Combination therapy

Abstract

To provide a combination therapy of an antibiotic and an alkylating agent, and uses thereof as a medicament and for the control of pathogens.SOLUTION: The present invention provides a therapeutic combination for use in treating an infection with a pathogen. The therapeutic combination comprises an aminoglycoside antibiotic and a nucleic acid monoalkylating agent that substitutes a single nucleotide of a nucleic acid with an alkyl group, wherein the therapeutic combination provides an enhanced suppression of a pathogen when compared with an otherwise identical composition lacking the aminoglycoside antibiotic, or wherein the therapeutic combination provides an enhanced suppression of a pathogen when compared with an otherwise identical composition lacking the nucleic acid monoalkylating agent, and kits for executing the method.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to combination therapy of antibiotics and alkylating agents and their use as medicines and for the control of pathogens. [Background technology]

[0002] DNA alkylation is an important regulatory mechanism in eukaryotes, with important functions such as transposable elements (TEs), silencing, and gene regulation. In particular, DNA methylation at the 5th position of cytosine (5-methylcytosine, 5meC) is a key epigenetic mark in eukaryotes, which can be maintained throughout DNA replication via DNA methyltransferase (DNMT) activity.

[0003] Chemotherapy has taken advantage of the fact that certain DNA alkylation events are toxic (e.g., 3-methylcytosine, 3meC) and thus has used alkylating drugs to induce the introduction of DNA damage that results in cytotoxicity in cancer cells. Such chemotherapy has focused almost exclusively on the use of alkylating antineoplastic drugs that usually result in the replacement of guanine by an alkyl group, for example at the seventh nitrogen atom of the purine ring.

[0004] However, such alkylating agents have never been routinely used as antibacterial agents, mainly due to the hazardous nature of representative alkylating agents, which limited their use to prescribed therapies (e.g., cancer therapy). To the inventors' knowledge, there have been no reports of alkylating agents being used as routine treatments for infectious diseases (e.g., bacterial infections), particularly where such treatments do not require dosages that similarly produce the side effects of alkylating agents seen in chemotherapy. In an attempt to demonstrate this, the inventors found that wild-type bacterial growth was not inhibited in the presence of alkylating agents (see Example 1). The latter may be exacerbated by the presence of enzymes in cells (e.g., bacterial cells) that control / reverse toxic DNA alkylation, and doses of alkylating agents that can be affected by such enzymes may be less effective. Indeed, toxic 3-methylcytosine (3meC) alkylation is reversed by AlkB (and its homologs), and we recently discovered that this mechanism appears to have surprisingly co-evolved with DNA methyltransferase activities that result in "off-target" 3meC alkylation (controlled by AlkB) and 5meC alkylation.

[0005] Therefore, there is a need for means and methods to enhance the cytotoxic effects of compositions with alkylating agents, enabling their utility in therapies beyond chemotherapy.

[0006] The present invention addresses one or more of the problems set forth above. Summary of the Invention [Means for solving the problem]

[0007] The present invention is based on the surprising discovery that antibiotics (e.g., aminoglycoside antibiotics) can be used to act synergistically with monofunctional alkylating agents (e.g., monoalkylating agents) resulting in a toxic combination and increased cytotoxic activity following exposure to a combination of these agents. Thus, the seminal discovery of the present invention is that combination therapy can be used not only to increase the efficacy of existing chemotherapeutic agents, but also to target pathogenic infections, pathogens in general. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In one aspect, the present invention provides a therapeutic combination for use in treating an infection by a pathogen, comprising: Antibiotics and b. Nucleic acid monoalkylating agents that replace a single nucleotide of a nucleic acid with an alkyl group (e.g., a methyl group) The present invention provides a therapeutic combination comprising:

[0009] Another aspect of the invention is a method for treating a subject having an infection by a pathogen, comprising administering to the subject a therapeutic combination, said therapeutic combination comprising: a. an antibiotic (preferably an aminoglycoside antibiotic); and b. Nucleic acid monoalkylating agents that replace a single nucleotide of a nucleic acid with an alkyl group (e.g., a methyl group) The present invention provides a method comprising:

[0010] In another embodiment, a. an antibiotic (preferably an aminoglycoside); and B. Nucleic acid monoalkylating agents that replace a single nucleotide in a nucleic acid with an alkyl group Provided is a therapeutic combination (e.g., composition) comprising:

[0011] In one embodiment, the antibiotic is one or more selected from aminoglycoside antibiotics, penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, fluoroquinolone antibiotics, sulfonamide antibiotics, and tetracycline antibiotics, or a combination thereof. In one embodiment, the antibiotic is one or more selected from aminoglycoside antibiotics, penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, fluoroquinolone antibiotics, sulfonamide antibiotics, or a combination thereof.

[0012] In a preferred embodiment, the antibiotic is an aminoglycoside antibiotic.

[0013] In one aspect, the present invention provides a therapeutic combination for use in treating an infection by a pathogen, said therapeutic combination comprising: Aminoglycoside antibiotics and B. Nucleic acid monoalkylating agents that replace a single nucleotide in a nucleic acid with an alkyl group The present invention provides a therapeutic combination comprising:

[0014] In another embodiment, Aminoglycoside antibiotics and B. Nucleic acid monoalkylating agents that replace a single nucleotide in a nucleic acid with an alkyl group Provided is a therapeutic combination (e.g., composition) comprising:

[0015] The following embodiments and definitions may apply to any aspect or embodiment of the invention described herein, for example to any aspect or embodiment of any method, use, composition, therapeutic combination, therapeutic combination for use, pharmaceutical combination for use, kit, or kit for use described herein.

[0016] In one embodiment, the therapeutic combination provides improved inhibition of pathogens when compared to an otherwise identical composition lacking said antibiotic (preferably, the antibiotic is an aminoglycoside antibiotic). In one embodiment, the therapeutic combination provides improved inhibition of pathogens when compared to an otherwise identical composition lacking said nucleic acid monoalkylating agent (which replaces a single nucleotide of a nucleic acid with an alkyl group).

[0017] In one embodiment, the therapeutic combination provides improved inhibition of infection by a pathogen when compared to an otherwise identical composition lacking said antibiotic (preferably, the antibiotic is an aminoglycoside antibiotic). In one embodiment, the therapeutic combination provides improved inhibition of infection by a pathogen when compared to an otherwise identical composition lacking said nucleic acid monoalkylating agent (which replaces a single nucleotide of a nucleic acid with an alkyl group).

[0018] The term "infection by a pathogen" may be used interchangeably with the term "infection caused by a pathogen."

[0019] The term "nucleic acid monoalkylating agent" is used interchangeably herein with the term "monofunctional alkylating agent." The term refers to an agent that replaces a nucleic acid (or more specifically, a nucleotide of said nucleic acid) with an alkyl group, such as a methyl group. Preferably, a nucleic acid monoalkylating agent (e.g., a monofunctional alkylating agent) replaces a single nucleotide with a single alkyl group (e.g., a methyl group).

[0020] A "nuclear monoalkylating agent" as described herein is a nuclear monoalkylating agent that replaces a single nucleotide of a nucleic acid with an alkyl group.

[0021] Nucleotide alkylation caused by such monoalkylating agents is a form of DNA damage that can be permanent, such that the agent causes cytotoxicity. Monoalkylating agents are one broad classification of nucleic acid alkylating agents, and another classification is "bifunctional alkylating agents". Bifunctional alkylating agents usually replace at least two nucleotides with alkyl groups.

[0022] Bifunctional alkylating agents are structurally and functionally distinct from monofunctional alkylating agents. For example, monoalkylating agents usually have a single alkyl group (by said alkyl group) to substitute for a nucleotide, whereas bifunctional alkylating agents usually have at least two such alkyl groups to substitute for a nucleotide. Furthermore, upon contact with DNA, bifunctional alkylating agents usually cause intrastrand and / or interstrand crosslinking, which is understood to be due to the interaction between two different alkyl substituents present on two different nucleotides (e.g., on two different strands).

[0023] Different types of DNA damage caused by such alkylating drugs also require different modes of DNA repair (e.g., different DNA repair pathways) to remove or tolerate the damage. For example, single alkylated nucleotides (monoalkylating drugs), such as 3meC, can be removed (e.g., by base excision repair) or repaired (e.g., via AlkB-catalyzed removal of the alkyl group), allowing cell survival. In contrast, interstrand crosslink repair mechanisms are complex and are understood to be very formidable obstacles to the DNA replication machinery. For example, efficient repair of crosslinked DNA (caused by bifunctional alkylating drugs) can require a combination of FA repair, NER, translesion synthesis (TLS), and HR repair.

[0024] In this way, monoalkylating agents may advantageously be less toxic to the subject in whom the therapeutic combination of the present invention is used (compared to bifunctional alkylating agents), and the subject's cells are usually better able to remove / repair the resulting DNA damage, whereas the target cells (e.g., pathogens) are less able to remove / repair such resulting DNA damage (e.g., when the pathogen lacks an alkb homolog). In this way, the effect of the therapeutic combination may be specific to the pathogen (e.g., bacteria) and may cause less "off-target" effects on the (infected) subject's cells (compared to an identical combination that includes a bifunctional alkylating agent instead of a monoalkylating agent).

[0025] In one embodiment, a nucleic acid monoalkylating agent contains a single alkyl group (eg, a single reactive alkyl group).

[0026] In one embodiment, a nucleic acid monoalkylating agent described herein does not cause DNA intrastrand and / or interstrand crosslinks (eg, upon contact with DNA).

[0027] In one embodiment, the nucleic acid monoalkylating agents described herein are N 1 and / or S N For example, nucleic acid monoalkylating agents are S N Alternatively (or in addition), the nucleic acid monoalkylating agent may be a type 1 drug. N It may also be a type 2 drug.

[0028] The term "treating" may mean that an infection is treated (e.g., an infection is eliminated), suppressed, or reduced, or that the symptoms of an infection are reduced. Preferably, said treating is used to provide a clinical endpoint in which the subject to whom the therapeutic combination of the invention is administered has a lower level of infection than immediately prior to administration of the therapeutic combination.

