Antimicrobial Combinations

Abstract

The present invention relates to a combination comprising at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof. The present invention also provides a combination for use in the treatment of bacterial infections.

Description

[Technical Field]

[0001] The present invention relates to combinations of at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof. In particular, the present invention relates to the use of such combinations to kill growth-phase (i.e., logarithmic) microorganisms associated with bacterial infections, such as gram-negative bacterial infections. [Background technology]

[0002] Before the introduction of antibiotics, patients with acute microbial infections (e.g., tuberculosis or pneumonia) had a low chance of survival. For example, the mortality rate from tuberculosis was nearly 50%. This situation changed rapidly with the introduction of antimicrobial agents in the 1940s and 1950s; today, approximately 100 antibiotics are used to treat a variety of bacterial infections. This led to the birth of modern medicine, as it effectively prevents and treats bacterial infections in millions of patients undergoing cancer, organ transplants, kidney dialysis, immunosuppression, and surgery.

[0003] However, in response to widespread antibiotic use, bacteria have gradually developed resistance. Antibiotic-resistant bacteria now exist in every country in the world, and this resistance is increasing every year, reducing the effectiveness of all antibiotics. By 2040-2050, antimicrobial-resistant infections are predicted to cause more than 10 million deaths per year (The Review on Antimicrobial Resistance, Chaired by Jim O'Neill, May 2016).

[0004] The rise of antimicrobial resistance is expected to occur sooner in poorer countries than 2040–2050 and is already a real and practical healthcare problem in Europe and the United States. In fact, over 70% of bacteria causing hospital-acquired infections in the United States are resistant to at least one of the major antimicrobial drugs commonly used to fight infections (Nature Reviews, Drug Discovery, 1, 895–910 (2002)). For this reason, the World Health Organization has classified antimicrobial resistance as “no longer a future prediction, but a current threat in every region of the world, potentially affecting anyone, of any age, in every country” ("Antimicrobial resistance: global report on surveillance", The World Health Organization, April 2014). If left unaddressed, life expectancy could fall to pre-antibiotic levels, approximately 20 years shorter than it is today.

[0005] Therefore, a solution to the growing problem of resistant bacteria is desperately needed. Essentially, the medical field needs to replace approximately 100 antibiotics with products that are effective against both antimicrobial-resistant infections while preventing future emergence of antimicrobial resistance.

[0006] Current efforts to solve this problem focus primarily on the development of new chemical entities (NCEs). Each NCE requires more than 10 years to develop and costs over $600 million to complete the necessary safety and clinical testing. Because a significant number of NCEs fail, it typically costs approximately $3.8 billion to provide a single NCE antibiotic. Ironically, because increased antibiotic use leads to faster development of antimicrobial resistance, all NCEs have a limited useful life, often less than 10 years. Therefore, replacing currently used antibiotics with NCEs would require approximately $3.8 trillion over a 10-year period. Even if successful, these products would require ongoing planning to replace them within their 10-year useful life due to the emergence of antimicrobial resistance. This is clearly unsustainable, even in high-income countries.

[0007] The present applicant has identified a solution to this serious global problem. Specifically, a combination of four or more known antibiotics has surprisingly been identified to have a synergistic effect against Gram-negative and / or Gram-positive bacteria. Such combinations have been found to kill antimicrobial-resistant bacteria, also known as drug-resistant bacteria, and prevent the development of antimicrobial resistance. The four or more known antibiotics are defined in the appended claims and described herein.

[0008] The demonstrated synergy means that the combination is more bioactive than the expected additive effect of each drug at a given dosage level, meaning that lower doses, e.g., 1 / 5 to 1 / 20 the amount of antibiotic, are more effective in combination therapy than either antibiotic alone.

[0009] Furthermore, by combining previously approved drugs (CADs), approval times and costs are much less than those for NCEs. For example, the time required is approximately five years, and each new product requires less than $50 million to produce. Because far fewer drugs fail in development, the cumulative cost of success / failure is approximately $130 million for a single CAD, compared with $3.8 billion for each NCE. The number of combinations needed to replace the approximately 100 single antibiotics used today could be reduced by CADs with activity against a broad range of different infectious species. Single antibiotics that become unavailable due to antimicrobial resistance could be reinstated in CADs with much greater activity against these antimicrobial-resistant strains, preventing future emergence of antimicrobial resistance. As a percentage of GDP, a shift from NCEs representing 4% of high-income countries' GDP to CAD technology representing approximately 0.005% would allow high-, middle-, and even low-income countries to contribute and benefit.

[0010] Currently, the world is focused on repetitive NCE programs that are far more costly, time-consuming, and prone to failure, with short durations of use, leading to the prediction that effective antibiotics will be depleted within 20 years, and that deaths from antimicrobial resistance will return to levels seen 200 years ago. Investing just 0.1% of NCE development costs in a CAD approach would provide a practical, affordable, and long-lasting solution that would become a realistic option for the continued provision of effective, affordable antibiotic products worldwide, offering the hope of "affordable antibiotics for all, forever."

[0011] Over the past decade, the applicant, a small UK company, and a non-profit organization (GARDP) have been actively developing CAD antibiotic combinations of existing drugs. In this application, the applicants have developed a new approach to replace all antibiotics by discovering a synergistic combination of three antimicrobial agents. This combination is active against at least extended-spectrum beta-lactamase (ESBL), carbapenemase-producing (CPE), and carbapenem-resistant Gram-negative bacteria. Even more surprisingly, the applicants have discovered that the concentration of each antimicrobial agent in the combination is sufficient to reduce, for example, the MIC of the agent. mono They found that the combination was active at very low concentrations, 1 / 16 of the usual dose. Therefore, the combination has the advantage of requiring less of each antimicrobial agent than, for example, monotherapy, reducing the potential burden of toxicity.

[0012] WO2015 / 114340 describes the use of zidovudine in combination with a polymyxin selected from colistin or polymyxin B; an antituberculosis antibiotic selected from rifampicin, rifapentine or rifabutin; and optionally piperine, for treating microbial infections. WO2018 / 011562 describes a combination comprising zidovudine and a carbapenem, optionally comprising a polymyxin selected from polymyxin B and polymyxin E. Thus, the present invention does not encompass these combinations previously identified by the applicant.

[0013] When two or more active agents are used in combination, synergy is neither predictable nor expected. Synergy in the context of antimicrobial agents is measured in a number of ways, consistent with the generally accepted view that synergy is greater than additive. One way to assess whether synergy is present is to use the "checkerboard" technique, an accepted method that generates a value called the fractional inhibitory concentration index (FICI). Orhan et al., J. Clin. Microbiol. 2005, 43(1):140, describes the checkerboard method and analysis in paragraphs 140-141, explaining that the FICI value is the ratio of the sum of the MIC (minimum inhibitory concentration) levels of each component alone and in the mixture.

[0014] The FICI, or partial inhibitory concentration index, is the sum of the FICs of each antimicrobial agent when used in combination. The FIC, or partial inhibitory concentration, of an antimicrobial agent in combination is the MIC of the antimicrobial agent in combination divided by the MIC of the same antimicrobial agent when used alone. The minimum inhibitory concentration is defined in the art as the lowest concentration of an antimicrobial agent that inhibits visible growth of a microorganism after overnight incubation.

[0015] The combinations are effective against resistant bacteria (see the Examples herein). Individual antimicrobial agents in these combinations are often remarkably effective at concentrations significantly below their MICs when used alone. However, there appears to be no method in the art for defining quaternary (combination containing four antimicrobial agents) synergistic effects expressed as fractional inhibitory concentrations. Indeed, very little research has been done in this area to date. Therefore, the present inventors have devised such a method. This method is applicable to any combination containing "n" antimicrobial agents.

[0016] In this method, two antibiotics were "fixed" as part of the backbone, and the other two antibiotics were varied over a doubling concentration scale starting from the MIC (×1) of the effective monotherapy dose against the microorganism under test. ΣFIC was then calculated as follows:

[0017] The FIC was obtained by dividing the ΣFIC by "0.5 / n" (n is the number of antimicrobial agents in the combination). We chose this expression because it more closely approximates the level of 2-way synergy. The same FIC scale used for 2-way was applied. An FIC of <0.5 indicates synergy. An FIC of 0.5 to <1 indicates an "additive" effect. An FIC of 1 to <2 indicates indifference. An FIC of 2 to 4 indicates antagonism.

[0018] The synergistic effect can be expressed as "ΣFIC≦0.25×n".

[0019] [Table 1]

[0020] The above method was used in the examples herein. Summary of the Invention

[0021] In one aspect, the present invention provides a combination as defined in the appended claims, which comprises at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof. In various embodiments, the combination comprises ceftazidime, doxycycline, or a pharmaceutically acceptable derivative thereof, meaning that the remaining antimicrobial agents in the combination are selected from polymyxin E, polymyxin B, levofloxacin, and rifampicin, or a pharmaceutically acceptable derivative thereof. Pharmaceutically acceptable derivatives of polymyxin E may be defined as colistin sulfate, colistin methanesulfonate, or colistin methanesulfonate sodium.

[0022] In another aspect, the present invention provides a combination as defined herein for use in the treatment of a bacterial infection.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising a combination as defined herein and a pharmaceutically acceptable adjuvant, diluent or carrier, which can be used in the treatment of a bacterial infection.

[0024] In another aspect, the present invention provides a product comprising at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof, as a combination drug for simultaneous, separate, or sequential use in the treatment of bacterial infections. In various embodiments, the product comprises ceftazidime, doxycycline, or a pharmaceutically acceptable derivative thereof, meaning that the remaining antimicrobial agents in the product are selected from polymyxin E, polymyxin B, levofloxacin, and rifampicin, or a pharmaceutically acceptable derivative thereof. Pharmaceutically acceptable derivatives of polymyxin E can be defined as colistin sulfate, colistin methanesulfonate, or colistin methanesulfonate sodium.

[0025] In another aspect, the present invention provides use of a first antimicrobial agent in combination with at least second, third, and fourth antimicrobial agents in the manufacture of a medicament for synergistically treating a Gram-negative or Gram-positive bacterial infection, wherein the first antimicrobial agent is rifampicin or a pharmaceutically acceptable derivative thereof, and the second, third, and fourth antimicrobial agents are selected from levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof.

[0026] In another aspect, the present invention provides use of a first antimicrobial agent in combination with at least second, third, and fourth antimicrobial agents in the manufacture of a medicament for synergistically treating a Gram-negative or Gram-positive bacterial infection, wherein the first antimicrobial agent is levofloxacin or a pharmaceutically acceptable derivative thereof, and the second, third, and fourth antimicrobial agents are selected from rifampicin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof.

[0027] In another aspect, the present invention provides the use of a first antimicrobial agent in combination with at least second, third, and fourth antimicrobial agents in the manufacture of a medicament for the synergistic treatment of a Gram-negative or Gram-positive bacterial infection, wherein the first antimicrobial agent is polymyxin E or B, or a pharmaceutically acceptable derivative thereof, and the second, third, and fourth antimicrobial agents are selected from levofloxacin, rifampicin, doxycycline, ceftazidime, and a pharmaceutically acceptable derivative thereof.

[0028] In another aspect, the present invention provides use of a first antimicrobial agent in combination with at least second, third, and fourth antimicrobial agents in the manufacture of a medicament for synergistically treating a Gram-negative or Gram-positive bacterial infection, wherein the first antimicrobial agent is doxycycline or a pharmaceutically acceptable derivative thereof, and the second, third, and fourth antimicrobial agents are selected from levofloxacin, polymyxin E, polymyxin B, rifampicin, ceftazidime, and pharmaceutically acceptable derivatives thereof.

