posttest in at least five (n = >5) biological replicates.

[0205] Chequerboard assays were conducted against Cu(II)GTSM to determine the optimal concentration of Cu to supplement the growth media for ionophore activity.

[0206] To determine the effect on metal homeostasis, S. pneumoniae 4496 was grown in CA-MHB supplemented with 8 pM Cu (untreated) and treated with Cu(II)GTSM (0.25 pM). Whole-cell metal accumulation was measured by ICP-MS. Statisticalsignificance was determined by one-way ANOVA with Tukey posttest in at least four (n = >4) biological replicates.

[0207] Samples for ICP-MS were prepared for elemental analysis by preparing a dessicated sample of bacterial cells, i.e. S. pneumoniae 4496 grown in CA-MHB supplemented with 8 pM Cu (untreated) and treated with Cu(II)GTSM (0.25 pM) and dilution into suprapure nitric acid (HNO3, catalogue number 100441, Merck KGaA, Darmstadt, Germany). For media analyses, samples were diluted 1 :2 with 65% (v / v) HNO3 and incubated at 95 °C for 20 min. Media, i.e. CA-MHB, was analysed directly via heating at 95 °C in an equal volume of suprapure nitric acid for 15 min in a dry block. Samples were diluted 10-fold with ultra-high purity water (MilliQ system, >18.2 Ohm) and heated for a further 15 min at 95 °C, then cooled on ice for 10 min. Samples were centrifuged for 5 min at 5,000 g and the supernatants (top 950 pL) were transferred to a fresh tube (Technoplas 1.5 mL polypropylene tube that had been rinsed with 1% (v / v) HNO3) for immediate analysis. Elemental measurements were performed using an Agilent 8900 inductively coupled plasma-mass spectrometer (ICP-MS; Agilent Technologies, Lexington, Massachusetts, USA). The instrument was calibrated for elements of interest using mixed calibrator solutions (Multi-element Calibration Standard 2A and 4, Agilent Technologies, Lexington, Massachusetts, USA) diluted in 3.25% (v / v) suprapure HNO3, acquiring signals for24Mg,44Ca,55Mn,56Fe,59Co,60Ni,63Cu,66Zn,78Se,95Mo andinCd. Yttrium (89Y; 100 pg L-1in 1% v / v HNO3) was introduced as the internal standard via a T-piece positioned after the peristaltic pump. The instrument was tuned using commercially sourced tuning solution (part number 5185-5959, Agilent Technologies, Lexington, Massachusetts, USA) according to the manufacturer's instructions. All element signals were acquired in He collision mode (4.3 mL min-1), except for Fe and Se, which were acquired in H2 mode (4.0 mL min-1). Raw data analysis was performed using MassHunter v5.3 (Agilent Technologies), exported for stoichiometry calculations in Microsoft Excel (v2502, build 18526.20168) and GraphPad Prism 10.Results

[0208] The analysis of the elemental composition of CA-MHB revealed very low (< 1 pM) concentrations of metal ions such as manganese (Mn), cobalt (Co), nickel (Ni), and Cu (Table 5). While metal ions such as magnesium (Mg) and calcium (Ca) were the most abundant followed by zinc (Zn) and iron (Fe) which have similar levels.TABLE 5CONCENTRATIONS (pM) OF BIOLOGICALLY RELEVANT METAL IONS IN CA-MHB AS DETERMINED BY ICP-MS

[0209] Various clinical S. pneumoniae isolates were then tested for their susceptibility to copper sulfate (CuSC ) or Cu and Cu(II)GTSM. These isolates include those that have been routinely used in literature such as D39, 23F, G54, and 4496, as well as isolates belonging to serotypes found in the vaccines. All tested isolates were highly resistant to Cu with a minimum inhibitory concentration (MIC) >256 pM. The intracellular Cu concentration in the serotype 1 isolate S pneumoniae 4496 treated with 500 pM Cu showed significant increase in Cu accumulation relative to untreated (~5-fold) without significant growth perturbation (Figure 5).

[0210] Given the low Cu concentration in CA-MHB, chequerboard assays were conducted against Cu(II)GTSM to determine the optimal concentration of Cu to supplement the growth media for ionophore activity. It was found that 8 pM Cu was the minimum concentration that enhanced susceptibility to Cu(II)GTSM and resulted in greater than twofold decrease in its MIC for most isolates (Table 6). Accumulation of Cu at this concentration was not significantly different to that in non-supplemented media indicating that this concentration does not change or contribute to the accumulation of Cu in the cell (Figure 5).TABLE 6SUSCEPTIBILITY OF VARIOUS MDR S. PNEUMONIAE ISOLATES TO CU(II)GTSM WITH AND WITHOUT Cuwherein ND = not determined.