[0029] The term "treat" or "treating" as used herein encompasses prophylactic treatment (e.g., to prevent the onset of a disease) and corrective treatment (treatment of a subject already suffering from a disease). Preferably, "treat" or "treating" as used herein refers to corrective treatment. The term "treat" or "treating" encompasses treating both a disease and / or infection as well as symptoms thereof. In some embodiments, "treat" or "treating" refers to the symptoms of a disease and / or infection.

[0030] As such, the therapeutic combinations of the present invention may be administered to a subject in therapeutically effective or prophylactically effective amounts.

[0031] A "therapeutically effective amount" is any amount of a therapeutic combination that, when administered alone or in combination to a subject for treating a disease and / or infection (or a symptom thereof), is sufficient to effect said treatment of the disease and / or infection or a symptom thereof.

[0032] A "prophylactically effective amount" is any amount of a therapeutic combination that, when administered alone or in combination to a subject, inhibits or delays the onset or recurrence of a disease and / or infection (or symptoms thereof). In some embodiments, a prophylactically effective amount prevents the onset or recurrence of a disease and / or infection altogether. "Inhibiting" onset means reducing the likelihood of onset of a disease and / or infection (or symptoms thereof) or preventing onset altogether.

[0033] Without wishing to be bound by theory, it is believed that antibiotics (aminoglycoside antibiotics) enhance the activity of nucleic acid monoalkylating drugs, or vice versa. Thus, cells or pathogens (e.g., bacteria) treated with a combination of these drugs (e.g., pharmaceuticals) undergo increased levels of nucleic acid (e.g., nucleotide) alkylation (e.g., causing 3meC) compared to cells or pathogens treated with monoalkylating drugs alone, and cell or pathogen growth is consequently inhibited due to toxic nucleic acid damage. Thus, treatment with antibiotics (e.g., aminoglycoside antibiotics) may increase the sensitivity of cells / pathogens to monoalkylating drugs and improve their efficacy (or vice versa). This synergy is not limited to bacterial cells that are sensitive to aminoglycoside antibiotics (e.g., via a conical mechanism), but has actually been observed in bacterial cells that are resistant (e.g., containing genes that confer resistance) to the exemplary aminoglycoside antibiotics tested (see Example 1).

[0034] Thus, in one embodiment, there is provided the use of an antibiotic to enhance alkylation of a nucleic acid (e.g., of a nucleotide of said nucleic acid) by a nucleic acid monoalkylating agent that replaces a single nucleotide with an alkyl group, said nucleic acid being either RNA or DNA (preferably DNA).

[0035] In one embodiment, there is provided a use of an aminoglycoside antibiotic to enhance alkylation of a nucleic acid (e.g., of a nucleotide of said nucleic acid) by an additional nucleic acid monoalkylating agent, said nucleic acid being either RNA or DNA (preferably DNA).

[0036] This newly discovered utility for antibiotics (e.g., aminoglycoside antibiotics) that improve (e.g., synergistically improve) the activity of nucleic acid monoalkylating drugs is particularly surprising, since aminoglycoside antibiotics, for example, have been used up until now mainly as disruptors of ribosomal activity (a mechanism other than inducing cytotoxicity). Thus, the inventors have not only identified a new technical field of application (i.e., beyond the field of conventional antibiotic therapy), but also an unexpected technical effect in said field (i.e., improving the activity of nucleic acid monoalkylating drugs).

[0037] Advantageously, this combination therapy (therapeutic combination for use) provides for the deliberate use of such antibiotics to target cells / organisms (e.g., non-bacterial cells / organisms or antibiotic-resistant bacteria) that are normally inactive (non-responsive) to such antibiotics. Furthermore, due to the synergistic nature of the combination, lower doses of antibiotics and alkylating agents may be used, thus reducing the risk of development of antibiotic resistance (a persistent threat to public health) due to overuse. Indeed, the present invention reduces the need to prescribe long-term antibiotic regimens for immunocompromised subjects.

[0038] Because many antibiotics are relatively inexpensive, the improved therapeutic combinations of the present invention represent a significant cost savings to public health agencies and patients due to the reduction in the amount of typically expensive monoalkylating drugs required for therapy.

[0039] Furthermore, the present invention allows for the clinical use of antibiotics that are rarely used due to their toxicity at conventional doses, and in effect, therapeutic combinations broaden the use of such antibiotics (by lowering the required therapeutic dose).

[0040] The order of dosing / administration of the components of the combination therapy can be changed. The antibiotic (e.g., aminoglycoside antibiotic) and the nucleic acid monoalkylating agent can be administered simultaneously (e.g., both at their specific optimal doses to achieve synergy) as part of a single composition or in separate compositions. For example, the monoalkylating agent can be present in a first composition (e.g., for intravenous administration to a subject) and the antibiotic can be present in a second composition (e.g., for oral administration to a subject).

[0041] Additionally, the antibiotic (preferably an aminoglycoside antibiotic) and the nucleic acid mono-alkylating agent may be administered at different times (e.g., the aminoglycoside antibiotic may be administered first to increase the sensitivity of the cells to the alkylating agent). Thus, in a further embodiment, the aminoglycoside antibiotic and the nucleic acid mono-alkylating agent are administered to the subject at different times in separate compositions.

[0042] In one embodiment, the antibiotic (preferably an aminoglycoside antibiotic) is administered prior to the nucleic acid monoalkylating agent. In one embodiment, the antibiotic is administered simultaneously with the nucleic acid monoalkylating agent. In one embodiment, the antibiotic is administered subsequent to the monoalkylating agent.

[0043] In one embodiment, the aminoglycoside antibiotic is administered prior to the nucleic acid monoalkylating agent. In one embodiment, the aminoglycoside antibiotic is administered simultaneously with the nucleic acid monoalkylating agent. In one embodiment, the aminoglycoside antibiotic is administered subsequent to the nucleic acid monoalkylating agent.

[0044] Suitably, the therapeutic combination is administered to a subject. The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammalian subject. In one embodiment, the "subject" is a human, a companion animal (e.g., a pet such as a dog, cat, and / or rabbit), a livestock animal (e.g., a pig, sheep, cow, and / or goat), and / or a horse. In a preferred embodiment, the subject is a human.

[0045] In one embodiment, the infection is an acute infection. In one embodiment, the infection is a chronic infection.

[0046] In the method of the present invention, the subject may not have been previously diagnosed as having an infectious disease. Alternatively, the subject may have been previously diagnosed as having an infectious disease. The subject may also be a subject that exhibits disease risk factors or has no symptoms of an infectious disease. The subject may also be a subject suffering from an infectious disease or at risk of contracting an infectious disease. In one embodiment, the subject has previously been administered a therapy for an infectious disease.

[0047] Advantageously, given the synergistic nature of the therapeutic combinations of the present invention, minimal doses of antibiotics may be administered, allowing for greater flexibility in the use of antibiotics (e.g., aminoglycoside antibiotics) that have traditionally been prescribed only for specific uses and / or administrations.

[0048] Suitably, a dose of the therapeutic combination may be chosen that balances the efficacy of the therapeutic combination to suppress infection with damage to non-infected cells of the host (e.g., healthy cells of an infected subject). Preferably, a dose of the therapeutic combination that is not toxic to a human subject (e.g., to human cells) may be used. This is particularly advantageous since some antibiotics can cause side effects at high doses - for example, certain aminoglycoside antibiotics (e.g., kanamycin) cause ototoxicity at high doses. Thus, the therapeutic combination of the present invention may be used to prevent the risk of ototoxicity occurring.

[0049] To illustrate the breadth of usefulness of antibiotics (e.g., aminoglycoside antibiotics) and nucleic acid monoalkylating agents when used as a synergistic combination, the present invention encompasses combinations of antibiotics (e.g., aminoglycoside antibiotics) and nucleic acid monoalkylating agents for general use, e.g., as disinfectants, to inhibit pathogens. Synergy between the components means that a relatively dilute composition / solution (or any other formulation) containing relatively low concentrations of antibiotics and nucleic acid monoalkylating agents (or alkylating-like agents) may be administered to a pathogen and still achieve a similar (or better) level of inhibition that would otherwise be possible only with much higher concentrations of the individual components.

[0050] In one aspect, the use of a drug combination as a disinfectant (preferably an in vitro disinfectant), said drug combination (e.g. a disinfectant) comprising: Antibiotics and B. Nucleic acid monoalkylating agents that replace a single nucleotide in a nucleic acid with an alkyl group Uses are provided, including

[0051] In one embodiment, the use of a drug combination as a disinfectant, said drug combination (e.g., a disinfectant) comprising: Aminoglycoside antibiotics and B. Nucleic acid monoalkylating agents that replace a single nucleotide in a nucleic acid with an alkyl group Uses are provided, including

[0052] In one embodiment, the drug combination provides improved inhibition of a pathogen when compared to an otherwise identical composition lacking said aminoglycoside antibiotic. Additionally or alternatively, the drug combination may provide improved inhibition of a pathogen when compared to an otherwise identical composition lacking said nucleic acid monoalkylating agent.

[0053] Suitably, the use may include dispensing the disinfectant (e.g. therapeutic combination) to a location containing, suspected of containing, or at risk of containing a pathogen.

[0054] The term "disinfectant" refers to a formulation (e.g., a composition or solution) that inhibits pathogens. Such use as a disinfectant is not limited to use to destroy the cell walls of pathogens (e.g., microorganisms) or to interfere with the metabolism of pathogens. Indeed, the present invention provides a convenient means of disinfection by causing toxic alkylating damage to the nucleic acids of pathogens, thus widening the range of available disinfecting solutions.

[0055] The term "inhibit" or "inhibiting" may mean "inhibiting the growth of" or "killing" a pathogen (or other target cell). The term "inhibit" or "inhibiting" is synonymous with the term "interfering with the growth of" a pathogen (or other target cell / organism). In one embodiment, the therapeutic combination of the invention may "kill" or may be "used to kill" a pathogen (or other target cell / organism). The term "inhibiting" also encompasses preventing the growth of a pathogen (or other target cell / organism), for example, when administered as a pretreatment to prevent contamination by a pathogen (or other target cell / organism).