[0029] In another aspect, the present invention provides use of a first antimicrobial agent in combination with at least second, third, and fourth antimicrobial agents in the manufacture of a medicament for synergistically treating a Gram-negative or Gram-positive bacterial infection, wherein the first antimicrobial agent is ceftazidime or a pharmaceutically acceptable derivative thereof, and the second, third, and fourth antimicrobial agents are selected from levofloxacin, polymyxin E, polymyxin B, doxycycline, rifampicin, and pharmaceutically acceptable derivatives thereof.

[0030] In another aspect, the present invention provides a method for treating a Gram-negative or Gram-positive bacterial infection, comprising administering a pharmaceutically effective amount of a combination comprising at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof.

[0031] These aspects and embodiments thereof are set out in the accompanying independent and dependent claims. It will be understood that features of the dependent claims may be combined with each other and with features of the independent claims in combinations other than those explicitly set out in the claims. Furthermore, the present disclosure is not limited to the particular embodiments described below, but rather includes and contemplates any combination of features provided herein.

[0032] These and other aspects, embodiments, features, and advantages of the present disclosure will be apparent from the following detailed description, and in this regard, no particular section of the description should be read in isolation from other sections. DETAILED DESCRIPTION OF THE INVENTION

[0033] Although various exemplary embodiments have been described or suggested herein, other exemplary embodiments utilizing various methods and materials similar or equivalent to those described or suggested herein are encompassed by the general inventive concept. Conventionally practiced aspects and features thereof may not be discussed or described in detail in the interest of brevity. Accordingly, it will be understood that features of the apparatus, products, and processes described herein that are not specifically described may be implemented in accordance with any conventional techniques for implementing such features in the particular context.

[0034] As used herein, the terms "combination of" and "in combination with" encompass separate, sequential, and simultaneous administration of agents. Unless otherwise specified, these terms are also intended to exclude additional active agents. For example, a "combination comprising at least four antimicrobial agents" means that the defined agents are administered separately, sequentially, or simultaneously, but no other active agents are administered.

[0035] When the agents are administered sequentially, either rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, or ceftazidime can be administered first. When administration is simultaneous, the agents can be administered in the same or different pharmaceutical compositions. In a preferred embodiment, the agents are administered sequentially or simultaneously.

[0036] The combination of the present invention can be used to treat Gram-positive or Gram-negative bacterial infections. In particular, it can be used to kill vegetative and / or clinically latent bacteria associated with such infections, preferably vegetative bacteria associated with such infections, such as vegetative bacteria associated with Gram-negative bacterial infections. Thus, when referring to the treatment of bacterial infections herein, it includes killing vegetative and / or clinically latent microorganisms associated with such infections.

[0037] As used herein, "kill" means loss of viability as assessed by lack of metabolic activity.

[0038] As used herein, "clinically latent bacteria" refers to bacteria that are metabolically active but have a growth rate below the threshold for infectious disease manifestation, which refers to the growth rate threshold below which there are no symptoms of infection in the host.

[0039] The metabolic activity of clinically latent bacteria can be measured by several methods known to those skilled in the art, for example by measuring the mRNA levels in the bacteria or by measuring the uridine incorporation rate of the bacteria. In this respect, clinically latent bacteria exhibit a lower, but still significant, metabolic activity compared to bacteria under logarithmic growth conditions (in vitro or in vivo). (I) mRNA levels (e.g., 0.0001 to 50%, e.g., 1 to 30%, 5 to 25%, or 10 to 20% of the mRNA levels); and / or (II) uridine (e.g., [H]uridine) incorporation level (e.g., 0.0005 to 50%, e.g., 1 to 40%, 15 to 35%, or 20 to 30% of the level of [H]uridine incorporation). It has.

[0040] Clinically latent bacteria typically have many distinguishing characteristics. For example, they may be viable but non-culturable; that is, they are usually undetectable by standard culture techniques, but are detectable and quantifiable by techniques such as broth dilution counting, microscopy, or molecular techniques such as polymerase chain reaction. Furthermore, clinically latent bacteria are phenotypically resistant and therefore susceptible (in log phase) to the bacteriostatic effects of conventional antimicrobial agents (i.e., bacteria for which the minimum inhibitory concentration (MIC) of a conventional antimicrobial agent remains substantially unchanged), but are significantly reduced in susceptibility to drug-induced killing (e.g., bacteria for which the ratio of minimum bactericidal concentration (e.g., minimum bactericidal concentration, MBC) to MIC is 10 or greater for any given conventional antimicrobial agent).

[0041] In various embodiments of the invention, one or more of the above combinations are used to treat bacterial infections, and in particular, the combinations may be used to kill vegetative and / or clinically latent bacteria associated with bacterial infections. As used herein, the term "bacteria" (and derivatives thereof, such as "bacterial infection") includes, but is not limited to, reference to the following classifications and specific types of organisms (or infections by organisms):

[0042] Gram-positive cocci, for example, Staphylococci (e.g., Staph. aureus, Staph. epidermidis, Staph. saprophyticus, Staph. auricularis, Staph. capitis capitis, Staph. c. ureolyticus, Staph. caprae, Staph. cohnii cohnii, Staph. c. urealyticus, Staph. aequorum, Staph. Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis hominis, Staphylococcus h. novobiosepticius, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus saccharolyticus, Staphylococcus schleiferi schleiferi Staphylococcus schleiferi, Staphylococcus schleiferi coagulans, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus.xylosus, Streptococci (e.g., β-hemolytic Streptococcus pyogenes (Streptococcus agalactiae, Streptococcus canis, Streptococcus dysgalactiae dysgalactiae, Streptococcus dysgalactiae equisimilis, Streptococcus equi equi, Streptococcus equi zooepidemicus, Streptococcus iniae, Streptococcus porcinus, and Streptococcus pyogenes) pyogenes), microaerophilic pyogenic streptococci (Streptococcus "milleri", e.g., Streptococcus anginosus, Streptococcus constellatus constellatus, Streptococcus constellatus pharyngidis and Streptococcus intermedius), "mitis" (α-haemolytic Streptococcus "viridans", e.g., Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus cristatus, etc.). cristatus, Streptococcus gordonii and Streptococcus parasanguinis), "salivarius" (non-hemolytic, e.g., Streptococcus salivarius and Streptococcus vestivelis).Oral streptococci of the group "mutans" (streptococci on the tooth surface, e.g., Streptococcus vestibularis) and "mutans" (streptococci on the tooth surface, e.g., Streptococcus criceti, Streptococcus mutans, Streptococcus ratti and Streptococcus sobrinus), Streptococcus acidominimus, Streptococcus bovis, Streptococcus faecalis, Streptococcus equinus, Streptococcus pneumoniae and Streptococcus suis.suis, or streptococci otherwise classified as group A, B, C, D, E, G, L, P, U, or V streptococci; enterococci (e.g., Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus and Enterococcus solitarius; Bacillaceae, such as Bacillus anthracis, Bacillus subtilis, Bacillus thuringiensis, Bacillus stearothermophilus and Bacillus cereus;