[0211] There was variable efficacy of Cu(II)GTSM with or without additional Cu between isolates (Table 6). Of the pneumococcal isolates tested, S. pneumoniae 4496 showed the greatest resistance to Cu(II)GTSM with a MIC >256 pM (± Cu) (Table 6). Concentration of metal ions in S. pneumoniae treated with a sub-micromolar concentration of Cu(II)GTSM (0.25 pM) showed specific and significantly enhanced accumulation of Cu which was accompanied by significant perturbation in growth relative to untreated (8 pM) and 500 pM Cu (Figures 5 and 6). This demonstrates the Cu-dependent activity of Cu(II)GTSM can overwhelm the cellular mechanisms within S. pneumoniae that maintain Cu homeostasis and cause toxicity. In addition to Cu, there was also a significant decrease in the cellular concentration of Co in both 500 pM Cu and Cu(II)GTSM and a significant decrease in Cd in Cu(II)GTSM alone (Figure 6).EXAMPLE 8 - COMBINED EFFICACY OF CU(II>GTSM WITH B-LACTAM, TETRACYCLINE, AND MACROLIDE ANTIBIOTICS AGAINST RESISTANT S. PNEUMONIAEMaterials and Methods

[0212] Synergy between Cu(II)GTSM and ampicillin, tetracycline, and azithromycin, were determined through chequerboard assays in resistant S. pneumoniae isolates, namely 23F, 4496, and 68062, respectively. Drug interaction was determined through the fractional inhibitory concentration index (FICI) where a FICI value <0.5 is synergistic, between 0. 5-0.4 as additive or no interaction, and >4 as antagonistic. These values were calculated as follows: FICI = FICcompound A + FICcompound B, where the FIC of each compound is derived by dividing their MIC in combination than alone.

[0213] To provide a dynamic representation of the interaction between these compounds, growth was measured by absorbance at 600 nM (Asoo) in a 96-well plate for each of these combinations and averaged between two biological replicates.Results

[0214] The combination of Cu(II)GTSM with ampicillin, tetracycline, and azithromycin antibiotics showed significant reduction in their MIC by at least four-fold in these respective isolates (Table 7), with synergistic FICI values (<0.5) for Cu(II)GTSM with tetracycline and azithromycin in S. pneumoniae strains 4496 and 68086, respectively.

[0215] The synergistic interaction between tetracycline, and azithromycin with Cu(II)GTSM is indicated by the ladder-like pattern of growth inhibition whereas the additive interaction between Cu(II)GTSM and ampicillin is shown in the more of a square-like pattern of growth inhibition (Figure 7). This data shows Cu(II)GTSM potentiating the in vitro efficacy of tetracycline and azithromycin antibiotics against S. pneumoniae strains.TABLE 7ANALYSIS OF SYNERGY BETWEEN ANTIBIOTICS AND CU(II)GTSM IN MULTI-DRUG RESISTANT S.PNEUMONIAEEXAMPLE 9 - SPONTANEOUS DEVELOPMENT OF RESISTANCE TO TETRACYCLINE WITH CU(II)GTSM IN S. PNEUMONIAEMaterials and Methods

[0216] The development of resistance to tetracycline with Cu(II)GTSM in S. pneumoniae 4496 was assessed by determining the rate of spontaneous resistant mutants that occur under these treatments. Specifically, a dense culture of S. pneumoniae 4496 (IO9-10CFU) was grown in agar containing the respective treatments alone and in combination at multiples of their MICs. The frequency of resistance is derived from growth (%) on treated relative to untreated media. Spontaneous mutants indicate the rate at which S. pneumoniae can quickly adapt to the treatments following overnight incubation(single-step mutants). The combined concentration of two different compounds that show no growth is then determined as the maximum concentration at which the combined stress exceeds the adaptation rate termed the mutant prevention concentration (MPC). The MPC is the lowest concentration of the drug(s) that prevents the growth of the first resistant mutant and is typically higher than the MIC. If the MIC is the same as the MPC, this indicates that the concentration that inhibits growth is sufficient to prevent the emergence of resistant mutants and that the mutant selection window (MSW) is likely narrow or nonexistent. The MSW represents the concentrations between the MIC and MPC where resistant mutants are likely to be selected.Results