[0056] In a further aspect, an in vitro method for inhibiting the growth of a pathogen comprises administering to a locus a disinfectant comprising a combination of an antibiotic and a nucleic acid monoalkylating agent (which replaces a single nucleotide of a nucleic acid with an alkyl group), said locus being: i. Location containing pathogens ii. Any location suspected of containing a pathogen; or iii. Locations that pose a risk of containing pathogens An in vitro method is provided, wherein

[0057] In a further aspect, an in vitro method for inhibiting the growth of a pathogen comprises administering to a locus an antiseptic comprising a combination of an aminoglycoside antibiotic and a nucleic acid monoalkylating agent (which substitutes a single nucleotide with an alkyl group), said locus comprising: i. Location containing pathogens ii. Any location suspected of containing a pathogen; or iii. Locations that pose a risk of containing pathogens An in vitro method is provided, wherein

[0058] In one embodiment, the drug combination and / or disinfectant of the present invention may be dispensed or used on one or more locations selected from medical equipment, bedding, furniture, walls, or floors, such as hospital floors, or combinations thereof.

[0059] Suitable medical instruments may be one or more selected from graspers, forceps and / or occluders (e.g., for blood vessels and other organs), needle holders (e.g., for holding suture needles), retractors (e.g., for opening skin), mechanical cutters (e.g., scalpels, lancets, drill bits, files, trocars, Ligasures, ultrasonic vibrating scalpels, surgical scissors, rongeurs, etc.), injection needles, intubators, or combinations thereof.

[0060] In one embodiment, the use or method of the invention comprises providing a local concentration of an antibiotic (preferably an aminoglycoside antibiotic) and a nucleic acid monoalkylating agent at a location of about 1 mM to about 200 mM (e.g., about 20 mM to 180 mM, about 40 mM to 160 mM, about 60 mM to 140 mM, or about 80 mM to 120 mM).

[0061] Suitably, a local concentration of antibiotic (preferably an aminoglycoside antibiotic) at about 80 mM to about 120 mM (e.g., about 85 mM to 115 mM, about 90 mM to 110 mM, or about 95 mM to 100 mM) may be provided. In one embodiment, a local concentration of nucleic acid monoalkylating agent at about 1 mM to about 40 mM (e.g., about 5 mM to 35 mM, 10 mM to 30 mM, or 15 mM to 25 mM) may be provided.

[0062] In a preferred embodiment, a local concentration of an antibiotic (preferably an aminoglycoside antibiotic) at a location of about 80 mM to about 120 mM (e.g., about 85 mM to 115 mM, about 90 mM to 110 mM, or about 95 mM to 100 mM) and a local concentration of an alkylating agent at a location of about 1 mM to about 40 mM (e.g., about 5 mM to 35 mM, 10 mM to 30 mM, or 15 mM to 25 mM) may be provided.

[0063] In one aspect, there is provided a use of a nucleic acid monoalkylating agent to improve inhibition of a pathogen by an antibiotic (preferably an aminoglycoside antibiotic), wherein the nucleic acid monoalkylating agent is a nucleic acid monoalkylating agent that replaces a single nucleotide of a nucleic acid with an alkyl group.

[0064] In one aspect, there is provided a use of an antibiotic (preferably an aminoglycoside antibiotic) to improve inhibition of a pathogen by a nucleic acid monoalkylating agent, the nucleic acid monoalkylating agent being a nucleic acid monoalkylating agent that replaces a single nucleotide of a nucleic acid with an alkyl group.

[0065] In one aspect, there is provided a use of a nucleic acid monoalkylating agent to improve the bactericidal activity of an antibiotic, wherein the nucleic acid monoalkylating agent is a nucleic acid monoalkylating agent that replaces a single nucleotide of a nucleic acid with an alkyl group.

[0066] In one aspect, there is provided a use of a nucleic acid monoalkylating agent to improve the bactericidal activity of an aminoglycoside antibiotic, wherein the nucleic acid monoalkylating agent is a nucleic acid monoalkylating agent that replaces a single nucleotide of a nucleic acid with an alkyl group.

[0067] In another aspect, there is provided a use of an antibiotic to promote (e.g., enhance) the introduction of alkylating lesions (e.g., 3meC) into a nucleic acid by a nucleic acid monoalkylating agent, where the nucleic acid monoalkylating agent replaces a single nucleotide of a nucleic acid with an alkyl group.

[0068] In another aspect, there is provided a use of an aminoglycoside antibiotic to promote (e.g., enhance) the introduction of alkylating lesions (e.g., 3meC) into a nucleic acid by a nucleic acid monoalkylating agent, where the nucleic acid monoalkylating agent replaces a single nucleotide with an alkyl group.

[0069] The therapeutic combination or antiseptic of the present invention may suitably be provided as part of a kit, which may, for example, be labelled or include instructions outlining the proper use of the kit.

[0070] In one embodiment, Antibiotics and B. Nucleic acid monoalkylating agents that replace a single nucleotide in a nucleic acid with an alkyl group A kit is provided comprising:

[0071] In one embodiment, Aminoglycoside antibiotics and B. Nucleic acid monoalkylating agents that replace a single nucleotide in a nucleic acid with an alkyl group A kit is provided comprising:

[0072] The kit may further comprise a case for containing an antibiotic (preferably an aminoglycoside antibiotic) and a nucleic acid monoalkylating agent.

[0073] In one embodiment, the kit may be used to treat an infection caused by a pathogen. In another embodiment, the kit may be used as a disinfectant. In a further embodiment, the kit may be used in an in vitro method for inhibiting a pathogen.

[0074] In one embodiment, the aminoglycoside antibiotic and the nucleic acid monoalkylating agent are contained within the same composition. In another embodiment, the aminoglycoside antibiotic and the nucleic acid monoalkylating agent are contained within separate compositions.

[0075] In one embodiment, the kit may include instructions for using the kit in a method for treating an infection by a pathogen, such as the optimal dose of the therapeutic combination to be administered, the optimal number of times to administer the therapeutic combination, the optimal method of administration, or any other information relevant to using the therapeutic combination of the present invention to treat a pathogenic infection (e.g., to treat an infectious disease).

[0076] The instructions may be provided as part of a label or as part of an instruction manual provided with the kit or separately.

[0077] In one embodiment, the kit may include instructions for using the kit as a disinfectant, such as the optimal concentration of the therapeutic combination to be administered as a disinfectant, the optimal number of times to administer the disinfectant, the optimal method of administration, or any other information related to using the therapeutic combination of the present invention as a disinfectant.

[0078] In one embodiment, the kit may include instructions for using the kit in an in vitro method for inhibiting a pathogen.

[0079] The kit provided for use as a disinfectant or in an in vitro method for inhibiting pathogens may suitably be a ready-to-use (RTU) kit. Thus, in one embodiment, the kit may be an RTU kit for use as a disinfectant or in an in vitro method for inhibiting pathogens. Disinfectants (including, for example, aminoglycoside antibiotics and nucleic acid monoalkylating agents) may be administered by spraying, injecting, painting, or rubbing the disinfectant into the location of interest.

[0080] The therapeutic combination for infection may also be provided in materials for protecting or suturing a wound. For example, the therapeutic combination of the present invention may be provided in a wound dressing (e.g., a bandage and / or plaster), a gauge, a suture, or a combination thereof.

[0081] Because nucleic acid alkylation damage can result in toxicity in many organisms / microorganisms, the present invention surprisingly enables the use of aminoglycoside antibiotics in organisms beyond bacteria.

[0082] Thus, in one embodiment, the pathogen may be one or more selected from a bacterium, a virus, a protozoan, a fungus, a helminth, or a combination thereof.

[0083] In one embodiment, the pathogen may be one or more selected from bacteria, protozoa, fungi, helminths, or combinations thereof.

[0084] In a preferred embodiment, the pathogen is a bacterium.

[0085] The bacteria may be one or more selected from Salmonella spp., Pseudomonas spp., Mycobacterium spp., Escherichia spp., Proteus spp., Serratia spp., Klebsiella spp., Acinetobacter spp., or combinations thereof.

[0086] Preferably, the pathogen is one or more selected from Salmonella spp., Pseudomonas spp., Mycobacterium spp., or combinations thereof.

[0087] The Salmonella species may be one or more selected from S. bongori, S. enterica, or a combination thereof. Suitably, the Salmonella species may be S. enterica (e.g. S. enterica serovar Typhimurium).

[0088] The Pseudomonas species may be one or more selected from P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, or combinations thereof. Suitably, the Pseudomonas may be P. aeruginosa (eg P. aeruginosa PAO1).

[0089] The Mycobacterium species may be one or more selected from M. africanum, M. bovis, M. bovis BCG, M. canetti, M. caprae, M. microti, M. mungi, M. orygis, M. pinnipedii, M. suricattae, M. tuberculosis. Suitably, the Mycobacterium may be M. tuberculosis.

[0090] The Escherichia species may be one or more selected from E. albertii, E. coli, E. fergusonii, E. hermannii, E. marmotae, E. vulneris, or combinations thereof. Suitably, the Escherichia species may be E. coli.

[0091] The Proteus species may be one or more selected from P. hauseri, P. mirabilis, P. myxofaciens, P. penneri, P. vulgaris, or a combination. Suitably, the Proteus species may be P. vulgaris.

[0092] The Serratia species may be S. marcescens.

[0093] The Klebsiella species may be one or more selected from K. aerogenes, K. granulomatis, K. oxytoca, K. michiganensis, K. pneumoniae, K. quasipneumoniae, K. grimontii, K. variicola, or combinations thereof. Suitably, the Klebsiella species may be K. pneumoniae.

[0094] The Acinetobacter species include A. albensis, A. apis, A. baumannii, A. beijerinckii, A. bereziniae, A. bohemicus, A. boissieri, A. bouvetii, A. brisouii, A. calcoaceticus, A. A. celticus, A. colistiniresistens, A. courvalinii, A. defluvii, A. disperses, A. dijkshoorniae, A. equi, A. gandensis, A. gerneri, A. guangdongensis, A. guillouiae, A. guillouiae, A. gyllenbergii, A. haemolyticus, A. harbinensis, A. indicus, A. junii, A. kookii, A. lactucae, A. larvae, A. lwoffii, A. modestus, A. nectaris, A. nosocomialis .nosocomialis, A.parvus, A.pakistanensis, A.populi, A.proteolyticus, A.pittii, A.piscicola, A.pragensis, A.proteolyticus, A.puyangensis, A.qingfengensis, A.The Acinetobacter species may be one or more selected from A. radioresistens, A. rudis, A. schindleri, A. seifertii, A. soli, A. tandoii, A. tjernbergiae, A. towneri, A. ursingii, A. variabilis, A. venetianus, A. vivianii, or combinations thereof. Suitably, the Acinetobacter species may be A. baumannii.