[0043] Gram-negative cocci, for example, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria sicca, Neisseria subflava and Neisseria weaveri; Enterobacteriaceae, for example, Escherichia coli, Enterobacter species (e.g., Enterobacter aerogenes, Enterobacter agglomerans), agglomerans and Enterobacter cloacae), Citrobacter spp. (such as Citrobacter freundii and Citrobacter divernis), Hafnia spp. (e.g., Hafnia alvei), Erwinia spp. (e.g., Erwinia persicinus), Morganella spp. (e.g., Morganella morganii), Salmonella spp. (Salmonella enterica and Salmonella typhi), Shigella spp. (e.g., Shigella dysenteriae, Shigella flexneri), flexneri, Shigella boydii and Shigella sonnei), Klebsiella species (e.g., Klebs. pneumoniae, Klebs. oxytoca, Klebs. ornitrichica, Klebs.ornitholytica, Klebs. planticola, Klebs. ozaenae, Klebs. terrigena, Klebs. granulomatis (Calymmatobacterium granulomatis and Klebs. rhinoscleromatis), Proteus (e.g., Proteus mirabilis, Proteus rettgeri and Proteus vulgaris), Providencia (e.g., Providencia alcalifaciens), alcalifaciens, Providencia rettgeri, and Providencia stuartii), Serratia (e.g., Serratia marcescens and Serratia liquifaciens), and Yersinia (e.g., Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis); Helicobacter (e.g., Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fenella). fennellii); Acinetobacter species (e.g., Acinetobacter baumanii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter ruwofii, Acinetobacter spp. ...lwoffi and Acinetobacter radioresistens; Pseudomonas species (e.g., Pseudomonas aeruginosa, Pseudomonas maltophilia (Stenotrophomonas maltophilia)), Pseudomonas alcaligenes, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas luteola, Pseudomonas mendocina, Pseudomonas monteilii, Pseudomonas oryzihabitans (Ps. oryzihabitans, Pseudomonas pertocinogena, Pseudomonas pseudalcaligenes, Pseudomonas putida, and Pseudomonas stutzeri; Bacteriodes fragilis; Peptococcus species (e.g., Peptococcus niger); Peptostreptococcus species; Clostridium species (e.g., Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tetani, Clostridium absonum, C. absonum, Clostridium argentinense (C. argentinense), Clostridium baratii (C. baratii), Clostridium bifermentans (C. bifermentans), Clostridium beijerinckii (C. beijerinckii), Clostridium butyricum (C. butyricum), Clostridium cadaveris (C. cadaveris), Clostridium carnis (C.carnis, Clostridium celatum, Clostridium clostridioforme, Clostridium cochlearium, Clostridium cocleatum, Clostridium fallax, Clostridium ghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, Clostridium leptum, Clostridium limosum limosum, Clostridium malenominatum, Clostridium novyi, Clostridium oroticum, Clostridium paraputrificum, Clostridium piliforme, Clostridium putrefasciens, Clostridium ramosum, Clostridium septicum, Clostridium sordelii, Clostridium sphenoides, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, and Clostridium tertium. tertium); Mycoplasma species (e.g., Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, and Mycoplasma urealyticum)Mycobacterium species (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium leprae, Mycobacterium smegmitis, Mycobacterium africanum) africanum, Mycobacterium alvei, Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium bohemicum, Mycobacterium bovis, Mycobacterium branderi, Mycobacterium brumae, Mycobacterium celatum, Mycobacterium chubense, Mycobacterium confluentis, Mycobacterium conspicum conspicuum, Mycobacterium cookii, Mycobacterium flavescens, Mycobacterium gaudiumgadium, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium hassicum, Mycobacterium intracellulare, Mycobacterium interjectum, Mycobacterium heidelberense, Mycobacterium lentiflavum lentiflavum, Mycobacterium malmoense, Mycobacterium microgenicum, Mycobacterium microti, Mycobacterium mucogenicum, Mycobacterium neoaurum, Mycobacterium nonchromogenicum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium simoidei shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium terraeterrae, Mycobacterium thermoresistabile, Mycobacterium triplex, Mycobacterium triviale, Mycobacterium tusciae, Mycobacterium ulcerans, Mycobacterium vaccae, Mycobacterium wolinskyi, and Mycobacterium xenopi; Haemophilus species (e.g., Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus; Actinobacillus (e.g., Actinobacillus actinomycetemcomitans, Actinobacillus equuli, Actinobacillus hominis, Actinobacillus lignieresii, Actinobacillus suis, and Actinobacillus ureae); Actinomycetes Bacillus spp. (e.g., Actinomyces israelii); Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melintensis, and Brucella suis); Campylobacter spp. (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus); Listeria monocytogenes; Vibrio spp. (e.g., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus, and Vibrio vulnificus; Erysipelothrix rhusopathiae; Corynebacterium species (e.g., Corynebacterium diphtheriae, Corynebacterium jeikeum, and Corynebacterium urealyticum);Spirochetaceae, for example the genus Borrelia (e.g., Borrelia recurrentis, Borrelia burgdorferi, Borrelia afzelii, Borrelia andersonii, Borrelia bissettii, Borrelia garinii, Borrelia japonica, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, Borrelia valaisiana, Borrelia caucasiaca, Borrelia caucasica, Borrelia crocidurae, Borrelia duttoni, Borrelia graingeri, Borrelia hermsii, Borrelia hispanica, Borrelia latyschewii, Borrelia mazzottii, Borrelia parkeri, Borrelia persica, Borrelia turicatae, and Borrelia venezuelensis) and Treponema spp. (Treponema pallidum ssp. pallidum, Treponema pallidum ssp. endemicum, Treponema pallidum ssp. pertenue and Treponema carateum);Pasteurella species (e.g., Pasteurella aerogenes, Pasteurella bettyae, Pasteurella canis, Pasteurella dagmatis, Pasteurella gallinarum, Pasteurella haemolytica, Pasteurella multocida multocida, Pasteurella multocida gallicida, Pasteurella multocida septica, Pasteurella pneumotropica, and Pasteurella stomatis) stomatis); Bordetella genus (e.g., Bordetella bronchiseptica, Bordetella hinzii, Bordetella holmseii, Bordetella parapertussis, Bordetella pertussis and Bordetella trematum); Nocardiaceae family, for example, Nocardia genus (e.g., Nocardia asteroides and Nocardia brasiliensis); Rickettsia genus (e.g., Ricksettsii or Coxiella burnetii);Legionella species (e.g., Legionella anisa, Legionella birminghamensis, Legionella bozemanii, Legionella cincinnatiensis, Legionella dumoffii, Legionella feeleii, Legionella gormanii, Legionella hackeliae, Legionella israelensis, Legionella jordanis, Legionella lansingensis, Legionella longbice) longbeachae, Legionella maceachernii, Legionella micdadei, Legionella oakridgensis, Legionella pneumophila, Legionella sainthelensi, Legionella tucsonensis, and Legionella wadsworthii; Moraxella catarrhalis; Cyclospora cayetanensis; Entamoeba histolytica; Giardia lamblia; Trichomonas vaginalis vaginalis); Toxoplasma gondii; Stenotrophomonas maltophilia;Burkholderia stenotrophomonas; Burkholderia cepacia; Burkholderia mallei and Burkholderia pseudomallei; Francisella tularensis; Cardnerella species (e.g., Gardneralla vaginalis and Gardneralla mobiluncus); Streptobacillus moniliformis; Flavobacteriaceae, e.g., Capnocytophaga species (e.g., Capnocytophaga canimorsus, Capnocytophaga cynodegumi), cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica, Capnocytophaga ochracea, and Capnocytophaga sputigena; Bartonella spp. (Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella elizabethae, Bartonella henselae, Bartonella quintana, and Bartonella vinsonii alpensis) vinsonii arupensis));Leptospira (e.g., Leptospira biflexa, Leptospira borgpetersenii, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira santarosai, and Leptospira weilii); Spirillium (e.g., Spirillum minus);Bacteroides genus (e.g., Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides merdae, Bacteroides ovatus, Bacteroides putredinis, Bacteroides pyogenes) pyogenes, Bacteroides splanchinicus, Bacteroides stercoris, Bacteroides tectus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus and Bacteroides vulgatus);Species of the genus Prevotella (e.g., Prevotella bivia, Prevotella buccae, Prevotella corporis, Prevotella dentalis (Mitsuokella dentalis), Prevotella denticola, Prevotella disiens, Prevotella enoeca, Prevotella heparinolytica, Prevotella intermedia, Prevotella loeschii, Prevotella melaninogenica, Prevotella nigrescens) nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulora, Prevotella tannerae, Prevotella venoralis and Prevotella zoogleoformans);Porphyromonas spp. (e.g., Porphyromonas asaccharolytica, Porphyromonas cangingivalis, Porphyromonas canoris, Porphyromonas cansulci, Porphyromonas catoniae, Porphyromonas circumdentaria, Porphyromonas crevioricanis, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gingivalis, Porphyromonas gingivicanis, Porphyromonas levii, and Porphyromonas macacae); Fusobacterium species (e.g., Fusobacterium gonadiaformans, F. mortiferum, F. naviforme, F. necrogenes, F. necrophorum necrophorum, F. necrophorum fundiliforme, F. nucleatum nucleatum; tum, Fusobacterium nucleatum fusiforme, Fusobacterium nucleatum polymorphum, Fusobacterium nucleatum vincentii, Fusobacterium periodonticum, Fusobacterium russii, Fusobacterium ulcerans, and Fusobacterium varium; Chlamydia species (e.g., Chlamydia trachomatis); Cryptosporidium species (e.g., C. parvum, C. hominis, C. hominis, Cryptosporidium canis (C. canis), Cryptosporidium felis (C. felis), Cryptosporidium meleagridis (C. meleagridis) and Cryptosporidium muris (C.muris); Chlamydophila genus (e.g., Chlamydophila abortus (Chlamydia psittaci), Chlamydophila pneumoniae (Chlamydia pneumoniae) and Chlamydophila psittaci (Chlamydia psittaci)); Leuconostoc genus (e.g., Leuconostoc citreum, Leuconostoc cremoris, Leuconostoc dextranicum, Leuconostoc lactis) lactis, Leuconostoc mesenteroides, and Leuconostoc pseudomesenteroides; Gemella genus (e.g., Gemella bergeri, Gemella haemolysans, Gemella morbillorum, and Gemella sanguinis); Aeromonas genus (e.g., Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii biovar sobria); and Ureaplasma genus (e.g., Ureaplasma parvum and Ureaplasma urealyticum). urealyticum)).

[0044] Preferably, the combination of the present invention is synergistic against Gram-positive or Gram-negative bacteria selected from:

[0045] Gram-negative bacteria: Enterobacteriaceae, Enterobacter, Pseudomonas, Acinetobacter, Shigella, Salmonella, Burkholderia stenotrophomonas, Citrobacter, Serratia, Proteus, Morganella, Providencia, Haemophilus, Aeromonas, Pasteurella, Brucella, Helicobacter, Campylobacter, Franciella tularensis, Legionella, Vibrio, Neisseria, Mycobacterium, Yersinia pestis, Rickettsia.

[0046] Gram-positive bacteria: Staphylococcus species, Enterococcus species, Streptococcus weekii, Bacillus anthracis, Bacillus anthracis.

[0047] Examples of Gram-negative bacteria include, for example, Enterobacteriaceae, such as Escherichia coli, Enterobacter (e.g., Enterobacter aerogenes, Enterobacter agglomerans, and Enterobacter cloacae), Citrobacter (e.g., Citrobacter freundii and Citrobacter divernis); Pseudomonas (e.g., Ps. aeruginosa, Ps. maltophilia, Stenotrophomonas maltophilia), Pseudomonas alcaligenes (e.g., Ps. alcaligenes, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas luteola, Pseudomonas mendocina, Pseudomonas monteilii, Pseudomonas oryzihabitans, Pseudomonas pertocinogena, Pseudomonas pseudalcaligenes, Pseudomonas putida, and Pseudomonas stutzeri; Yersinia species (e.g., Yersinia enterocolitica, Yersinia pestis, Yersinia pestis and Yersinia pseudotuberculosis); Helicobacter species (e.g., Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fenellia)fennellii); Acinetobacter spp. (e.g., Acinetobacter baumanii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter lwoffi, and Acinetobacter radioresistens); Morganella spp. (e.g., Morganella morganii), Salmonella spp. (Salmonella enterica and Salmonella typhi), Shigella spp. (e.g., Shigella dysenteriae), dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Klebsiella species (e.g., Klebs. pneumoniae, Klebs. oxytoca, Klebs. ornitholytica, Klebs. planticola, Klebs. ozaenae, Klebs. terrigena, Klebs. granulomatis, and Calymmatobacterium granulomatis) granulomatis and Klebsiella rhinoscleromatis); Burkholderia stenotrophomonas; Francisella tularensis; Serratia species (e.g., Serratia marcescens and Serratia liquifaciens);liquifaciens); Proteus (e.g., Proteus mirabilis, Proteus rettgeri, and Proteus vulgaris), Providencia (e.g., Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii); Haemophilus (e.g., Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, haemolyticus and Haemophilus parahaemolyticus; Aeromonas spp. (e.g., Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii biovar sobria); Pasteurella spp. (e.g., Pasteurella aerogenes, Pasteurella bettyae, Pasteurella canis, Pasteurella dagmatis, Pasteurella gallinarum, Pasteurella haemolytica, Pasteurella multocida, Pasteurella multocida multocida, Pasteurella multocida gallicida, Pasteurella multocida septicaseptica, Pasteurella pneumotropica, and Pasteurella stomatis; Brucella (e.g., Brucella abortus, Brucella canis, Brucella melintensis, and Brucella suis); Campylobacter (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus); Legionella (e.g., Legionella anisa, Legionella virmingamensis, and Legionella birminghamensis, Legionella bozemanii, Legionella cincinnatiensis, Legionella dumoffii, Legionella feeleii, Legionella gormanii, Legionella hackeliae, Legionella israelensis, Legionella jordanis, Legionella lansingensis, Legionella longbeachae, Legionella maceachernii, Legionella mikudadei micdadei), Legionella oakridgensis, Legionella pneumophila, Legionella saintellensisainthelensi, Legionella tucsonensis, and Legionella wadsworthii); Vibrio species (e.g., Vibrio cholerae and Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus, and Vibrio vulnificus). vulnificus); Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria sicca, Neisseria subflava, and Neisseria weaveri; Mycobacterium species (e.g., Mycobacterium tuberculosis, Mycobacterium avium ... avium, Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium sheronaechelonae, Mycobacterium abscessus, Mycobacterium leprae, Mycobacterium smegmitis, Mycobacterium africanum, Mycobacterium alvei, Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium bohemicum, Mycobacterium bovis, Mycobacterium branderi branderi, Mycobacterium brumae, Mycobacterium celatum, Mycobacterium chubense, Mycobacterium confluentis, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium flavescens, Mycobacterium gadium, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordona gordonae, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium hassicum, Mycobacterium intracellulareintracellulare, Mycobacterium interjectum, Mycobacterium heidelberense, Mycobacterium lentiflavum, Mycobacterium malmoense, Mycobacterium microgenicum, Mycobacterium microti, Mycobacterium mucogenicum, Mycobacterium neoaurum, Mycobacterium nonchromogenicum, Mycobacterium peregrinum peregrinum, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium thermoresistabile, Mycobacterium triplex, Mycobacterium triviale, Mycobacterium tsciae tusciae, Mycobacterium ulcerans, Mycobacterium vaccae, Mycobacterium wolinskiiwolinskyi and Mycobacterium xenopi ; Rickettsia species (e.g., Ricksettsii or Coxiella burnetii) may be present.