[0217] No spontaneous mutants were able to grow at the MIC of tetracycline and at 0.25x MIC of Cu(II)GTSM indicating that both these compounds individually can suppress the development of spontaneous resistance at or below their MIC (Table 8). At 0.5x MIC of tetracycline and 0.125x MIC of Cu(II)GTSM, the frequency of resistance was >l.O8xlO10as confluent growth was observed at these concentrations of compounds individually (Table 8). The combination of these concentrations completely suppressed growth (frequency of resistance <l.O8xlO'10) (Table 8). These data show that both tetracycline and Cu(II)GTSM can suppress the development of spontaneous mutants at or below their MICs. Further, the combination of both compounds was able to suppress the emergence of spontaneously resistant mutants at fold-lower concentrations of each compound demonstrating their synergy.TABLE 8FREQUENCY OF SPONTANEOUS RESISTANCE TO TETRACYCLINE ± Cu(II)GTSM IN S. PNEUMONIAE 4496EXAMPLE 10 - IMPACT OF CU(II)GTSM AND TETRACYCLINE TREATMENT ON METAL HOMEOSTASIS IN S. PNEUMONIAEMaterials and Methods

[0218] To examine the further contribution of tetracycline to alteration of cellular metal homeostasis in the presence or absence of Cu(II)GTSM, ICP-MS analyses were conducted. S. pneumoniae 4496 was grown in CA-MHB supplemented with 8 pM Cu andtreated with tetracycline (1 pg / mL) and Cu(II)GTSM (0.25 pM). Whole-cell accumulation was measured by ICP-MS. Statistical significance was determined by one-way ANOVA with Tukey posttest in at least four (n >4) biological replicates.Results

[0219] Significant accumulation of Mn occurred under tetracycline treatment relative to untreated, Cu(II)GTSM, and the combined treatment (Figure 8). The combination of Cu(II)GTSM with tetracycline resulted in the intermediate accumulation of Mn relative to the individual treatments The combination of tetracycline with Cu(II)GTSM magnified the intracellular accumulation of Ca and Cu (Figure 8).

[0220] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0221] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0222] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.EMBODIMENTS