[0095] In one embodiment, the pathogen is a fungus. Suitably, the fungus is a Candida species.

[0096] The Candida species include C. albicans, C. ascalaphidarum, C. amphixiae, C. Antarctica, C. argentea, C. atlantica, C. atmosphaerica, C. auris, C. blattae, C. bromeliacearum, C. brome ... arum, C.carpophila, C.carvajalis, C.cerambycidarum, C.chauliodes, C.corydalis, C.dosseyi, C.dubliniensis, C.ergatensis, C.fructus, C.glabrata, C.fermentatii entati, C. guilliermondii, C. haemulonii, C. humilis, C. insectamens, C. insectorum, C. intermedia, C. jeffresii, C. kefyr, C. keroseneae, C. krusei, C. lusitaniae aniae, C. lyxosophila, C. maltose, C. marina, C. membranifaciens, C. mogii, C. oleophila, C. oregonensis, C. parapsilosis, C. quercitrusa, C. rugose, C. sake, C. shehatea, C.The Candida species may be one or more selected from C. shehatea, C. temnochilae, C. tenuis, C. theae, C. tolerans, C. tropicalis, C. tsuchiyae, C. sinolaborantium, C. sojae, C. subhashii, C. viswanathii, C. utilis, C. ubatubensis, and C. zemplinina. Suitably, the Candida species may be C. albicans.

[0097] In another embodiment, the pathogen is a helminth. The helminth may be one or more selected from the phyla Annelida, Platyhelminthes, Nematoda, and Acanthocephala.

[0098] In one embodiment, the helminth is a parasitic flatworm. The parasitic flatworm may be one or more selected from the classes Cestoda, Trematoda, and Monogenea.

[0099] In one embodiment, the helminth is a parasitic nematode, which may be one or more selected from roundworms (Ascaris), filariasis, hookworms, pinworms (Enterobius), and whipworms (Trichuris trichiura).

[0100] In a preferred embodiment, the pathogen lacks one or more genes encoding a polypeptide that removes an alkyl group from an alkylated nucleotide adduct. For example, the pathogen may lack an alka or alkb gene that encodes a polypeptide that removes an alkyl group from an alkylated nucleotide adduct. In a preferred embodiment, the pathogen may lack an alkb gene that encodes a polypeptide that removes an alkyl group from an alkylated nucleotide adduct.

[0101] Reference to a pathogen or cell lacking one or more genes described herein includes pathogens or cells that contain a mutated form of said gene that encodes a non-functional polypeptide (or a polypeptide with reduced function).

[0102] In one embodiment, the alkylated nucleotide adduct is one or more selected from 3meC, 3meG, 3meA, 8-oxo-G, O2-alkylthymines, O4-alkylthymines, O6-methylguanine, and O6-ethylguanine. In one embodiment, the alkylated nucleotide adduct is one or more selected from 3meC, 3meG, 3meA, O2-alkylthymines, O4-alkylthymines, O6-methylguanine, and O6-ethylguanine.

[0103] The AlkB protein is a protein involved in the direct repair of alkylation damage (including the repair of 3meC, as explained by the inventors). The AlkB gene was originally discovered in E. coli. Genes that express proteins with high sequence identity to the E. coli AlkB gene and / or have the same or similar function are called "AlkB homologs". Thus, the term alkB as used herein includes alkB homologs. Thus, reference to a pathogen or cell lacking alkb includes a pathogen or cell lacking its homolog (alkb homolog). Similarly, reference to a pathogen or cell lacking alkb includes a cell lacking its homolog (alkb homolog). The term alka also includes alka homologs.

[0104] In one embodiment, the pathogen may lack one or more genes encoding a polypeptide that promotes cellular resistance to the presence of alkylated nucleotide adducts in the nucleic acid of the cell. For example, the pathogen may lack the Ada, OGT, RecA, DinB, and / or UmuCD genes that encode polypeptides that promote cellular resistance to the presence of alkylated nucleotide adducts in the nucleic acid of the cell.

[0105] Ada and OGT are enzymes that repair 8-oxoguanine (8-oxo-G), which results from alkylation damage to guanine; AlkA repairs 3-methylguanine (3meG) and 3-methyladenine (3meA); RecA promotes tolerance of DNA damage; and DinB and UmuCD (translesion DNA polymerase) enable cells to bypass alkylation damage.

[0106] As used herein, the terms Ada, OGT, RecA, DinB, and UmuCD include Ada, OGT, RecA, DinB, and UmuCD homologues, respectively.

[0107] The Escherichia coli AlkB protein is an iron- and 2-oxoglutarate-dependent oxygenase that is primarily responsible for repairing 1-methyladenine and 3-methylcytosine lesions in DNA (e.g., removing alkyl groups from nucleic acids, as shown in Figure 2). AlkB homologs also exist in many prokaryotic and eukaryotic species. For example, nine human homologs of AlkB are known to exist, namely, Alkb homolog 1 (NCBI Gene ID: 8846), histone h2a dioxygenase, ALKBH2 (GeneID: 121642), ALKBH3 (GeneID: 221120), ALKBH4 (NCBI GeneID: 54784), ALKBH5 (NCBI GeneID: 54890), ALKBH6 (NCBI GeneID: 84964), ALKBH7 (NCBI GeneID: 84266), ALKBH8 (NCBI GeneID: 91801), and FTO (NCBI GeneID: 79068).

[0108] DNMTs (DNA methyltransferases) are enzymes that catalyze the alkylation (eg, methylation) of nucleic acids (eg, at CpG sites in DNA).

[0109] For example, the nucleotides of the nucleic acid may be alkylated to result in nucleotide additions such as 5meC and / or 3meC. [ka]

[0110] The inventors have recently discovered that organisms in which DNA is known to be methylated (e.g. as part of epigenetic control of gene regulation) often have homologs of the bacterial enzyme AlkB (e.g. ALKB2) and that such homologs are lost in organisms (or species) that do not have DNA methylation activity.

[0111] Interestingly, P. aeruginosa has lost AlkB relatively recently in evolution and may therefore be a particularly suitable target for the therapeutic combination of the present invention. Because P. aeruginosa represents a major health problem for subjects with chronic lung disease (e.g., cystic fibrosis), the therapeutic combination of the present invention may be particularly suitable for treating infections in such patients. Thus, in one embodiment, the therapeutic combination may be used to treat pathogen infections in patients with chronic lung disease, such as cystic fibrosis.

[0112] Similarly, the therapeutic combinations of the invention may be particularly suitable for treating tuberculosis (TB) because M. tuberculosis lacks an AlkB homolog and is able to alkylate (e.g., methylate) cytosines at the 3' position.

[0113] Any pathogen (e.g. infectious organism) lacking a gene for control / reversal / resistance to nucleic acid alkylation (e.g. monoalkylation) may be particularly susceptible to attack by the therapeutic combination of the invention. Thus, in one embodiment, the pathogen (e.g. bacteria, viruses, protozoa, fungi, and / or helminths) targeted by the therapeutic combination of the invention may lack a gene selected from AlkA, AlkB, Ada, OGT, RecA, DinB, UmuCD, or a combination thereof. Suitably, the pathogen lacks the AlkB gene.

[0114] Thus, subjects with a pathogenic infection may be stratified based on whether the pathogen of the infection lacks one or more of the above genes, and said subjects may be selected as particularly suitable recipients of the therapeutic combination of the present invention.

[0115] Reference to the E. coli genes AlkA, AlkB, Ada, OGT, RecA, DinB, UmuCD includes homologues thereof (e.g., homologues present in different domains, phyla, classes, orders, families, or genera). The skilled artisan will be aware of suitable tools and techniques for identifying and / or classifying homologous genes (see, e.g., the "Sequence Identity" paragraph below).

[0116] An exemplary Alkb sequence is provided in SEQ ID NO: 1 (corresponding to NCBI Reference Sequence: NC_000913.3). An Alkb homologue may exhibit at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to the sequence of SEQ ID NO: 1. For example, an Alkb homologue may exhibit at least 80% or at least 90% sequence identity to the sequence of SEQ ID NO: 1. Suitably, an Alkb homologue may exhibit at least 90% sequence identity to the sequence of SEQ ID NO: 1.

[0117] In a preferred embodiment, the pathogen (eg, a bacterium, a virus, a protozoan, a fungus, and / or a helminth) targeted by the therapeutic combination of the present invention may lack AlkB or a homolog thereof.

[0118] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more metazoans selected from Trichoplax adherens, Schistosoma mansoni, Brugia malayi, and Ixodes scapularis.

[0119] Non-pathogenic metazoan organisms to which the therapeutic combinations of the present invention may be administered include Enoplus brevis, Caenorhabditis elegans, Hypsibium dujardini, Daphnia magna, Apis mellifera, Dendroctonus ponderosae, Danaus plexippus, and Drosophila melanogaster.

[0120] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more protozoans selected from Entamoeba histolytica, Trichomonas vaginalis, Blastocystis hominis, Leishmania species, and Giardia lamblia.

[0121] Non-pathogenic protist organisms to which the therapeutic combinations of the present invention may be administered include Fonticula alba, Dictyostelium discoideum, Chlamydomonas reinhardtii, Crytomonas paramedium, Paulinella chromatophora, Nannochloropsis gaditana, and Tetrahymena species.

[0122] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is selected from the group consisting of Encephalitozoon cuniculi, Nosema apis, Namema ceranae, Vittaforma carneae, Enterocytosoan bieneusi, Spraguea lophii, Vavra culiculis, Edharzardia aedes, Nematocida parisii, Razella spp., Parasitella parasitica, Lichteimia ramosei, and the like. ramose, Sporisorium scitamineum, Trametes versicolor, and Punctularia strigosozonata.

[0123] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more ascomycetes selected from Drechslera stenobrocha and Candida albicans, or a combination thereof.

[0124] Non-pathogenic Ascomycete organisms to which the therapeutic combination of the present invention may be administered include Schizosaccharomyces pombe and Saccharomyces cerevisiae, or combinations thereof.