[0048] Gram-positive bacteria include, for example, Staphylococcus aureus (e.g., Staphylococcus aureus, Staph. epidermidis, Staph. saprophyticus, Staph. auricularis, Staph. capitis capitis, Staph. c. ureolyticus, Staph. caprae, Staph. cohnii cohnii, Staph. c. urealyticus, Staph. aequorum, Staph. Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis hominis, Staphylococcus h. novobiosepticius, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus saccharolyticus, Staphylococcus schleiferi schleiferi Staphylococcus schleiferi, Staphylococcus schleiferi coagulans, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus.xylosus, Streptococci (e.g., β-hemolytic Streptococcus pyogenes (Streptococcus agalactiae, Streptococcus canis, Streptococcus dysgalactiae dysgalactiae, Streptococcus dysgalactiae equisimilis, Streptococcus equi equi, Streptococcus equi zooepidemicus, Streptococcus iniae, Streptococcus porcinus, and Streptococcus pyogenes) pyogenes), microaerophilic pyogenic streptococci (Streptococcus "milleri" e.g., Streptococcus anginosus, Streptococcus constellatus constellatus, Streptococcus constellatus pharyngidis and Streptococcus intermedius), "mitis" (α-haemolytic Streptococcus "viridans" e.g., Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus cristatus, Streptococcus gordonii, etc.). gordonii and Streptococcus parasanguinis), "salivarius" (non-hemolytic, e.g., Streptococcus salivarius and Streptococcus vestivelis),Oral streptococci of the group "mutans" (streptococci on the tooth surface, e.g., Streptococcus vestibularis) and "mutans" (streptococci on the tooth surface, e.g., Streptococcus criceti, Streptococcus mutans, Streptococcus ratti and Streptococcus sobrinus), Streptococcus acidominimus, Streptococcus bovis, Streptococcus faecalis, Streptococcus equinus, Streptococcus pneumoniae and Streptococcus suis.suis, or streptococci otherwise classified as group A, B, C, D, E, G, L, P, U, or V streptococci; enterococci (e.g., Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, and Enterococcus solitarius; Bacillus anthracis.

[0049] Preferably, the bacterial infections treated by the combinations described herein are gram-negative infections. Particular gram-negative bacteria that can be treated using the combinations of the present invention include:

[0050] Enterobacteriaceae, such as Escherichia coli, Klebsiella (e.g., Klebs. pneumoniae and Klebs. oxytoca) and Proteus (e.g., Proteus mirabilis, Proteus rettgeri, and Proteus vulgaris); Haemophilus influenzae; Mycobacterium, such as Mycobacterium tuberculosis; and Enterobacter (e.g., Enterobacter cloacae). Preferably, the bacterium is a bacterium of the family Enterobacteriaceae, such as Escherichia coli and Klebsiella (e.g., Klebs. pneumoniae and Klebs. oxytoca). Particularly preferred are Escherichia coli and Klebs. pneumoniae (e.g., Klebs. pneumoniae subsp. pneumoniae).

[0051] The combinations of the present invention are particularly useful in the treatment of (multiple) drug-resistant ((M)DR) bacteria. For Enterobacteriaceae, drug resistance is most often directed against carbapenemases, i.e., carbapenemase-resistant strains and "extended-spectrum β-lactamase" (ESBL) strains, such as New Delhi metallo-β-lactamase-1 (NDM-1)-resistant Klebsiella pneumoniae and NDM-1 Escherichia coli. The combinations of the present invention are also particularly effective against carbapenemase-producing Enterobacteriaceae (CPE). Other drug-resistant strains, such as colistin-resistant strains and carbapenemase-resistant strains of bacteria outside of Enterobacteriaceae, including carbapenem-resistant Acinetobacter and carbapenem-resistant Pseudomonas harboring the blaKPC gene, may also be used.

[0052] In various embodiments, the combinations of the invention are beneficial against ESKAPE pathogens, which are six highly virulent and typically antibiotic-resistant bacterial pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. This group of Gram-positive and Gram-negative bacteria is increasingly multidrug-resistant, allowing them to evade or "escape" commonly used antibiotics. Thus, the combinations of the invention are beneficial against (M)DR strains of ESKAPE pathogens.

[0053] Advantageously, in various embodiments, the combinations of the invention can have a broader spectrum of activity than monotherapies or combinations of only two active agents. In particular, various combinations are effective against at least Acinetobacter, Pseudomonas, and Enterobacteriaceae, which have been identified by the World Health Organization as including multidrug-resistant bacteria for which new antibiotics are urgently needed.

[0054] It should be noted that while the claimed combinations can be initially shown to be functional in the treatment of (M)DR strains, they can subsequently be used to treat non-resistant strains. This is particularly beneficial because the primary therapy for Enterobacteriaceae, such as Escherichia coli and Klebsiella species (e.g., Klebsiella pneumoniae and Klebsiella oxytoca), is expensive, patent-protected antimicrobial drugs. Replacing such "prescription" drugs with "generic" antibiotic combinations would be beneficial from a therapeutic standpoint, as well as from a financial / economic standpoint at a time when governments are seeking to reduce healthcare costs.

[0055] The combinations of the present invention can be used to treat infections associated with any of the above bacterial organisms, and in particular they can be used to kill proliferative and / or clinically latent microorganisms associated with such infections, such as ESKAPE pathogen bacterial infections.

[0056] In various embodiments, the combinations of the invention are effective in treating infections caused by (1) carbapenem-resistant E. coli, Klebsiella spp., Acinetobacter spp., Pseudomonas aeruginosa, Serratia spp., or Proteus spp., (2) MRSA, vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococcus faecium (VRE), clarithromycin-resistant Helicobacter pylori, or quinolone-resistant Salmonella spp., or (3) penicillin-resistant Streptococcus pneumoniae, ampicillin-resistant Haemophilus influenzae, or quinolone-resistant Shigella spp. In various embodiments, the combinations of the invention are effective in treating infections caused by Acinetobacter baumannii, Pseudomonas aeruginosa, or MRSA.

[0057] Particular diseases that can be treated using the combinations of the invention include tuberculosis (e.g., pulmonary tuberculosis, non-pulmonary tuberculosis (lymphatic tuberculosis, urogenital tuberculosis, bone and joint tuberculosis, tuberculous meningitis) and miliary tuberculosis), anthrax, abscesses, acne vulgaris, actinomycosis, asthma, bacterial shigellosis, bacterial conjunctivitis, bacterial keratitis, bacterial vaginosis, botulism, Buruli ulcer, bone and joint infections, bronchitis (acute or chronic), brucellosis, burns, cat scratch fever, cellulitis, chancroid, cholangitis, cholecystitis, cutaneous diphtheria, cystic fibrosis, cystitis, diffuse panbronchiolitis, diphtheria, dental caries, upper respiratory tract infections, and the like. Respiratory tract diseases, eczema, empyema, endocarditis, endometritis, typhoid, enteritis, epididymitis, epiglottitis, erysipelas, erysipelas, erysipelas-like disease, erythrasma, eye infections, furuncles, Gardnerella vaginitis, gastrointestinal infections (gastroenteritis), genital infections, gingivitis, gonorrhea, granuloma inguinale, Haverhill fever, infected burns, infections after dental surgery, infections of the oral region, infections associated with prosthetic devices, intra-abdominal abscesses, Legionnaires' disease, leprosy, leptospirosis, listeriosis, liver abscess, Lyme disease, lymphogranuloma venereum, mastitis, mastoiditis, meningitis and nervous system infections, mycetoma, nodules Cardiosis (e.g., madura's foot), nonspecific urethritis, ophthalmia (e.g., neonatal ophthalmia), osteomyelitis, otitis (e.g., otitis externa and otitis media), orchitis, pancreatitis, paronychia, pelvic peritonitis, peritonitis, peritonitis associated with appendicitis, pharyngitis, cellulitis, pinta, infectious diseases, pleural effusion, pneumonia, postoperative wound infection, postoperative gas gangrene, prostatitis, pseudomembranous colitis, psittacosis, emphysema, pyelonephritis, pyoderma (e.g., impetigo), Q fever, rat-bite fever, reticulosis, ricin poisoning, Litter's disease, salmonellosis, salpingitis, septic arthritis, septic infection, sepsis, sinusitis, skin infections (e.g., cutaneous granuloma, impetigo) rash, folliculitis and furunculosis), syphilis, systemic infections, tonsillitis, toxic shock syndrome, trachoma, tularemia, typhoid, typhus (e.g. epidemic typhus, typhus fever, grassland fever and spotted fever), urethritis, wound infections, yaws, aspergillosis, candidiasis (e.g. oropharyngeal candidiasis, vaginal candidiasis or balanitis), cryptococcosis, jaundice, histoplasmosis, intertrigo, mucormycosis, tinea (e.g. tinea corporis, tinea capitis, tinea toughensis, tinea pedis and tinea unguium), onychomycosis, pityriasis iridum, ringworm and sporotrichosis;or infections caused by MSSA, MRSA, Staph. epidermidis, Streptococcus agalactiae, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Haemophilus influenzae, Enterococcus faecalis, and Enterococcus faecium;

[0058] Particular diseases that can be treated using the combinations of the present invention include those caused by gram-negative bacteria, such as abscesses, asthma, bacterial dysentery, bacterial conjunctivitis, bacterial keratitis, bacterial vaginosis, bone and joint infections, bronchitis (acute or chronic), brucellosis, burns, cat scratch fever, cellulitis, chancroid, cholangitis, cholecystitis, cystic fibrosis, cystitis, diffuse panbronchiolitis, dental caries, upper respiratory tract disease, empyema, endocarditis, endometritis, typhoid, enteritis, epididymitis, epiglottitis, eye infections, furuncles, Gardnerella vaginitis, gastrointestinal infections (gastroenteritis), genital infections, gingivitis, gonorrhea, granuloma inguinale, Haverhill fever, infected burns, infections after dental surgery, infections of the oral region, prosthetic devices, and the like. infections associated with the liver, intra-abdominal abscess, legionellosis, leptospirosis, listeriosis, liver abscess, Lyme disease, lymphogranuloma venereum, mastitis, mastoiditis, meningitis and nervous system infections, nonspecific urethritis, ophthalmia (e.g., neonatal ophthalmia), osteomyelitis, otitis (e.g., otitis externa and otitis media), orchitis, pancreatitis, paronychia, pelvic peritonitis, peritonitis, peritonitis associated with appendicitis, pharyngitis, pleural effusion, pneumonia, postoperative wound infection, postoperative gas gangrene, prostatitis, pseudomembranous colitis, psittacosis, pyelonephritis, Q fever, Litter's disease, salmonellosis, salpingitis, septic arthritis, septic infection, sepsis, systemic infection, tonsillitis, trachoma, typhoid fever, urethritis, urinary tract infection, wound infection, or Escherichia coli coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Haemophilus influenzae, Enterococcus faecalis, Enterococcus faecium, and Enterobacter cloacae.

[0059] Preferably, the combinations of the invention are used to treat acute or complicated urinary tract infections, acute or complicated skin and soft tissue infections, intra-abdominal infections, upper respiratory tract infections, community-acquired pneumonia, hospital-acquired pneumonia, ventilator-associated pneumonia, or bloodstream infections.

[0060] It will be clear that references herein to "treatment" extend to prophylaxis as well as the treatment of existing diseases or conditions.

[0061] As used herein, the term "pharmaceutically acceptable derivative" means (a) a pharmaceutically acceptable salt; (b) a solvate (such as a hydrate); and / or (c) a prodrug (where appropriate).

[0062] Pharmaceutically acceptable salts of the compounds included in the combination of the present invention include suitable acid addition or base salts thereof. A comprehensive list of suitable pharmaceutical salts can be found in Berge et al., J Pharm Sci, 66, 119 (1977).