[0223] Exemplary embodiments include, but are not limited to:1. A method of conferring antibiotic sensitivity to a resistant bacterium, comprising contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and wherein the bacterium is a gram-positive bacterium.2. A method of inhibiting the development of resistance of a bacterium to an antibiotic, comprising contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and wherein the bacterium is a gram-positive bacterium.3. A method of increasing the susceptibility of a bacterium to an antibiotic, comprising contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and wherein the bacterium is a gram-positive bacterium.4. The method according to embodiment 3, wherein the bacterium is resistant to the antibiotic.5. The method according to any one of embodiments 1-4, further comprising contacting the bacterium with an antibiotic.6. The method according to any one of embodiments 1-5, wherein the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a glycopeptide, a lincosamide, a macrolide, a nitrofuran, an oxazolidinone, a penicillin, a polypeptide, a fluoroquinolone, a sulfonamide or a tetracycline.7. The method according to embodiment 6, wherein the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a penicillin, a macrolide or a tetracycline.8. The method according to embodiment 7, wherein the antibiotic is a macrolide or a tetracycline.9. The method according to embodiment 8, wherein the antibiotic is selected from the group consisting of azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin and spiramycin.10. The method according to embodiment 8, wherein the antibiotic is selected from the group consisting of doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline and tigecycline.11. The method according to embodiment 5, wherein the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.12. The method according to any one of embodiments 1-11, wherein the thiosemicarbazone is selected from the group consisting of glyoxal-bis(N(4)- methylthiosemicarbazone), di-2-pyridyl ketone 4-methyl-4-cyclohexyl-3- thiosemicarbazone and pharmaceutically acceptable salts and solvates thereof.13. The method according to embodiment 12, wherein the thiosemicarbazone is glyoxal-bis(N(4)-methylthiosemicarbazone).14. The method according to any one of embodiments 1-11, wherein the ionophore is nitroxoline.15. The method according to any one of embodiments 1-11, wherein the ionophore is elesclomol.16. The method according to any one of embodiments 1-15, wherein the bacterium is selected from the group consisting of a Streptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species.17. The method according to embodiment 16, wherein the bacterium is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile and Mycobacterium tuberculosis.18. The method according to embodiment 17, wherein the bacterium is S. pneumoniae, E. faecium, S. pyogenes or S. aureus.19. The method according to any one of embodiments 1-18, wherein the copper ionophore is a selective copper ionophore.20. A method of treating or inhibiting the development of a bacterial infection in a subject, comprising administering a copper ionophore and an antibiotic to the subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl- / V'^ / V^-di phenylcarbonothioyljmalonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and wherein the bacterial infection is caused by a gram-positive bacterium.21. The method according to embodiment 20, wherein the infection is a lung, blood or brain infection.22. The method according to embodiment 21, wherein the infection is a lung infection.23. The method according to any one of embodiments 20-22, wherein the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a glycopeptide, a lincosamide, a macrolide, a nitrofuran, an oxazolidinone, a penicillin, a polypeptide, a fluoroquinolone, a sulfonamide or a tetracycline.24. The method according to embodiment 23, wherein the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a penicillin, a macrolide or a tetracycline.25. The method according to embodiment 24, wherein the antibiotic is a macrolide or a tetracycline.26. The method according to embodiment 25, wherein the antibiotic is selected from the group consisting of azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin and spiramycin.27. The method according to embodiment 25, wherein the antibiotic is selected from the group consisting of doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline and tigecycline.28. The method according to any one of embodiments 20-27, wherein the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.29. The method according to any one of embodiments 20-28, wherein the thiosemicarbazone is selected from the group consisting of glyoxal-bis(N(4)- methylthiosemicarbazone), di-2-pyridyl ketone 4-methyl-4-cyclohexyl-3- thiosemicarbazone and pharmaceutically acceptable salts and solvates thereof.30. The method according to embodiment 29, wherein the thiosemicarbazone is glyoxal-bis(N(4)-methylthiosemicarbazone).31. The method according to any one of embodiments 20-28, wherein the ionophore is nitroxoline.32. The method according to any one of embodiments 20-28, wherein the ionophore is elesclomol.33. The method according to any one of embodiments 20-32, wherein the bacterial infection is caused by a bacterium selected from the group consisting of a Streptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species.34. The method according to embodiment 33, wherein the bacterium is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile and Mycobacterium tuberculosis.35. The method according to embodiment 34, wherein the bacterium is S. pneumoniae, E. faecium, S. pyogenes or S. aureus.36. The method according to any one of embodiments 20-35, wherein the copper ionophore is a selective copper ionophore.37. A pharmaceutical composition comprising a copper ionophore and an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl- / V' / V^-di phenylcarbonothioyDmalonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.38. The pharmaceutical composition according to embodiment 37, wherein the composition further comprises a carrier or diluent.39. Use of a copper ionophore as an antibiotic adjuvant or antibiotic potentiator, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl- / V'^ / V^-di phenylcarbonothioyljmalonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.40. A method of potentiating an activity of an antibiotic against a gram-positive bacterium, comprising contacting the bacterium with a copper ionophore concurrently with the antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V' / V'3- dimethyl-yV'^ / V^-di phenylcarbonothioyljmalonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.41. The method according to embodiment 40, wherein the bacterium is resistant to the antibiotic.42. The method according to embodiment 40 or embodiment 41, wherein the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a glycopeptide, a lincosamide, a macrolide, a nitrofuran, an oxazolidinone, a penicillin, a polypeptide, a fluoroquinolone, a sulfonamide or a tetracycline.43. The method according to embodiment 42, wherein the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a penicillin, a macrolide or a tetracycline.44. The method according to embodiment 43, wherein the antibiotic is a macrolide or a tetracycline.45. The method according to embodiment 44, wherein the antibiotic is selected from the group consisting of azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithomycin and spiramycin.46. The method according to embodiment 44, wherein the antibiotic is selected from the group consisting of doxycycline, chlortetracycline, tetracycline, demeclocycline, lymecycline, methacycline, minocycline, rolitetracycline, oxytetracycline, eravacycline, sarecycline, omadacycline and tigecycline.47. The method according to embodiment 40 or embodiment 41, wherein the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.48. The method according to any one of embodiments 40-47, wherein the thiosemicarbazone is selected from the group consisting of glyoxal-bis(N(4)- methylthiosemicarbazone), di-2-pyridyl ketone 4-methyl-4-cyclohexyl-3- thiosemicarbazone and pharmaceutically acceptable salts and solvates thereof.49. The method according to embodiment 48, wherein the thiosemicarbazone is glyoxal-bis(N(4)-methylthiosemicarbazone).50. The method according to any one of embodiments 40-47, wherein the ionophore is nitroxoline. 51. The method according to any one of embodiments 40-47, wherein the ionophore is elesclomol.52. The method according to any one of embodiments 40-51, wherein the bacterium is selected from the group consisting of a Streptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species.53. The method according to embodiment 52, wherein the bacterium is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile and Mycobacterium tuberculosis. 54. The method according to embodiment 53, wherein the bacterium is S. pneumoniae, E. faecium, S. pyogenes or S. aureus.55. The method according to any one of embodiments 40-54, wherein the copper ionophore is a selective copper ionophore.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of conferring antibiotic sensitivity to a resistant bacterium, comprising contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and wherein the bacterium is a gram-positive bacterium.