[0125] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more Actinobacteria selected from Mycobacterium tuberculosis NCGM2209, Mycobacterium leprae TN, Tropheryma whipplei TW08 / 27, and Mycobacterium ulcerans Agy99, or a combination thereof.

[0126] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more Alphaproteobacteria selected from Rickettsia rickettsii strain Hauke, Ehrlichia canis strain Jake, Bartonella henselae strain Houston-1, Ehrlichia chaffeensis strain Arkansas, and Bartonella quintana strain Toulouse, or a combination thereof.

[0127] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is selected from the group consisting of Streptococcus pneumoniae GA04375, Streptococcus agalactiae BSU247, Staphylococcus aureus subsp. aureus MSHR1132, Staphylococcus epidermidis NIH051668, Listeria monocytogenes FSL J2-071, Enterococcus faecalis ERV103, Enterococcus faecium TX0133C, Listeria innocua Clip11262, Bacillus anthracis. and one or more Bacilli selected from Bacillus anthracis strain A0442, Bacillus cereus Rock1-3, Streptococcus pyogenes MGAS315, and Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305, or a combination thereof.

[0128] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is Bacteroides fragilis HMW 615.

[0129] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more Betaproteobacteria selected from Neisseria meningitidis M04-240196 and Neisseria gonorrhoeae PID1, or a combination thereof.

[0130] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more Chlamydiae selected from Chlamydophila pneumoniae J138, Chlamydia trachomatis F / SotonF3, and Chlamydia psittaci NJ1, or a combination thereof.

[0131] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more Clostridia selected from Clostridium botulinum strain A ATCC 3502, Clostridium difficile NAP08, Clostridium perfringens strain 13, and Clostridium tetani E88, or a combination thereof.

[0132] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is selected from the group consisting of Acinetobacter baumannii ATCC 17978, Pseudomonas aeruginosa LESB58, Haemophilus influenzae 22.4-21, Francisella tularensis subsp. tularensis SCHU S4, Stenotrophomonas maltophilia R551-3, Yersinia pseudotuberculosis IP 31758, Yersinia pestis biovar Orientalis strain BA200901799, Legionella pneumophila pneumophila strain Lens, Stenotrophomonas species SKA14, and Vibrio cholera CIRS 101, or a combination thereof.

[0133] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more Mollicutes selected from Ureaplasma urealyticum serotype 4 strain ATCC 27816 and Mycoplasma pneumoniae M129, or a combination thereof.

[0134] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is one or more Proteobacteria selected from Campylobacter jejuni subsp. jejuni LMG 23218 and Helicobacter pylori South Africa 7, or a combination thereof.

[0135] In one embodiment, the pathogen (preferably a pathogen lacking an AlkB homolog) is Treponema pallidum subsp. pallidum strain Nichols, Leptospira santarosai strain 2000027870, Borrelia burgdorferi 29805, Leptospira interrogans serovar Copenhageni strain Fiocruz LV2791, Leptospira weilii serovar Topaz strain LT2116, Leptospira noguchii strain 2006001870, Borrelia afzelii afzelii PKo, Borrelia recurrentis A1, and Borrelia garinii PBr, or a combination thereof.

[0136] For the avoidance of doubt, the pathogen may contain an AlkB homologue. Given the protective role of AlkB / AlkB homologues (in removing alkylating damage from nucleic acids) and the alkylating (e.g. methylating) role of DNMT enzymes (e.g. DNMT1 and / or DNMT2), pathogens with high levels of DNMT expression relative to AlkB expression may be particularly susceptible to the therapeutic combination of the present invention. The skilled artisan will understand methods for determining gene expression, which may be achieved, for example, via microarray analysis, reverse transcriptase polymerase chain reaction (RT-PCT), Northern blot, Serial Analysis of Gene Expression (SAGE), fluorescent in situ hybridization (FISH), and / or RNA-sequencing (RNA-seq).

[0137] The gene expression may be absolute gene expression or relative gene expression. Determining the relative expression may involve analyzing the expression of DNMT genes and / or the expression of AlkB homolog genes compared to the expression of a suitable standard / reference gene (e.g. a housekeeping gene).

[0138] In one embodiment, the pathogen comprises a high ratio of DNMT gene expression to AlkB homolog gene expression, hi one embodiment, the ratio of DNMT gene expression to AlkB homolog gene expression is selected from one or more of about 2:1, about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, and about 50:1.

[0139] Furthermore, many strains of bacteria (especially M. tuberculosis) have been reported to be resistant to certain aminoglycoside antibiotics (especially kanamycin), thus making such resistant strains particularly suitable targets, as such antibiotics may be purposefully used as part of the therapeutic combination of the invention (e.g., improving the therapeutic combination).

[0140] The assessment of "improved pathogen inhibition" is demonstrated by reference to the following examples and may be assessed using the techniques described in the examples (e.g., Example 1). For example, Example 1 describes a method to measure the number of colony forming units (CFU) in samples treated with the therapeutic combination along with samples not treated with one (or neither) of the components of the therapeutic combination. This allows for a direct comparison of pathogen inhibition between the therapeutic combination of the present invention and an otherwise identical composition lacking an aminoglycoside antibiotic or a nucleic acid monoalkylating agent (or both).

[0141] In one embodiment, inhibition of B cell malignancies is considered to be improved if the "CFU" value obtained for a combination of two primary active compounds (e.g., an aminoglycoside antibiotic and a nucleic acid monoalkylator) is lower than the "CFU" value obtained in the absence (but otherwise under identical conditions) of the aminoglycoside antibiotic or nucleic acid monoalkylator of the invention.

[0142] Thus, in one embodiment, improved pathogen inhibition may be determined by comparing the "CFU" value obtained for a combination of an aminoglycoside antibiotic and a nucleic acid monoalkylator of the present invention with the "CFU" value obtained for the same formulation / composition in the absence of the aminoglycoside antibiotic or nucleic acid monoalkylator of the present invention under the same conditions.

[0143] In one embodiment, the present invention provides a CFU value that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% (preferably at least 5%, 10%, 15%, 20%, 25%, 30%; more preferably at least about 10%) lower than the CFU value provided by the same formulation (e.g., composition) absent the aminoglycoside antibiotic or absent the nucleic acid monoalkylating agent of the present invention but under otherwise the same conditions.

[0144] In one embodiment, the therapeutic combination of the present invention may exhibit synergistic inhibition of pathogens.

[0145] As used herein, the term "synergistic" means that the inhibition of the depicted pathogens is greater than the sum of their parts. In other words, the inhibition of the pathogens is more than additive.

[0146] Any cell (e.g., tumor) or organism in which nucleic acid (e.g., DNA) may be alkylated may be targeted by the therapeutic combination of the present invention. Thus, the therapeutic combination of the present invention may be advantageously used to inhibit tumors in subjects suffering from cancer.

[0147] More particularly, the therapeutic combination of the present invention may further be used to increase the efficacy of chemotherapy involving administration of a nucleic acid monoalkylating agent, which is highly surprising since aminoglycosides have not traditionally been used as part of (e.g., as an adjunct to) such cancer therapy.

[0148] Thus, in a further embodiment, there is provided a therapeutic combination for use in treating cancer, comprising: Antibiotics and b. Additional nucleic acid monoalkylating agents that substitute a single nucleotide of a nucleic acid with an alkyl group. A therapeutic combination is provided comprising:

[0149] In another embodiment, a therapeutic combination for use in treating cancer, comprising: Aminoglycoside antibiotics and b. Additional nucleic acid monoalkylating agents that substitute a single nucleotide of a nucleic acid with an alkyl group. A therapeutic combination is provided comprising:

[0150] In one embodiment, the therapeutic combination provides improved inhibition of cancer (preferably cancer cells, more preferably tumors) when compared to an otherwise identical composition lacking said aminoglycoside antibiotic. In one embodiment, the therapeutic combination provides improved inhibition of cancer (preferably cancer cells, more preferably tumors) when compared to an otherwise identical composition lacking said nucleic acid monoalkylating agent.

[0151] Suitably, the therapeutic combination may be administered to a subject. The subject may not have been previously diagnosed as having cancer. Alternatively, the subject may have been previously diagnosed as having cancer. The subject may also be a subject who exhibits disease risk factors or has no symptoms of cancer. The subject may also be a subject who suffers from or is at risk of developing cancer. In one embodiment, the subject has previously been administered a therapy for cancer.

[0152] In embodiments where the method of therapy is a method for treating cancer, a therapeutic combination dosage may be chosen that maximizes damage to cancer cells and minimizes damage to non-cancerous cells.

[0153] In one embodiment, the cancer is selected from one or more of glioblastoma (e.g., glioblastoma multiforme), acute myeloid leukemia, breast cancer, leukemia, lymphoma, Hodgkin's disease, multiple myeloma, sarcoma, lung cancer (small and / or non-small cell lung cancer), ovarian cancer, astrocytoma, osteosarcoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, myeloma, Waldenstrom's macroglobulinemia, small cell lung cancer, neuroblastoma, testicular cancer, soft tissue sarcoma, osteogenic sarcoma, bladder cancer, small cell lung cancer, cervical cancer, pancreatic cancer, neuroendocrine tumors, polycythemia vera, myeloid metaplasia, fibrosarcoma, rhabdomyosarcoma, islet cell carcinoma, medullary thyroid carcinoma, anaplastic astrocytoma, head and neck cancer, esophageal cancer, gastric cancer, prostate cancer, mesothelioma, and endometrial cancer.

[0154] In a preferred embodiment, the cancer may be one or more selected from glioblastoma, acute myeloid leukemia, and breast cancer.

[0155] Suitably, the breast cancer may be BRCA defective (eg BRCA1 and / or BRCA2 defective).

[0156] Interestingly, DNMT activity is important in the pathogenesis of acute myeloid leukemia (AML), so AML may be a particularly suitable target for the therapeutic combination of the present invention. Thus, in one embodiment, the cancer is acute myeloid leukemia.

[0157] The cancer cells (e.g., tumors) of the cancer may contain a higher expression level of DNMTs than the expression level of DNMTs in a reference standard, the reference standard being derived from a healthy cell. Preferably, the healthy cell is a non-cancerous cell.