[0063] Suitable acid addition salts include carboxylates (e.g., formate, acetate, trifluoroacetate, propionate, isobutyrate, heptanoate, decanoate, caprate, caprylate, stearate, acrylate, caproate, propionate, ascorbate, citrate, glucuronate, glutamate, glycolate, α-hydroxybutyrate, lactate, tartrate, phenylacetate, mandelate, phenylpropionate, phenylbutyrate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, dinitrobenzoate, o-acetoxybenzoate, salicylate, nicotinate, isonicotinate, cinnamate, oxalate, malonate, succinate, Suberate, sebacate, fumarate, malate, maleate, hydroxymaleate, hippurate, phthalate, or terephthalate salts), halide salts (e.g., chloride, bromide, or iodide salts), sulfonate salts (e.g., benzenesulfonate, methyl-, bromo-, or chloro-benzenesulfonate, xylenesulfonate, methanesulfonate, ethanesulfonate, propanesulfonate, hydroxyethanesulfonate, 1- or 2-naphthalenesulfonate, or 1,5-naphthalenedisulfonate), or sulfate, pyrosulfate, bisulfite, hydrogensulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, or nitrate salts. Suitable base salts include metal salts, such as sodium salts, calcium salts, and amine salts.

[0064] For example, ceftazidime pentahydrate, colistin sulfate, polymyxin B sulfate, doxycycline hyclate (doxycycline hydrochloride hemiethanolate hemihydrate), doxycycline hydrochloride, doxycycline monohydrate, levofloxacin hemihydrate, and rifampicin N-oxide are commercially available from Sigma Aldrich. Other suppliers are known in the art.

[0065] As used herein, the term "prodrug" refers to an antimicrobial compound in which one or more groups have been modified such that the modifications are reversible upon administration to a human or mammalian subject. Such reversal is typically effected by enzymes naturally present in such subjects, although a second agent can be administered along with such a prodrug to effect reversal in vivo. Examples of such modifications include ester formation (e.g., any of those described above), where reversal can be effected by esterases, etc. Other such systems will be known in the art.

[0066] Polymyxin E, or colistin, is commercially available as its methanesulfonic acid derivative, colistimethate sodium, or colistin sodium methanesulfonate (CMS). Colitimethate sodium is a prodrug. It is produced by the reaction of colistin with formaldehyde and sodium bisulfite, which adds a sulfomethyl group to the primary amine of colistin. In aqueous solution, it hydrolyzes to form a complex mixture with partially sulfomethylated derivatives.

[0067] The present invention includes the use of these pharmaceutically acceptable derivatives and prodrugs. In particular, the present invention includes the use of colistin and its pharmaceutically acceptable derivatives, including colistin sulfate, colistimethate sodium, and colistin sodium methanesulfonate.

[0068] The present invention also includes, where appropriate, all enantiomers and tautomers of the compounds. Compounds having optical properties (one or more chiral carbon atoms) or tautomeric characteristics will be recognized by those skilled in the art. The corresponding enantiomers and / or tautomers can be isolated or prepared by methods known in the art.

[0069] Some of the compounds included in the combinations of the present invention can exist as stereoisomers and / or geometric isomers, e.g., they can have one or more asymmetric and / or geometric centers and therefore can exist in more than one stereoisomeric and / or geometric isomeric form. The present invention contemplates the use of all individual stereoisomers and geometric isomers of the inhibitors, as well as mixtures thereof. The terms used in the claims encompass these forms, provided that the appropriate functional activity is retained (though not necessarily to the same degree).

[0070] The present invention also encompasses all suitable isotopic forms of the compound or its pharmaceutically acceptable salts. An isotopic form or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass normally found in nature. Examples of isotopes that can be incorporated include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, for example, 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 31 P, 32 P, 35 S, 18 F and 36 Cl. Certain isotopic forms, e.g. 3 H or 14 Incorporation of radioactive isotopes such as 1C is useful in drug and / or substrate tissue distribution studies. 3 H, and carbon-14, i.e. 14 C isotopes are particularly preferred for their ease of preparation and detectability. 2 Substitution with isotopes such as H may be preferable in some circumstances due to the potential for greater metabolic stability and certain therapeutic advantages, such as increased in vivo half-life or reduced required dosage. Isotopic forms can be prepared by conventional procedures using appropriate isotopic forms of suitable reagents.

[0071] The compounds, including pharmaceutically acceptable derivatives or prodrugs, used in the combinations of the present invention are commercially available and / or can be prepared by synthetic methods known in the art. Ceftazidime, polymyxin E, polymyxin B, doxycycline, levofloxacin, doxycycline, rifampicin, ceftazidime pentahydrate, colistin sulfate, colistimethate sodium, colistin sodium methanesulfonate, polymyxin B sulfate, doxycycline hyclate (doxycycline hydrochloride hemiethanolate hemihydrate), doxycycline hydrochloride, doxycycline monohydrate, levofloxacin hemihydrate, and rifampicin N-oxide are commercially available from Sigma-Aldrich, e.g., Sigma-Aldrich®. Other commercial suppliers are known in the art.

[0072] Ceftazidime, sold under brand names such as Fortaz, is a third-generation cephalosporin antibiotic used to treat many bacterial infections. Its chemical structure is:

[0073] [ka]

[0074] Polymyxin E, also known as colistin, is an antibiotic used as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections, such as pneumonia. These infections may be caused by bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter. It is available in two forms: colistimethate sodium, which can be injected intravenously, intramuscularly, or inhaled, and colistin sulfate, which is primarily applied to the skin or taken orally. Its chemical structure is as follows:

[0075] [ka]

[0076] Polymyxin B, sold under trade names such as Poly-Rx, is an antibiotic used to treat meningitis, pneumonia, sepsis, and urinary tract infections. It can be administered by injection into a vein, muscle, or cerebrospinal fluid, or by inhalation. It has the following chemical structure:

[0077] [ka]

[0078] Doxycycline is a broad-spectrum antibiotic in the tetracycline family used to treat bacterial and certain parasitic infections. It is used to treat bacterial pneumonia, acne, chlamydial infections, Lyme disease, cholera, typhoid, and syphilis. Doxycycline can be taken orally or intravenously. It has the following chemical structure:

[0079] [ka]

[0080] Levofloxacin, sold under brand names such as Levaquin, is an antibiotic used to treat many bacterial infections, including acute bacterial sinusitis, pneumonia, urinary tract infections, chronic prostatitis, and some forms of gastroenteritis. It is available orally, intravenously, or in eye drop form. It is the (S)-isomer of ofloxacin and has the following chemical structure:

[0081] [ka]

[0082] Rifampicin, also known as rifampin, is an ansamycin antibiotic used to treat several bacterial infections, including tuberculosis, Mycobacterium avium complex infections, leprosy, and Legionnaires' disease. It is administered orally or intravenously and has the following chemical structure:

[0083] [ka]

[0084] The synergistic combination of the present invention includes four antimicrobial agents, which are grouped in the appended claims to most effectively cover the exemplified combinations.

[0085] In various embodiments, the at least four antimicrobial agents are selected from rifampicin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof. Alternatively, the at least four antimicrobial agents are selected from levofloxacin, polymyxin B, polymyxin E, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof. A further alternative is where the at least four antimicrobial agents are selected from rifampicin, levofloxacin, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof.

[0086] In various embodiments, the at least four antimicrobial agents are rifampicin, polymyxin E, doxycycline, and ceftazidime, or pharmaceutically acceptable derivatives thereof.

[0087] In various embodiments, the at least four antimicrobial agents are levofloxacin, polymyxin E, doxycycline, and ceftazidime, or pharmaceutically acceptable derivatives thereof.

[0088] In various embodiments, the at least four antimicrobial agents are rifampicin, levofloxacin, doxycycline, ceftazidime, or pharmaceutically acceptable derivatives thereof.

[0089] Preferably, the at least four antimicrobial agents include at least ceftazidime, doxycycline, or pharmaceutically acceptable derivatives thereof.

[0090] More preferably, the at least four antimicrobial agents include (i) ceftazidime or a pharmaceutically acceptable derivative thereof, (ii) doxycycline or a pharmaceutically acceptable derivative thereof, (iii) polymyxin E, polymyxin B, levofloxacin or a pharmaceutically acceptable derivative thereof, and (iv) levofloxacin, rifampicin or a pharmaceutically acceptable derivative thereof, with the proviso that (iii) and (iv) are different.

[0091] The compounds for use according to the present invention can be administered as bulk substances, but are preferably provided in the form of pharmaceutical compositions. The compounds can be used as separate formulations or as a single combined formulation. When combined in the same formulation, it will be clear that the two compounds must be stable and compatible with each other and with the other components of the formulation.

[0092] The formulations of the present invention include those suitable for oral, parenteral (e.g., subcutaneous, intrathecal, intramuscular, and intravenous administration, e.g., by injection or depot tablet), and rectal administration, or in a form suitable for inhalation or insufflation administration. The most suitable route of administration may depend on the condition and disorder of the patient. Preferably, the compositions of the present invention are formulated for oral administration.

[0093] The formulations can conveniently be presented in unit dosage forms and can be prepared by any method known in the pharmaceutical industry, for example, as described in "Remington: The Science and Practice of Pharmacy", Lippincott Williams and Wilkins, 21st Edition, (2005). A suitable method includes combining the active ingredient with a carrier, which constitutes one or more excipients. In general, the formulations are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the mixture into the desired formulation. It will be apparent that when these two active ingredients are administered independently, they can be administered by different means.

[0094] When formulated with excipients, the active ingredient may be present in a concentration of 0.1 to 99.5% by weight (such as 0.5 to 95% by weight) of the total mixture, conveniently 30 to 95% for tablets and capsules, and 0.01 to 50% (e.g., 3 to 50%) for liquid formulations.

[0095] The concentration of each antimicrobial agent in a synergistic combination is determined by the minimum inhibitory concentration (i.e., MIC) of the agent in monotherapy against the bacteria for which the combination is used. mono ) below. Therefore, any reference to "MIC" in this specification means MIC unless otherwise specified. mono Preferably, the concentration of at least one of the antimicrobial agents in the synergistic combination is less than the MIC mono and more preferably, the concentrations of at least two of the antimicrobial agents in the synergistic combination are less than the MIC mono The use of such concentrations is advantageous because it avoids toxicity issues and reduces the likelihood of the development of antimicrobial resistance to one or more of the agents in the combination.

[0096] In various embodiments, the concentration of ceftazidime is less than or equal to 1×MIC against the bacteria with which the combination is used, where MIC is the minimum inhibitory concentration of ceftazidime when used alone against the bacteria, i.e., MIC mono Preferably, the concentration of ceftazidime is 0.5 x MIC against the bacteria for which the combination is used. mono More preferably, the concentration of ceftazidime is 0.25 x MIC against the bacteria for which the combination is used. mono Most preferably, the concentration of ceftazidime is 0.125 x MIC or less against the bacteria for which the combination is used. mono In some embodiments, the concentration of ceftazidime is less than or equal to 0.0625 x MIC against the bacteria against which the combination is used. mono (1 / 16 MIC mono In another embodiment, the concentration of ceftazidime is as low as 0.0039 x MIC against the bacteria for which the combination is used. mono (1 / 32 MIC mono This is a low value (equivalent to

[0097] For example, in a combination that exhibits a synergistic effect against ESBL E. coli, the ceftazidime concentration can be about 32 mg / L or less. In a preferred embodiment, in a combination that exhibits a synergistic effect against ESBL E. coli, the ceftazidime concentration can be about 1 to 32 mg / L.

[0098] As another example, in a combination that exhibits a synergistic effect against KPC Klebsiella pneumoniae (K. pneumoniae), the ceftazidime concentration can be about 32 mg / L or less. In a preferred embodiment, in a combination that exhibits a synergistic effect against KPC Klebsiella pneumoniae (K. pneumoniae), the ceftazidime concentration can be about 0.125 to about 32 mg / L.