2. A method of inhibiting the development of resistance of a bacterium to an antibiotic, comprising contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and wherein the bacterium is a gram-positive bacterium.

3. A method of increasing the susceptibility of a bacterium to an antibiotic, comprising contacting the bacterium with a copper ionophore, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and wherein the bacterium is a gram-positive bacterium.

4. The method according to claim 3, wherein the bacterium is resistant to the antibiotic.

5. The method according to any one of claims 1-4, further comprising contacting the bacterium with an antibiotic.

6. The method according to any one of claims 1-5, wherein the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a glycopeptide, a lincosamide, a macrolide, a nitrofuran, an oxazolidinone, a penicillin, a polypeptide, a fluoroquinolone, a sulfonamide or a tetracycline.

7. The method according to claim 5, wherein the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.

8. The method according to any one of claims 1-7, wherein the thiosemicarbazone is selected from the group consisting of glyoxal-bis(N(4)- methylthiosemicarbazone), di-2-pyridylketone 4-methyl-4-cyclohexyl-3- thiosemicarbazone and pharmaceutically acceptable salts and solvates thereof.

9. The method according to claim 8, wherein the thiosemicarbazone is glyoxal-bis(N(4)-methylthiosemicarbazone).

10. The method according to any one of claims 1-7, wherein the ionophore is nitroxoline.

11. The method according to any one of claims 1-7, wherein the ionophore is elesclomol.

12. The method according to any one of claims 1-11, wherein the bacterium is selected from the group consisting of a Streptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species.

13. The method according to claim 12, wherein the bacterium is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile and Mycobacterium tuberculosis.

14. A method of treating or inhibiting the development of a bacterial infection in a subject, comprising administering a copper ionophore and an antibiotic to the subject, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V' / V'3- dimethyl-yV'^ / V^-di phenylcarbonothioyljmalonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof; and wherein the bacterial infection is caused by a gram-positive bacterium.

15. The method according to claim 14, wherein the infection is a lung, blood or brain infection.

16. The method according to claim 15, wherein the infection is a lung infection.

17. The method according to any one of claims 14-16, wherein the antibiotic is a carbacephem, a carbapenem, a cephalosporin, a cephamycin, a glycopeptide, a lincosamide, a macrolide, a nitrofuran, an oxazolidinone, a penicillin, a polypeptide, a fluoroquinolone, a sulfonamide or a tetracycline.

18. The method according to any one of claims 14-17, wherein the antibiotic is tetracycline, doxycycline, erythromycin or azithromycin.

19. The method according to any one of claims 14-18, wherein the thiosemicarbazone is selected from the group consisting of glyoxal-bis(N(4)- methylthiosemicarbazone), di-2-pyridylketone 4-methyl-4-cyclohexyl-3- thiosemicarbazone and pharmaceutically acceptable salts and solvates thereof.

20. The method according to claim 19, wherein the thiosemicarbazone is glyoxal-bis(N(4)-methylthiosemicarbazone).

21. The method according to any one of claims 14-18, wherein the ionophore is nitroxoline.

22. The method according to any one of claims 14-18, wherein the ionophore is elesclomol.

23. The method according to any one of claims 14-22, wherein the bacterial infection is caused by a bacterium selected from the group consisting of aStreptococcus species, a Staphylococcus species, an Enterococcus species, a Clostridium species and a Mycobacterium species.

24. The method according to claim 23, wherein the bacterium is selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile and Mycobacterium tuberculosis.

25. A pharmaceutical composition comprising a copper ionophore and an antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V'3- dimethyl- / V'1, / V'3-di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

26. The pharmaceutical composition according to claim 25, wherein the composition further comprises a carrier or diluent.

27. Use of a copper ionophore as an antibiotic adjuvant or antibiotic potentiator, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'1, / V'3-dimethyl- / V'1, / V'3-di(phenylcarbonothioyl)malonod I hydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

28. A method of potentiating an activity of an antibiotic against a grampositive bacterium, comprising contacting the bacterium with a copper ionophore concurrently with the antibiotic, wherein the copper ionophore is a thiosemicarbazone, elesclomol ( / V'^ / V^-dimethyl-ZV'^ / V'3- di(phenylcarbonothioyl)malonodihydrazide) or nitroxoline or a pharmaceutically acceptable salt or solvate thereof.

29. The method according to claim 28, wherein the bacterium is resistant to the antibiotic.

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