[0158] The cancer cells (e.g., tumors) of the cancer may contain an expression level of alkB that is higher than the expression level of alkb in a reference standard, the reference standard being derived from a healthy cell. Preferably, the healthy cell is a non-cancerous cell.

[0159] Cancers may be stratified by the ratio of DNMT gene expression to AlkB homolog gene expression. For example, cancer cells of a cancer may have high levels of DNMT gene expression relative to the level of AlkB homolog gene expression.

[0160] In one embodiment, the cancer cells comprise a high ratio of DNMT gene expression to AlkB homolog gene expression, in one embodiment, the ratio of DNMT gene expression to AlkB homolog gene expression is selected from one or more of about 2:1, about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, and about 50:1.

[0161] In one embodiment, the cancer cell has decreased expression of an AlkB homolog gene compared to expression of an AlkB homolog gene in a non-cancerous cell (e.g., a healthy cell). In one embodiment, the cancer cell has increased expression of a DNMT gene compared to expression of a DNMT gene in a non-cancerous cell (e.g., a healthy cell). In one embodiment, the cancer cell has decreased expression of an AlkB homolog gene compared to expression of an AlkB homolog gene in a non-cancerous cell and increased expression of a DNMT gene compared to expression of a DNMT gene in a non-cancerous cell.

[0162] In one embodiment, the AlkB homolog is one or more selected from Alkb homolog 1, histone h2a dioxygenase, ALKBH2, ALKBH3, ALKBH4, ALKBH5, ALKBH6, ALKBH7, ALKBH8, and FTO, or a combination thereof. In a suitable embodiment, the AlkB homolog is ALKBH2.

[0163] In one embodiment, the DNMT is one or more selected from DNMT1, DNMT3A, and DNMT3B.

[0164] Suitably, the non-cancerous cells are cells derived from the same organ and / or tissue in which the cancer (eg the cancerous tumour) is present.

[0165] In one embodiment, Antibiotics and b. Further monoalkylating agents that substitute a single nucleotide of a nucleic acid with an alkyl group. A kit for use in a method for treating cancer is provided, comprising:

[0166] In one embodiment, Aminoglycoside antibiotics and b. Additional nucleic acid monoalkylating agents that substitute a single nucleotide of a nucleic acid with an alkyl group. A kit for use in a method for treating cancer is provided, comprising:

[0167] The kit may further include a case for containing the antibiotic (eg, an aminoglycoside antibiotic) and the monoalkylating drug.

[0168] In one embodiment, the antibiotic and the monoalkylating agent are included in the same composition. In another embodiment, the antibiotic and the monoalkylating agent are included in separate compositions. In one embodiment, the aminoglycoside antibiotic and the monoalkylating agent are included in the same composition. In another embodiment, the aminoglycoside antibiotic and the monoalkylating agent are included in separate compositions.

[0169] In one embodiment, the kit may include instructions for using the kit in a method for treating cancer, the instructions may include, for example, the optimal dosage of the therapeutic combination to be administered, the optimal number of times to administer the therapeutic combination, the optimal method of administration, or any other information relevant to using the therapeutic combination to treat cancer.

[0170] The instructions may be provided as part of a label or as part of an instruction manual provided with the kit or separately.

[0171] The following embodiments and definitions may apply to any aspect of the invention described herein (or other embodiments), such as to any method, use, composition, therapeutic combination, therapeutic combination for use, pharmaceutical combination for use, kit, or kit for use described herein.

[0172] In one embodiment, the therapeutic combination of the present invention may be administered by one or more selected from oral, intravenous, intramuscular, subcutaneous, and intradermal administration or a combination thereof. The antibiotic (e.g., aminoglycoside antibiotic) and the nucleic acid monoalkylating agent may be administered by the same or separate means. For example, the antibiotic (aminoglycoside antibiotic) may be administered orally and the nucleic acid monoalkylating agent may be administered intravenously.

[0173] The aminoglycoside class of antibiotics consists of about 10 different antibiotics, each with a common mechanism of action. Aminoglycosides act primarily by binding to the aminoacyl site of the 16S ribosomal RNA within the 30S ribosomal subunit, which results in misreading of the genetic code and thus inhibition of translocation. The initial steps required for peptide synthesis, such as binding of the mRNA and joining of the 50S ribosomal subunit, are not interrupted, but elongation does not occur due to the disruption of the mechanisms that ensure the accuracy of translation. The resulting antibacterial activity is usually bactericidal against susceptible aerobic gram-negative bacilli. The most common clinical use of aminoglycosides is for the treatment of serious infections caused by aerobic gram-negative bacilli.

[0174] In one embodiment, the aminoglycoside is one or more selected from kanamycin (e.g., kanamycin A), amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, streptomycin, or combinations thereof. Such antibiotics are often used / administered in salt form, and thus, any salt of an aminoglycoside antibiotic is encompassed by the present invention.

[0175] In one embodiment, the aminoglycoside is one or more selected from kanamycin (eg, kanamycin A), amikacin, tobramycin, dibekacin, sisomicin, netilmicin, neomycin, streptomycin, or a combination thereof.

[0176] The neomycin may be one or more of neomycin B, neomycin C, or neomycin E (eg, paromomycin).

[0177] In a preferred embodiment, the aminoglycoside antibiotic is one or more selected from kanamycin (e.g., kanamycin A) and neomycin. Suitably, the aminoglycoside antibiotic is kanamycin (e.g., kanamycin A).

[0178] In one embodiment, the penicillin antibiotic is one or more selected from benzylpenicillin (penicillin G), procaine benzylpenicillin (procaine penicillin), benzathine benzylpenicillin (benzathine penicillin), and phenoxymethylpenicillin (penicillin V), or a combination thereof.

[0179] In one embodiment, the cephalosporin antibiotic is one or more selected from cefazolin, cefaclor, cefdinir, ceftin, cefuroxime, cefadroxil, cephalexin, cefepime, ceftriaxone, cefixime, and ceftaroline fosamil, or combinations thereof.

[0180] In one embodiment, the macrolide antibiotic is one or more selected from azithromycin, clarithromycin, erythromycin, fidaxomicin, and telithromycin, or a combination thereof.

[0181] In one embodiment, the fluoroquinolone antibiotic is one or more selected from ciprofloxacin, delafloxacin, gemifloxacin, levofloxacin, moxifloxacin, norfloxacin, and ofloxacin, or combinations thereof.

[0182] In one embodiment, the sulfonamide antibiotic is one or more selected from sulfacetamide, sulfadiazine, sulfadimidine, sulfafurazole (sulfisoxazole), sulfisomidine, sulfamethoxazole, sulfamoxole, sulfanitran, sulfadimethoxine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfadoxine, sulfamethpyrazine, and terephtyl, and combinations thereof.

[0183] In one embodiment, the tetracycline antibiotic is one or more selected from tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, tigecycline, and doxycycline, or a combination thereof.

[0184] In one embodiment, the nucleic acid monoalkylating agent is one or more selected from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), dacarbazine, methylnitrosourea (MMU), ethyl methanesulfonate (EMS), temozolomide (TMZ), ethylnitrosourea (ENU), diethylnitrosamine (DEN), and N-nitroso-N-methylurea (NMU).

[0185] In one embodiment, the nuclear monoalkylating agent is one or more selected from dacarbazine, methylnitrosourea (MMU), ethyl methanesulfonate (EMS), temozolomide (TMZ), ethylnitrosourea (ENU), diethylnitrosamine (DEN), and N-nitroso-N-methylurea (NMU).

[0186] In a preferred embodiment, the alkylating agent is temozolomide (TMZ).

[0187] Methyl methanesulfonate (PubChem CID:4156) methylates DNA normally at N7-deoxyguanosine and N3-deoxyadenosine, and may also methylate other oxygen and nitrogen atoms in DNA bases and phosphodiester linkages of nucleic acids.

[0188] Methyl methanesulfonate (MMS) may have the following structure: [ka]

[0189] Ethyl methanesulfonate (PubChem CID: 6113) may have the following structure: [ka]

[0190] Temozolomide (PubChem CID:5394) may have the following structure: [ka]

[0191] Ethyl nitrosourea (PubChem CID: 12967) may have the following structure: [ka]

[0192] N-nitroso-N-methylurea (NMU) (PubChem CID: 12699) may have the following structure: [ka]

[0193] N-methyl-N'-nitro-N-nitrosoguanidine (PubChem CID:6261) may have the following structure: [ka]

[0194] Dacarbazine (PubChem CID:2942) may have the structure: [ka]

[0195] Diethylnitrosamine (PubChem CID:5921) may have the following structure: [ka]

[0196] Such nucleic acid monoalkylating agents typically result in alkyl substitution of a nucleic acid (eg, a base / nucleotide of a nucleic acid) when the nucleic acid is contacted with the monoalkylating agent.

[0197] An "alkyl" (synonymous with the terms "alkyl group" and "alkyl substituent") may be a saturated or unsaturated, straight or branched hydrocarbon chain. An alkyl group has 1 to 18 carbon atoms, e.g., an alkyl group may be a C1 to C18 group or is a C1 to C12 group, C1 to C6 group, or C1 to C4 group. A lower alkyl group may typically have 1 to 6 carbons (and may be a chain that may be straight or branched). An alkyl group may have zero branches (e.g., straight chain), one branch, two branches, or two or more branches. In one embodiment, an alkyl group may be saturated. In another embodiment, an alkyl group may be unsaturated. In one embodiment, an unsaturated alkyl group may have one double bond, two double bonds, more than two double bonds, and / or one triple bond, two triple bonds, or more than two triple bonds. The alkyl chain may be optionally substituted with one substituent (e.g., the alkyl group is single substituted) or with 1-2 substituents or with 1-3 substituents or with 1-4 substituents, etc. The substituents may be one or more selected from the group consisting of hydroxy, amino, alkylamino, boronyl, carboxy, nitro, cyano, or combinations thereof. Alkyl groups that incorporate one or more heteroatoms may be referred to as heteroalkyl groups.

[0198] In one embodiment, the alkyl group may be one or more selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, heptyl, nonyl, decyl, or a combination thereof. Suitably, the alkyl group may be one or more selected from methyl, ethyl, or a combination thereof (preferably methyl).