[0099] In various embodiments, the concentration of doxycycline is 1×MIC for the bacteria against which the combination is used. mono Preferably, the concentration of doxycycline is 0.5 x the MIC for the bacteria with which the combination is used. mono More preferably, the concentration of doxycycline is 0.25 x MIC against the bacteria for which the combination is used. mono Most preferably, the concentration of doxycycline is 0.125 x MIC against the bacteria for which the combination is used. mono It can be:

[0100] As an example, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the doxycycline concentration can be about 1 mg / L or less, preferably about 0.03 to about 1 mg / L.

[0101] As another example, in a combination that exhibits a synergistic effect against KPC Klebsiella pneumoniae (K. pneumoniae), the doxycycline concentration can be about 1 mg / L or less, preferably about 0.03125 to about 1 mg / L.

[0102] A range of doxycycline concentrations can be combined with a range of ceftazidime concentrations. In various embodiments, in combinations that demonstrate synergistic effects against ESBL Escherichia coli (E. coli), ceftazidime is used at a concentration of about 1 to about 32 mg / L and doxycycline is used at a concentration of about 0.03 to about 1 mg / L. In various embodiments, in combinations that demonstrate synergistic effects against Klebsiella pneumoniae (KPC), ceftazidime is used at a concentration of about 0.125 to about 32 mg / L and doxycycline is used at a concentration of about 0.03125 to about 1 mg / L, more preferably 0.25 to about 32 mg / L.

[0103] In various embodiments, the concentration of rifampicin is 1×MIC for the bacteria for which the combination is used. monoPreferably, the concentration of rifampicin is 0.5 x MIC against the bacteria for which the combination is used. mono More preferably, the concentration of rifampicin is 0.25 x MIC against the bacteria for which the combination is used. mono Most preferably, the concentration of rifampicin is 0.125 x MIC against the bacteria for which the combination is used. mono For example, in various embodiments, the concentration of rifampicin can be less than or equal to 0.0625 x MIC against the bacteria for which the combination is used. mono It can be.

[0104] For example, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the rifampicin concentration can be about 4 mg / L or less, preferably about 0.125 to about 4 mg / L. In another example, in a combination that exhibits a synergistic effect against Klebsiella pneumoniae (KPC), the rifampicin concentration can be about 4 mg / L or less, preferably about 0.125 to about 4 mg / L.

[0105] The concentration ranges of rifampicin can be combined with the respective concentration ranges of ceftazidime and doxycycline set forth above.

[0106] In various embodiments, in a combination that exhibits a synergistic effect against ESBL E. coli, ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, and rifampicin is used at a concentration of about 0.125 to about 4 mg / L. Preferably, in a combination that exhibits a synergistic effect against ESBL E. coli, the ceftazidime concentration can be about 1 to about 32 mg / L, the doxycycline concentration can be about 0.06 to about 1 mg / L, and the rifampicin concentration can be about 0.125 to about 4 mg / L.

[0107] In various embodiments, in combinations that demonstrate synergistic effects against ESBL E. coli, ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, and rifampicin is used at a concentration of 1 to 4 mg / L.

[0108] In various embodiments, in combinations that demonstrate synergistic effects against ESBL E. coli, ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, and rifampicin is used at a concentration of 0.125 to 4 mg / L.

[0109] In various embodiments, in combinations that exhibit a synergistic effect against ESBL E. coli, ceftazidime is used at a concentration of about 8 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, and rifampicin is used at a concentration of about 0.125 to about 4 mg / L. Preferably, in combinations that exhibit a synergistic effect against ESBL E. coli, ceftazidime can be used at a concentration of about 8 to about 32 mg / L, doxycycline can be used at a concentration of 0.125 to about 1 mg / L, and rifampicin can be used at a concentration of about 0.125 to about 4 mg / L.

[0110] In various embodiments, in a combination exhibiting a synergistic effect against KPC K. pneumoniae, ceftazidime is used at a concentration of about 0.125 to about 32 mg / L, doxycycline is used at a concentration of about 0.03125 to about 1 mg / L, and rifampicin is used at a concentration of about 0.125 mg / L to about 4 mg / L. Preferably, in a combination exhibiting a synergistic effect against KPC K. pneumoniae, ceftazidime is used at a concentration of about 0.125 to about 32 mg / L, doxycycline is used at a concentration of about 0.25 to about 1 mg / L, and rifampicin is used at a concentration of about 0.125 mg / L to about 4 mg / L. More preferably, in the combination that exhibits synergistic effects against KPC K. pneumoniae, ceftazidime is used at a concentration of about 0.125 to about 32 mg / L, doxycycline is used at a concentration of about 0.25 to about 1 mg / L, and rifampicin is used at a concentration of about 0.25 mg / L to about 4 mg / L.

[0111] The concentrations of ceftazidime, doxycycline, and rifampicin were determined based on the MIC mono For example, ceftazidime is 1 × MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: rifampicin at 1x MIC mono The following concentrations can be used: Preferably, ceftazidime is at 1×MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: rifampicin 0.125 to 1 × MIC mono It can be used at a concentration of

[0112] In various embodiments, ceftazidime is administered at a concentration of 0.5×MIC mono Doxycycline can be used at the following concentrations: 1x MIC mono The following concentrations can be used: rifampicin 1x MIC mono The following concentrations can be used: Alternatively, ceftazidime can be used at 1x the MICmono Doxycycline can be used at concentrations of 0.5x MIC. mono The following concentrations can be used: rifampicin 1x MIC mono The following concentrations are used:

[0113] In various embodiments, the concentration of polymyxin E or polymyxin B is 1×MIC for the bacteria for which the combination is used. mono Preferably, the concentration of polymyxin E or polymyxin B is 0.5 x MIC against the bacteria for which the combination is used. mono More preferably, the concentration of polymyxin E or polymyxin B is 0.25 x MIC against the bacteria for which the combination is used. mono Most preferably, the concentration of polymyxin E or polymyxin B is 0.125 x MIC against the bacteria for which the combination is used. mono It can be:

[0114] For example, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the concentration of polymyxin E or polymyxin B can be about 2 mg / L or less, preferably about 0.06 to about 2 mg / L.

[0115] In another example, in a combination that exhibits a synergistic effect against KPC Klebsiella pneumoniae (K. pneumoniae), the concentration of polymyxin E or polymyxin B can be about 1 mg / L or less, preferably about 0.125 mg / L to about 1 mg / L.

[0116] The polymyxin E / B concentration range can be combined with the respective concentration ranges of ceftazidime, doxycycline, and rifampicin listed above.

[0117] In various embodiments, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, rifampicin is used at a concentration of about 0.125 to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.06 to about 2 mg / L. Preferably, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the ceftazidime concentration can be about 1 to about 32 mg / L, the doxycycline concentration can be about 0.125 to about 1 mg / L, the rifampicin concentration can be about 0.125 to about 4 mg / L, and the polymyxin E / B concentration can be about 0.125 to about 2 mg / L.

[0118] In various embodiments, in combinations that exhibit a synergistic effect against ESBL E. coli, ceftazidime is used at a concentration of about 8 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, rifampicin is used at a concentration of about 0.125 to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.06 to about 2 mg / L. Preferably, in combinations that exhibit a synergistic effect against ESBL E. coli, ceftazidime can be used at a concentration of about 8 to about 32 mg / L, doxycycline can be used at a concentration of about 0.125 to about 1 mg / L, rifampicin can be used at a concentration of about 0.125 to about 4 mg / L, and polymyxin E / B can be used at a concentration of about 0.06 to about 2 mg / L.

[0119] In various embodiments, in combinations that demonstrate synergistic effects against ESBL Escherichia coli (E. coli), ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, rifampicin is used at a concentration of about 1 to 4 mg / L, and polymyxin E / B is used at a concentration of about 0.06 to about 2 mg / L.

[0120] In various embodiments, in a combination that exhibits a synergistic effect against ESBL E. coli, ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, rifampicin is used at a concentration of about 0.125 to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.25 to about 2 mg / L. Preferably, in a combination that exhibits a synergistic effect against ESBL E. coli, ceftazidime is used at a concentration of about 4 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, rifampicin is used at a concentration of about 0.25 to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.25 to about 2 mg / L.

[0121] In various embodiments, in combinations that demonstrate synergistic activity against KPC K. pneumoniae, ceftazidime is used at a concentration of about 0.125 to about 32 mg / L, doxycycline is used at a concentration of about 0.03125 to about 1 mg / L, rifampicin is used at a concentration of about 0.125 mg / L to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.125 mg / L to about 1 mg / L. Preferably, in the combinations that exhibit synergistic effects against KPC K. pneumoniae, ceftazidime is used at a concentration of about 0.125 to about 32 mg / L, doxycycline is used at a concentration of about 0.03125 to about 1 mg / L, rifampicin is used at a concentration of about 0.125 mg / L to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.125 mg / L to about 1 mg / L. More preferably, in the combination exhibiting a synergistic effect against KPC K. pneumoniae, ceftazidime is used at a concentration of about 0.125 to about 32 mg / L, doxycycline is used at a concentration of about 0.25 to about 1 mg / L, rifampicin is used at a concentration of about 0.125 mg / L to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.125 mg / L to about 1 mg / L. Most preferably, in the combination exhibiting a synergistic effect against KPC K. pneumoniae, ceftazidime is used at a concentration of about 0.125 to about 32 mg / L, doxycycline is used at a concentration of about 0.25 to about 1 mg / L, rifampicin is used at a concentration of about 0.25 mg / L to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.125 mg / L to about 1 mg / L.

[0122] The concentrations of ceftazidime, doxycycline, rifampicin, and polymyxin E / B were determined based on the MIC mono For example, ceftazidime is 1 × MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: rifampicin at 1x MIC monoIt can be used at the following concentrations: Polymyxin E / B is 1x MIC mono The following concentrations can be used: Preferably, ceftazidime is at 1×MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: rifampicin 0.0625 to 1 × MIC mono Polymyxin E / B can be used at concentrations of 0.0625 to 1 × MIC. mono More preferably, ceftazidime can be used at a concentration of 1×MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: rifampicin 0.0625 to 1 × MIC mono Polymyxin E / B can be used at concentrations of 0.125 to 1 × MIC. mono It can be used at a concentration of

[0123] In various embodiments, ceftazidime is administered at a concentration of 0.5×MIC mono Doxycycline can be used at the following concentrations: 1x MIC mono The following concentrations can be used: polymyxin E / B and rifampicin, 1x MIC each. mono The following concentrations can be used: Alternatively, ceftazidime can be used at 1x the MIC mono Doxycycline can be used at concentrations of 0.5x the MIC. mono The following concentrations can be used: polymyxin E / B and rifampicin, 1x MIC each. mono The following concentrations can be used:

[0124] In various embodiments, ceftazidime is administered at a concentration of 0.5×MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: rifampicin at 1x MIC mono It can be used at the following concentrations: Polymyxin E / B 0.125 to 1 x MIC mono It can be used at a concentration of

[0125] In various embodiments, the concentration of levofloxacin is 1×MIC against the bacteria for which the combination is used. mono Preferably, the concentration of levofloxacin is 0.5 x the MIC against the bacteria for which the combination is used. mono More preferably, the concentration of levofloxacin is 0.25 x MIC against the bacteria for which the combination is used. mono Most preferably, the concentration of levofloxacin is 0.125 x MIC against the bacteria for which the combination is used. mono The following is the result.

[0126] For example, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the levofloxacin concentration can be about 8 mg / L or less, preferably about 0.25 to about 8 mg / L.

[0127] The concentration range of levofloxacin can be combined with the respective concentration ranges of ceftazidime and doxycycline. In various embodiments, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, and levofloxacin is used at a concentration of about 0.25 to about 8 mg / L. Preferably, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the ceftazidime concentration can be about 8 to about 32 mg / L, the doxycycline concentration can be about 0.03 to about 1 mg / L, and the levofloxacin concentration can be about 0.25 to about 8 mg / L. Most preferably, in a combination that exhibits a synergistic effect against ESBL E. coli, the ceftazidime concentration can be about 16 to about 32 mg / L, the doxycycline concentration can be about 0.03 to about 1 mg / L, and the levofloxacin concentration can be about 0.25 to about 8 mg / L.

[0128] The concentration ranges of ceftazidime, doxycycline and levofloxacin can also be combined with the concentration ranges of polymyxin E / B or rifampicin as shown below.

[0129] In various embodiments, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, levofloxacin is used at a concentration of about 0.25 to about 8 mg / L, and polymyxin E / B is used at a concentration of about 0.06 to about 2 mg / L. Preferably, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the ceftazidime concentration can be about 4 to about 32 mg / L, the doxycycline concentration can be about 0.06 to about 1 mg / L, the levofloxacin concentration can be about 8 mg / L, and the polymyxin E / B concentration can be about 0.125 to about 2 mg / L.

[0130] In various embodiments, in combinations that demonstrate synergistic effects against ESBL Escherichia coli (E. coli), ceftazidime is used at a concentration of about 16 to about 32 mg / L, doxycycline is used at a concentration of about 0.03 to about 1 mg / L, levofloxacin is used at a concentration of about 0.125 to about 4 mg / L, and polymyxin E / B is used at a concentration of about 0.06 to about 2 mg / L.

[0131] The concentrations of ceftazidime, doxycycline, levofloxacin, and polymyxin E / B were determined based on the MIC mono For example, ceftazidime is 1 × MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: levofloxacin at 1x MIC mono It can be used at the following concentrations: Polymyxin E / B is 1x MIC mono The following concentrations can be used: Preferably, ceftazidime is at 1×MIC mono The following concentrations can be used: doxycycline at 1x MIC monoThe following concentrations can be used: levofloxacin 0.0625 to 1 × MIC mono Polymyxin E / B can be used at concentrations of 0.0625 to 1 × MIC. mono It can be used at a concentration of

[0132] In various embodiments, ceftazidime is 0.5×MIC mono ~1×MIC mono Doxycycline can be used at a concentration of 1x MIC mono The following concentrations can be used: polymyxin E / B and levofloxacin, 1x MIC each. mono The following concentrations are used:

[0133] In various embodiments, in a combination that exhibits a synergistic effect against ESBL E. coli, ceftazidime is used at a concentration of about 1 to about 32 mg / L, doxycycline is used at a concentration of about 0.06 to about 1 mg / L, levofloxacin is used at a concentration of about 0.25 to about 8 mg / L, and rifampicin is used at a concentration of about 0.03 to 1 mg / L. Preferably, in a combination that exhibits a synergistic effect against ESBL E. coli, ceftazidime can be used at a concentration of about 2 to about 32 mg / L, doxycycline can be used at a concentration of about 0.06 to about 1 mg / L, levofloxacin can be used at a concentration of about 2 to about 8 mg / L, and rifampicin can be used at a concentration of about 0.03 to 1 mg / L. More preferably, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the concentration of ceftazidime can be about 16 to 32 mg / L, the concentration of doxycycline can be about 0.06 to 1 mg / L, the concentration of levofloxacin can be about 4 to 8 mg / L, and rifampicin is used at a concentration of about 0.03 to 1 mg / L.

[0134] Alternatively, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the ceftazidime concentration can be about 2 to about 32 mg / L, the doxycycline concentration can be about 0.06 to about 0.5 mg / L, the levofloxacin concentration can be about 0.25 to about 8 mg / L, the doxycycline concentration can be about 0.06 to about 1 mg / L, the levofloxacin concentration can be about 2 to about 8 mg / L, and the rifampicin concentration can be about 0.03 to 1 mg / L. Preferably, in a combination that exhibits a synergistic effect against ESBL Escherichia coli (E. coli), the ceftazidime concentration can be about 16 to about 32 mg / L, the doxycycline concentration can be about 0.06 to about 1 mg / L, the levofloxacin concentration can be about 0.25 to about 8 mg / L, and the rifampicin concentration can be about 0.03 to 1 mg / L.

[0135] The concentrations of ceftazidime, doxycycline, levofloxacin, and rifampicin were determined based on the MIC mono For example, ceftazidime is 1 × MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: levofloxacin at 1x MIC mono The following concentrations can be used: rifampicin at 1x MIC mono The following concentrations can be used: Preferably, ceftazidime is at 1×MIC mono The following concentrations can be used: doxycycline at 1x MIC mono The following concentrations can be used: levofloxacin at 1x MIC mono The following concentrations can be used: rifampicin at 1x MIC mono The following concentrations can be used: Ceftazidime 0.5 to 1 x MIC mono and doxycycline at 1×MIC. mono and levofloxacin at 1×MIC. mono The following concentrations can be used: rifampicin at 1x MIC mono It can be used in the following concentrations:

[0136] MIC as defined herein mono The lower limit of the range is not limited. If not specified, it is preferably 1 / 512 MIC. mono , 1 / 256 MIC mono , 1 / 128 MIC mono , 1 / 64 MIC mono , 1 / 32 MIC mono , or 0.0625MIC mono For example, "0.5 x MIC mono "Less than" means "0.5 x MIC mono ~0.0625MIC mono "

[0137] Formulations suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets (e.g., chewable tablets, particularly for pediatric administration), each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. The active ingredient may also be presented as a bolus, electuary, or paste.

[0138] Tablets can be made by compression or molding, optionally with one or more excipients. Compressed tablets can be made by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with other conventional excipients such as binders (e.g., syrup, acacia, gelatin, sorbitol, tragacanth, starch paste, polyvinylpyrrolidone, and / or hydroxymethylcellulose), fillers (e.g., lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate, and / or sorbitol), lubricants (e.g., magnesium stearate, stearic acid, talc, polyethylene glycol, and / or silica), disintegrants (e.g., potato starch, croscarmellose sodium, and / or sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate). Molded tablets can be made by molding, in a suitable machine, a mixture of the powdered active ingredient and an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide controlled release of the active ingredient, for example delayed, sustained or pulsed release, or a combination of immediate and controlled release.

[0139] Alternatively, the active ingredient can be incorporated into oral liquid preparations such as aqueous or oily suspensions, solutions, emulsions, syrups or elixirs. Formulations containing the active ingredient can also be presented as a dry product for reconstitution with water or another suitable vehicle before use.

[0140] Such liquid formulations may contain conventional additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxymethylcellulose, carboxymethylcellulose, aluminum stearate gel, and / or hydrogenated edible fats and oils), emulsifiers (e.g., lecithin, sorbitan monooleate, and / or acacia), non-aqueous vehicles (e.g., edible oils such as almond oil, coconut oil, oily esters, propylene glycol, and / or ethyl alcohol), and preservatives (e.g., methyl or propyl p-hydroxybenzoate and / or sorbic acid).

[0141] The combinations for use according to the invention may be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredients. The pack may, for example, comprise metal or plastic foil, such as a blister pack. When the compositions are intended to be administered as two separate compositions, they may be presented in the form of a twin pack.

[0142] Pharmaceutical compositions may also be prescribed to patients in "patient packs" that contain the entire course of treatment in a single package, usually a blister pack. Patient packs have the advantage over traditional prescriptions, in which a pharmacist dispenses a patient's supply of medication from a bulk supply, that the patient always has access to the package insert contained in the patient pack, which is typically lost with traditional prescriptions. The inclusion of a package insert has been shown to improve patient compliance with physician instructions.

[0143] Administration of the combination of the present invention in a single patient pack or patient packs for each composition containing package inserts instructing the patient on the correct use of the present invention is a desirable feature of the present invention.

[0144] According to another embodiment of the invention there is provided a patient pack comprising at least one active agent of the combination according to the invention and an instruction leaflet providing instructions for the use of the combination according to the invention.In another embodiment of the invention there is provided a double pack comprising, in separate administrations, an antimicrobial agent, preferably biologically active against clinically latent microorganisms, and one or more compounds disclosed herein, preferably biologically active against clinically latent microorganisms.

[0145] The amount of active ingredient required for treatment will vary depending on the nature of the disease being treated and the age and condition of the patient, and is ultimately at the discretion of the attending physician. In general, however, doses used for adult treatment will typically range from 0.02 to 5000 mg / day, preferably 1 to 1500 mg / day. The desired dose can conveniently be expressed as a single dose or as divided doses administered at appropriate intervals, for example, as two, three or more subdoses per day.

[0146] Thus, this information would be readily available and understandable to one skilled in the art.

[0147] Biological testing Test procedures that can be used to determine the biological activity (e.g., bactericidal or antimicrobial activity) of an active ingredient include: (a) bactericidal activity against clinically latent bacteria; and (b) Antimicrobial activity against logarithmic-phase bacteria These include those known to those skilled in the art for measuring

[0148] With regard to (a) above, methods for measuring activity against clinically latent bacteria include measuring the minimum stationary bactericidal concentration ("MSC") or minimum dormant bactericidal concentration ("MDC") for a test compound under conditions known to those of skill in the art (such as those described in Nature Reviews, Drug Discovery 1, 895-910 (2002), the disclosure of which is incorporated herein by reference).

[0149] For example, WO2000 / 028074 describes a suitable method for screening compounds to determine their ability to kill clinically latent microorganisms. A typical method comprises the following steps: (1) growing the bacterial culture to stationary phase; (2) selecting for a phenotypically resistant subpopulation by treating the stationary phase culture with one or more antimicrobial agents at a concentration and / or for a time sufficient to kill the vegetative bacteria; (3) incubating samples of the phenotypically resistant subpopulation with one or more test compounds or drugs; and (4) Evaluating antimicrobial efficacy against phenotypically resistant subpopulations may include:

[0150] According to this method, the phenotypically resistant subpopulation can be viewed as representing clinically latent bacteria that remain metabolically active in vivo and can cause recurrent or progressive disease.

[0151] With regard to (b) above, methods for measuring activity against log-phase bacteria include determining the minimum inhibitory concentration ("MIC") or minimum bactericidal concentration ("MBC") for the test compound under standard conditions (i.e., conditions known to those of skill in the art, such as those described in WO2005 / 014585, the disclosure of which is incorporated herein by reference). Specific examples of such methods are described below. [Example]

[0152] Antimicrobial agents were commercially available. They were weighed and dissolved in water, PBS, DMSO, or acidified water to prepare final concentrations of 1–10 mg / mL. The antimicrobial solution was diluted 10-fold from the highest concentration used in the experiment, and then serially diluted 2-fold, not exceeding 11 serial dilutions. This allowed the operator to use up to 12 different, decreasing concentrations of the selected antimicrobial agent.

[0153] Bacteria were obtained from Ninewells Hospital and Medical School in Dundee, Scotland. They were obtained as patient strains and characterized by Vitek2 screening. To prepare for the following examples, bacteria were grown overnight in Mueller-Hinton cation-adjusted broth or grown to confluence in medium with or without supplements. The OD of the bacteria was 600 OD was measured and readings below 0.25 were returned to the incubator. 600 The bacterial culture was diluted in medium until the reading was = <0.01, which is the approximate value for the culture of 10 6 This indicated that the CFU / mL.