[0199] In one embodiment, the alkylating agent is one or more selected from methyl methanesulfonate (MMS), MMU, ethyl methanesulfonate (EMS), temozolomide (TMZ), ethylnitrosourea (ENU), and methylnitrosourea (NMU).

[0200] Many of such nucleic acid monoalkylating agents have previously only been used as part of chemotherapy, and the present invention therefore provides an advantageous additional use of such agents, namely for bacterial inhibition.

[0201] In one embodiment, the nucleic acid monoalkylating agent may be a nitrosourea. Suitably, the nitrosourea may be one or more selected from MMU, ENU, NMU, or a combination thereof.

[0202] In one embodiment, the nucleic acid monoalkylating agent is MNNG and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0203] In one embodiment, the nucleic acid monoalkylating agent is MMS and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0204] In one embodiment, the nucleic acid monoalkylating agent is dacarbazine and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0205] In one embodiment, the nucleic acid monoalkylating agent is MMU and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0206] In one embodiment, the nucleic acid monoalkylating agent is EMS and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0207] In one embodiment, the nucleic acid monoalkylating agent is TMZ and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0208] In one embodiment, the nucleic acid monoalkylating agent is ENU and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0209] In one embodiment, the nucleic acid monoalkylating agent is DEN and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0210] In one embodiment, the nucleic acid monoalkylating agent is NMU and the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin, and streptomycin or a salt thereof.

[0211] In a preferred embodiment, the nucleic acid monoalkylating agent of the present invention is a DNA monoalkylating agent. Such nucleic acid / DNA monoalkylating agents are not limited to monoalkylating agents that only alkylate nucleic acids (or, for example, only DNA). For example, the monoalkylating agent may be capable of alkylating another molecule, such as a polypeptide.

[0212] Suitably, the aminoglycoside antibiotic synergizes with the nucleic acid monoalkylating agent, resulting in an increased amount of nucleic acid alkylation (e.g. toxic DNA alkylation) occurring in a cell (e.g. bacterial cell) or organism to which such a drug combination is administered. Thus, the aminoglycoside antibiotic may enhance nucleic acid alkylation in the presence of the monoalkylating agent. In a further embodiment, the aminoglycoside enhances the nucleic acid alkylating activity of the nucleic acid monoalkylating agent, resulting in increased nucleic acid (e.g. nucleotide) alkylation. Preferably, the nucleic acid alkylation is toxic DNA alkylation.

[0213] In one embodiment, a nucleic acid monoalkylating agent substitutes an alkyl group at a nucleotide of a nucleic acid to form one or more nucleotide adducts, preferably 3-methylcytosine (3meC), 3-methylguanine (3meG), 3-methyladenine (3meA), 8-oxoguanine (8-oxo-G), O 2 -Alkylthymine, O 4 -Alkylthymine, O 6 -Methylguanine, O 6 -ethylguanine, or any combination thereof.

[0214] In one embodiment, a nucleic acid monoalkylator replaces a nucleotide (e.g., of a nucleic acid) with an alkyl group to form 3-methylcytosine (3meC), 3-methylguanine (3meG), 3-methyladenine (3meA), O 2 -Alkylthymine, O 4 -Alkylthymine, O 6 -Methylguanine, O 6 -ethylguanine, or any combination thereof.

[0215] In one embodiment, a nucleic acid monoalkylating agent substitutes (eg, methylates) a cytosine with a methyl group at the third position (eg, at the third nitrogen), resulting in the nucleotide adduct 3meC.

[0216] In one embodiment, a nucleic acid monoalkylating agent substitutes (eg, methylates) guanine at the third position (eg, at the third nitrogen) with an alkyl group, resulting in the nucleotide adduct 3meG.

[0217] In one embodiment, a nucleic acid monoalkylating agent replaces (eg, methylates) an adenine at the third position (eg, at the third nitrogen) with an alkyl group to yield 3meA.

[0218] In one embodiment, a nucleic acid monoalkylator is a thymine monoalkyl group that is substituted (e.g., alkylated) at the O2 position with an alkyl group to form a thymine monoalkyl group. 2 -Resulting in alkylthymine.

[0219] In one embodiment, a nucleic acid monoalkylating agent substitutes (e.g., alkylates) a thymine at the O4 position with an alkyl group to form a nucleotide adduct O 4 -Resulting in alkylthymine.

[0220] In one embodiment, a nucleic acid monoalkylating agent substitutes (e.g., alkylates) a thymine at the O6 position with an alkyl group to form a nucleotide adduct O 6 -Resulting in alkylthymine.

[0221] In one embodiment, a nucleic acid monoalkylating agent substitutes (e.g., methylates) guanine at the O6 position with an alkyl group to form a nucleotide adduct O 6 -Resulting in methylguanine.

[0222] In one embodiment, a nucleic acid monoalkylating agent substitutes (e.g., methylates) guanine at the O6 position with an alkyl group to form a nucleotide adduct O 6 -Ethylguanine results.

[0223] In one embodiment, the nucleic acid monoalkylator substitutes a nucleotide or nucleic acid to provide 3meC, 3meG, 3meA, 8-oxo-G, O 2 -Alkylthymine, O 4 -Alkylthymine, O 6 -methylguanine, and O 6 -ethylguanine, resulting in one or more nucleotide additions selected from

[0224] Any combination of antibiotics / aminoglycoside antibiotics and nucleic acid monoalkylating agents is encompassed by the present invention. For example, a therapeutic combination or composition may contain multiple aminoglycosides and / or multiple nucleic acid monoalkylating agents. Different combinations may be selected based on the disease / pathogen / infection to be treated, and different combinations may be optimal for a particular disease. Given the wide range of antibiotics / aminoglycoside antibiotics and nucleic acid monoalkylating agents encompassed within the therapeutic combinations / compositions of the present invention, this allows for improved treatment of many infections, as well as many infections within this category caused by many different pathogens (e.g., bacterial, viral, fungal, etc. infections).

[0225] In one embodiment, the therapeutic combination may include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoglycoside antibiotics. For example, the therapeutic combination may include at least 2, 3, 4, or 5 aminoglycoside antibiotics.

[0226] In one embodiment, the therapeutic combination may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acid monoalkylating agents. For example, the therapeutic combination may include at least 2, 3, 4, or 5 nucleic acid monoalkylating agents.

[0227] Sequence identity Reference to a gene or polypeptide described herein includes its homologues. Those skilled in the art also understand how to use appropriate sequence alignments to identify homologous / similar nucleic acid / polypeptide sequences.

[0228] Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global, local, and hybrid methods, such as segment approach methods. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994), and iterative refinement methods, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example, Match-box, see e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992), Gibbs sampling, see e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993), Align-M, see e.g., Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0229] Thus, percent sequence identity is determined by conventional methods, see, e.g., Altschul et al., Bull. Math. Bio. 48: 603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.), as shown below (amino acids are indicated by standard single-letter code):

[0230] Alignment scores for determining sequence identity [ka]

[0231] The percent identity is then calculated as follows:

number

[0232] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably of the minor kind, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, usually small deletions of from one to about 30 amino acids, and small amino- or carboxy-terminal extensions such as an amino-terminal methionine residue, small linker peptides of no more than about 20-25 residues, or affinity tags.

[0233] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary for many of the terms used in this disclosure.

[0234] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, each nucleic acid sequence is written left to right in 5' to 3' orientation, and each amino acid sequence is written left to right in amino to carboxy orientation.

[0235] The headings provided herein do not limit the various aspects or embodiments of the disclosure.

[0236] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation. The term "protein" as used herein includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter code for amino acids is as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0237] Other definitions of terms may be found throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and may thus be modified. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0238] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range is also expressly disclosed, unless the context clearly dictates otherwise. Each smaller range between any given or intervening value in a given range and any other given or intervening value in that given range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range, subject to any specifically excluded limit in the given range, is also encompassed within the disclosure. Where a given range includes one or both limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0239] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an alkylating agent" includes a plurality of such agents, a reference to "an aminoglycoside antibiotic" includes a reference to one or more aminoglycoside antibiotics and equivalents thereof known to those skilled in the art, and so forth.

[0240] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0241] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples. [Brief description of the drawings]

[0242] [Figure 1] Figure 1 shows the synergistic effect of the combination of alkylating agents and aminoglycoside antibiotics in inhibiting bacteria. Error bars indicate standard deviation for at least three replicates. CFU = colony forming units. The left four bars correspond to wild type (WT) cells. The right four bars correspond to alkb- / - cells (alkb). [Diagram 2] Figure 2 shows no difference in sensitivity between WT and alkB mutants to the bifunctional alkylating drug lomustine in combination with kanamycin. wt = wild type, alkb = alkb mutant (non-functional alkb). "Viable colonies lomustine / untreated" = ratio of colonies observed after lomustine treatment to colonies after DMSO treatment. [Diagram 3] Figure 3 shows a schematic illustrating a model for the introduction of alkylating lesions by DNA methyltransferase activity. The primary activity is the introduction of 5meC (dark arrow), but 3meC is also introduced as a minor by-product (light arrow). 3meC is removed in eukaryotes by the activity of ALKB2, which is homologous to bacterial AlkB. EXAMPLES

[0243] Working Example Materials and Methods Overexpression of mSSSI in E. coli The mSSSI expression plasmid pAIT2, which contains a kanamycin resistance gene (encoding neomycin phosphotransferase II), was a gift from New England Biolabs. This plasmid expresses mSSSI methyltransferase (DNMT) under the control of the IPTG-inducible pTac promoter.

[0244] Standard CaCl2 transformation was used to introduce the pAIT2 plasmid into E. coli strain C2353 (NEB) with or without (WT) a deletion of the alkB gene (ALKB). Basal activity of the mSSSI gene in both backgrounds was verified by using methyl-sensitive restriction digestion with HpaII and MspI.

[0245] To test the effect of alkylation damage on kanamycin resistance in the absence of DNMT overexpression, the standard protein expression plasmid pET28a, which also carries the kanamycin resistance gene, was used as a negative control. Transformation into C2353 was similar to pAIT2.

[0246] Example 1 - Alkylating Drug Susceptibility Assays Show Aminoglycoside Synergy with Alkylating Drugs in Inhibiting Bacterial Growth Synergy between the exemplary aminoglycoside kanamycin and the exemplary alkylating agent MMS is demonstrated.

[0247] E. coli strains were grown from single colonies in Luria Bertani (LB) medium at 37°C until an OD of 0.6 was reached. Bacteria were then separated into different aliquots for different treatments and centrifuged at 3000 revolutions per minute (rpm) for 5 min to pellet down. Bacteria were then resuspended in LB medium supplemented or not with 20 mM methyl methanesulfonate (MMS; Sigma-Aldrich) with or without 50 ug / ml kanamycin. After 30 min at 37°C, bacteria were washed thoroughly in LB to remove MMS and plated on LB with or without 50 ug / ml kanamycin. Sensitivity was assayed by calculating the number of colony forming units (CFU) and taking the base 10 logarithm. Experiments were repeated three times for each condition and the mean and standard deviation were plotted. Significance was calculated using a two-tailed t-test.

[0248] As shown in Figure 1, WT E. coli was resistant to low doses of 20 mM MMS, which had minimal effects on bacterial growth. Sensitivity to MMS increased when treated simultaneously with kanamycin, even more so in bacteria expressing DNMTs. Surprisingly, this sensitivity was significantly increased in alkB mutants, as was synergy between MMS and kanamycin, even when these bacteria carried a plasmid encoding kanamycin resistance. This sensitivity and synergy was even further increased upon overexpression of DNMTs (enzymes that can introduce toxic alkylating lesions into DNA, see Figure 2), suggesting that kanamycin synergizes with MMS to increase the introduction of toxic 3meC alkylating lesions into DNA.

[0249] Example 2 - No difference in sensitivity between WT and alkB mutants to the bifunctional alkylating drug lomustine in combination with kanamycin This experiment was carried out in a similar manner to Example 1, except that lomustine was used instead of MMS.

[0250] Cells were grown in LB+50ug / ml kanamycin to an OD of 0.8 and treated with 20mM lomustine (in a similar manner according to Example 1) or DMSO (control) for 2 hours. Cells expressed the kanamycin resistance gene (encoding neomycin phosphotransferase II).

[0251] Survival was measured as colony number relative to untreated control (DMSO control). Results are shown in Figure 2. Each bar is a biological replicate, p value from a two-tailed t-test was 0.93. No hypersensitivity of AlkB to the combination of lomustine + kanamycin was observed.

[0252] All publications mentioned herein above are incorporated herein by reference. Various modifications and variations of the methods and systems described for the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed is not unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

[0253] array SEQ ID NO:1 (E. coli strain, K-12 alkb; NCBI Reference Sequence: NC_000913.3) [ka]

Claims

1. A therapeutic combination for use in treating an infection by a pathogen. a. an aminoglycoside antibiotic; b. A nucleic acid monoalkylating agent that substitutes a single nucleotide of a nucleic acid with an alkyl group; Including, provides improved inhibition of pathogens when compared to an identical composition but lacking said aminoglycoside antibiotic; or A therapeutic combination that provides improved inhibition of a pathogen when compared to an otherwise identical composition lacking said nucleic acid monoalkylating agent.

2. 2. The therapeutic combination for use according to claim 1, wherein the aminoglycoside antibiotic is administered prior to, simultaneously with, or subsequent to the nucleic acid monoalkylating agent.

3. 3. The therapeutic combination for use according to claim 1 or claim 2, wherein the pathogen is a bacterium.

4. The therapeutic combination for use according to any one of claims 1 to 3, wherein the pathogen lacks one or more genes encoding a polypeptide that removes an alkyl group from an alkylated nucleotide adduct.

5. 5. The therapeutic combination for use according to claim 4, wherein the alkylated nucleotide adduct is one or more selected from 3meC, 3meG, 3meA, 8-oxo-G, O2-alkylthymine, O4-alkylthymine, O6-methylguanine, and O6-ethylguanine, preferably one or more selected from 3meC, 3meG, 3meA, O2-alkylthymine, O4-alkylthymine, O6-methylguanine, and O6-ethylguanine.

6. 6. The therapeutic combination for use according to claim 4 or claim 5, wherein the gene is one or more selected from alkA and alkB, preferably alkB.

7. 7. The therapeutic combination for use according to any one of claims 1 to 6, wherein the pathogen lacks one or more genes encoding a polypeptide which promotes resistance of the cell to the presence of an alkylated nucleotide adduct in the nucleic acid of the cell, preferably wherein the alkylated nucleotide adduct is one or more selected from 3meC, 3meG, 3meA, 8-oxo-G, O2-alkylthymine, O4-alkylthymine, O6-methylguanine, and O6-ethylguanine.

8. 8. The therapeutic combination for use according to claim 7, wherein said one or more genes are selected from Ada, OGT, RecA, DinB, and UmuCD.

9. 9. The therapeutic combination for use according to any one of claims 1 to 8, wherein the pathogen is one or more selected from Salmonella (preferably S. enterica), Pseudomonas (preferably P. aeruginosa), and Mycobacterium (preferably M. tuberculosis) bacteria.

10. The therapeutic combination for use according to any one of claims 1 to 9, wherein the aminoglycoside antibiotic is one or more selected from kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin and streptomycin or a salt thereof, preferably one or more selected from kanamycin, amikacin, tobramycin, dibekacin, sisomicin, netilmicin, neomycin and streptomycin or a salt thereof.

11. The therapeutic combination for use according to any one of claims 1 to 10, wherein said aminoglycoside antibiotic is kanamycin.

12. 12. The therapeutic combination for use according to any one of claims 1 to 11, wherein the nucleic acid monoalkylating agent is one or more selected from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), dacarbazine, methyl nitrosourea (MMU), ethyl methanesulfonate (EMS), temozolomide (TMZ), ethyl nitrosourea (ENU), diethylnitrosamine (DEN), and N-nitroso-N-methylurea (NMU).

13. The therapeutic combination for use according to any one of claims 1 to 12, wherein the nucleic acid monoalkylating agent is methyl methanesulfonate (MMS).

14. The therapeutic combination for use according to any one of claims 1 to 13, wherein said nucleic acid monoalkylator replaces a nucleotide of a nucleic acid with an alkyl group to result in one or more alkylated nucleotide adducts selected from 3meC, 3meG, 3meA, 8-oxo-G, O2-alkylthymine, O4-alkylthymine, O6-methylguanine, and O6-ethylguanine, preferably one or more alkylated nucleotide adducts selected from 3meC, 3meG, 3meA, O2-alkylthymine, O4-alkylthymine, O6-methylguanine, and O6-ethylguanine.

15. Use of a drug combination as a disinfectant in vitro, comprising: The drug combination is a. an aminoglycoside antibiotic; b. A nucleic acid monoalkylating agent that substitutes a single nucleotide of a nucleic acid with an alkyl group; Including, the drug combination provides improved inhibition of pathogens when compared to an otherwise identical composition lacking the aminoglycoside antibiotic; or The drug combination provides improved inhibition of a pathogen when compared to an otherwise identical composition lacking the nucleic acid monoalkylating drug.

16. 16. The use of claim 15, comprising administering the drug combination to a location that contains, is suspected of containing, or is at risk of containing a pathogen.

17. The dosing provides a local concentration of the aminoglycoside antibiotic and the nucleic acid monoalkylating agent at the location of about 1 mM to about 200 mM, preferably a local concentration of the aminoglycoside antibiotic of about 80 mM to about 120 mM; and / or b. a local concentration of said nucleic acid monoalkylating agent of about 1 mM to about 40 mM 17. The use of claim 16, comprising providing

18. 1. Use of a nucleic acid monoalkylating agent to improve inhibition of pathogens by aminoglycoside antibiotics, wherein the nucleic acid monoalkylating agent is a nucleic acid monoalkylating agent that replaces a single nucleotide of a nucleic acid with an alkyl group.

19. 1. A therapeutic combination for use in treating cancer, comprising: a. an aminoglycoside antibiotic; b. A nucleic acid monoalkylating agent that substitutes a single nucleotide of a nucleic acid with an alkyl group; Including, provides improved inhibition of cancer when compared to an identical composition but lacking said aminoglycoside antibiotic; or A therapeutic combination that provides improved inhibition of cancer when compared to an identical composition except lacking said nucleic acid monoalkylating agent.

20. 20. The therapeutic combination for use according to claim 19, wherein the cancer is selected from one or more of glioblastoma (e.g. glioblastoma multiforme), acute myeloid leukemia, breast cancer, leukemia, lymphoma, Hodgkin's disease, multiple myeloma, sarcoma, lung cancer (small and / or non-small cell lung cancer), ovarian cancer, astrocytoma, osteosarcoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, myeloma, Waldenstrom's macroglobulinemia, small cell lung cancer, neuroblastoma, testicular cancer, soft tissue sarcoma, osteogenic sarcoma, bladder cancer, small cell lung cancer, cervical cancer, pancreatic cancer, neuroendocrine tumors, polycythemia vera, myeloid metaplasia, fibrosarcoma, rhabdomyosarcoma, islet cell carcinoma, medullary thyroid carcinoma, anaplastic astrocytoma, head and neck cancer, esophageal cancer, gastric cancer, prostate cancer, mesothelioma, and endometrial cancer.

21. The therapeutic combination for use according to claim 19 or 20, wherein the cancer cells (e.g., tumor) of the cancer comprise an expression level of DNMT that is higher than the expression level of DNMT in a reference standard, the reference standard being derived from healthy cells.

22. 22. The therapeutic combination for use according to any one of claims 19 to 21, wherein cancer cells (e.g. tumour) of the cancer comprise an expression level of alkB that is higher than the expression level of alkB in a reference standard, the reference standard being derived from healthy cells.

23. a. an aminoglycoside antibiotic; b. A nucleic acid monoalkylating agent that substitutes a single nucleotide of a nucleic acid with an alkyl group; Kit including:

24. 24. The kit of claim 23, wherein the aminoglycoside antibiotic and the nucleic acid monoalkylating agent are contained within the same composition or in separate compositions.

25. a. instructions for using the kit in a method for treating an infection by a pathogen; b. Instructions for using the kit as an in vitro disinfectant; c. instructions for using the kit in an in vitro method for inhibiting a pathogen, and / or d. instructions for using the kit in a method for treating cancer; 25. The kit of claim 23 or 24, further comprising:

Images