[0154] All data herein was generated using the same checkerboard assay.

[0155] 20 μL of antibiotic A (backbone) and 20 μL of antibiotic B (backbone) of a single dilution were pipetted into all wells of the 96-well plate being used.

[0156] 20 μL of the lowest concentration of antibiotic C (first variable) was pipetted into column 1 of a 96-well plate. The second concentration (2x the concentration) was pipetted into column 2. This process was repeated until all concentrations of antibiotic B used were completed.

[0157] 20 μL of the lowest concentration of antibiotic D (second variable) was pipetted into row A of a 96-well plate. The second concentration (2x the concentration) was pipetted into row B. This process was repeated until all concentrations of antibiotic C used were completed.

[0158] 120 μL of sterile medium (BHI / MHB2) was added.

[0159] 20 μL of the prepared bacterial culture was added.

[0160] The plate was sealed with a lid and incubated overnight for 16 hours.

[0161] The plate was read at OD in a 96-well plate reader. 600 The OD values ​​are reported for each combination below.

[0162] For quaternary combinations, the concentrations of two antibiotics were varied and one was held constant, with the latter being the "backbone." This method is described above along with the calculation of FICI and index values ​​for synergy, indifference, and antagonism.

[0163] Example 1: Synergy between ceftazidime, doxycycline, colistin and rifampicin A four-way combination of ceftazidime, doxycycline, colistin, and rifampicin was tested in the above assay. Using colistin at 1 / 16 the MIC (0.125 mg / L) and rifampicin at 1 / 16 the MIC (0.25 mg / L) as the backbone, the combination was tested against Escherichia coli (ESBL) strains. The highest concentration in each experiment was equal to 1× the MIC for the isolate tested.

[0164] The isolates in these examples were resistant to cephalosporins and ceftazidime at ≥ 32 mg / L. The concentrations used are within the range of dosage guidelines recommended by EUCAST (European Committee on Antimicrobial Susceptibility Testing). EUCAST has produced breakpoint tables for the interpretation of MICs and zone diameters (see, e.g., Version 12).

[0165] The following concentrations (mg / L) of ceftazidime and doxycycline were tested:

[0166] [Table 2]

[0167] The results reported in the table above are OD600 values ​​determined according to the methods described herein. Bold values ​​in the table above (as well as the corresponding tables in subsequent examples) indicate bacterial growth that is believed to represent ineffective bacterial killing (i.e., no synergy).

[0168] The MICs of each drug alone and in combination were calculated according to the method described above.

[0169] [Table 3]

[0170] ΣFIC=0.813, FIC=0.406, which indicates a synergistic effect as explained above.

[0171] A four-way combination of ceftazidime, doxycycline, colistin, and rifampicin was also tested against ESBL Escherichia coli (E. coli) using colistin at 1 / 16 MIC (0.125 mg / L) and ceftazidime at 1 / 8 MIC (4 mg / L) as the backbone. The following concentrations (mg / L) of rifampicin (x-axis) and doxycycline (y-axis) were used:

[0172] [Table 4]

[0173] The results reported in the table above are OD600 values ​​determined according to the methods described herein.

[0174] The MICs of each drug alone and in combination were calculated according to the method described above.

[0175] [Table 5]

[0176] ΣFIC=0.44 and FIC=0.221, which indicates a synergistic effect as explained above.

[0177] A four-way combination of ceftazidime, doxycycline, colistin, and rifampicin was also tested against ESBL Escherichia coli (E. coli) using ceftazidime at 1 / 8 MIC (4 mg / L) and rifampicin at 1 / 16 MIC (0.25 mg / L) as the backbone. The following concentrations (mg / L) of colistin and doxycycline were tested:

[0178] [Table 6]

[0179] The results reported in the table above are OD600 values ​​determined according to the methods described herein.

[0180] The MICs of each drug alone and in combination were calculated according to the method described above.

[0181] [Table 7]

[0182] ΣFIC=0.34 and FIC=0.172, which indicates a synergistic effect as explained above.

[0183] Finally, a four-way combination of ceftazidime, doxycycline, colistin, and rifampicin was tested against ESBL Escherichia coli (E. coli) using ceftazidime at 1 / 8 MIC (4 mg / L) and doxycycline at 1 / 16 MIC (0.06 mg / L) as the backbone. The following concentrations (mg / L) of colistin and rifampicin were tested:

[0184] [Table 8]

[0185] The results reported in the table above are OD600 values ​​determined according to the methods described herein.

[0186] The MICs of each drug alone and in combination were calculated according to the method described above.

[0187] [Table 9]

[0188] ΣFIC=0.375 and FIC=0.188, which indicates a synergistic effect as explained above.

[0189] The various studies in Example 1 show how the four-way combination of colistin, rifampicin, doxycycline and ceftazidime is synergistic, even though all compounds are present at concentrations below the MICmono.

[0190] Synergy is not a predictable outcome, especially at concentrations below the MIC or against MDR bacteria (ESBL E. coli). This means that the combination is an important advance in the fight against antimicrobial resistance. Surprisingly, the combination works against bacteria that harbor enzymes (ESBLs) found in strains known to be resistant to many antibiotics used by those skilled in the art to treat infections.

[0191] Example 2: Synergistic Effect of Ceftazidime, Doxycycline, Colistin, and Levofloxacin A quaternary combination of ceftazidime, doxycycline, colistin, and levofloxacin was tested in the above assay. Using colistin at 1 / 16 the MIC (0.125 mg / L) and doxycycline at 1 / 16 the MIC (0.06 mg / L) as the backbone, the combination was tested against ESBL Escherichia coli (E. coli). The highest concentration in each experiment was equal to 1× the MIC of the isolate tested. The following concentrations (mg / L) of ceftazidime and levofloxacin were used:

[0192] [Table 10]

[0193] The results reported in the table above are OD600 values ​​determined according to the methods described herein.

[0194] The MICs of each drug alone and in combination were calculated according to the method described above.

[0195] [Table 11]

[0196] ΣFIC=0.719 and FIC=0.359, which indicates a synergistic effect as explained above.

[0197] Example 3: Synergy between ceftazidime, doxycycline, rifampicin and levofloxacin A quaternary combination of ceftazidime, doxycycline, rifampicin, and levofloxacin was tested in the above assay. Using rifampicin at 1 / 16 the MIC (0.06 mg / L) and doxycycline at 1 / 16 the MIC (0.06 mg / L) as the backbone, the combination was tested against ESBL Escherichia coli (E. coli). The highest concentration in each experiment was equal to 1 x the MIC of the isolate tested. The following concentrations (mg / L) of ceftazidime and levofloxacin were used:

[0198] [Table 12]

[0199] The results reported in the table above are OD600 values ​​determined according to the methods described herein.

[0200] The MICs of each drug alone and in combination were calculated according to the method described above.

[0201] [Table 13]

[0202] ΣFIC=0.656 and FIC=0.328, which indicates a synergistic effect as explained above.

[0203] Example 4: Synergy between ceftazidime (ceft), doxycycline (doxy), rifampicin (rif) and colistin (CSS) A four-way combination of ceftazidime, doxycycline, rifampicin, and colistin was tested in the above assay. Using a backbone of ceftazidime at 1 / 256 MIC (0.125 mg / L) and colistin at 1 / 8 MIC (0.125 mg / L), the combination was tested against KPC (Klebsiella pneumoniae carbapenemase)-producing K. pneumoniae. The highest concentration in each experiment was equal to 1× the MIC of the isolate tested. The following concentrations (mg / L) of ceftazidime and colistin were used:

[0204] [Table 14]

[0205] The results reported in the table above are reported as percent bacterial kill based on OD600 values ​​compared to the positive control.

[0206] [Table 15]

[0207] The MICs of each drug alone and in combination were calculated according to the method described above.

[0208] ΣFIC=0.441 and FIC=0.221, which indicates a synergistic effect as explained above.

[0209] These examples confirm that the combinations of the present invention are synergistic. Synergy is not an expected outcome when combining antimicrobial agents, and certainly not when combining four or more antimicrobial agents and / or against multidrug-resistant bacteria such as ESBL strains. These examples confirm that the combinations of the present invention are synergistic against drug-resistant bacteria, and therefore provide a solution to the global problem of antimicrobial resistance, as discussed above. This is a major advance in the art.

[0210] The various embodiments described herein are presented solely to aid in understanding and teaching the claimed features. These embodiments are provided only as a representative sample of embodiments and are not exhaustive and / or exclusive. It should be understood that the advantages, embodiments, examples, functions, features, structures, and / or other aspects described herein should not be construed as limitations on the scope of the invention as defined by the claims or limitations on the equivalents of the claims, and that other embodiments are available and modifications can be made without departing from the scope of the claimed invention. Various embodiments of the present invention may suitably comprise, consist of, or consist essentially of any suitable combination of the disclosed elements, components, features, parts, steps, means, etc., other than those specifically described herein. Furthermore, this disclosure may include other inventions not currently claimed but that may be claimed in the future.

Claims

1. A combination comprising at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime and pharmaceutically acceptable derivatives thereof.

2. 2. The combination of claim 1, wherein the at least four antimicrobial agents are selected from rifampicin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof.

3. 2. The combination of claim 1, wherein the at least four antimicrobial agents are selected from levofloxacin, polymyxin B, polymyxin E, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof.

4. 10. The combination of claim 1, wherein the at least four antimicrobial agents comprise ceftazidime and doxycycline or pharmaceutically acceptable derivatives thereof, and at least two of polymyxin E, polymyxin B, rifampicin, levofloxacin and pharmaceutically acceptable derivatives thereof.

5. 5. The combination of claim 4, wherein the at least four antimicrobial agents are levofloxacin, polymyxin E, doxycycline and ceftazidime, or pharmaceutically acceptable derivatives thereof.

6. 6. The combination of any one of claims 1 to 5, wherein the pharmaceutically acceptable derivative of polymyxin E is colistin sulfate, colistin methanesulfonate, or colistin methanesulfonate sodium.

7. 5. The combination of claim 4, wherein the at least four antimicrobial agents are rifampicin, levofloxacin, doxycycline, ceftazidime, or pharmaceutically acceptable derivatives thereof.

8. A combination according to any one of claims 1 to 7 for use in the treatment of a bacterial infection.

9. A pharmaceutical composition comprising a combination according to any one of claims 1 to 7 and a pharmaceutically acceptable adjuvant, diluent or carrier.

10. 10. The pharmaceutical composition of claim 9 for use in the treatment of a bacterial infection.

11. 11. The combination for use according to claim 8, or the pharmaceutical composition for use according to claim 10, wherein said use is in killing proliferative microorganisms associated with bacterial infections.

12. 11. The combination for use according to claim 8, or the pharmaceutical composition for use according to claim 10, wherein the infection is a gram-negative bacterial infection or a gram-positive bacterial infection.

13. 13. The combination for use according to any one of claims 8 or 11 to 12, or the pharmaceutical composition for use according to any one of claims 10 to 12, wherein the bacterial infection is caused by a bacterium of the family Enterobacteriaceae, preferably the bacterial infection is caused by E. coli or Klebsiella pneumoniae.

14. 13. The combination for use according to claim 8 or any one of claims 11 to 12, or the pharmaceutical composition for use according to any one of claims 10 to 12, wherein the bacterial infection is caused by Acinetobacter baumannii, Pseudomonas aeruginosa or MRSA.

15. The combination for use according to claim 8 or any one of claims 11 to 14, or the pharmaceutical composition for use according to any one of claims 10 to 14, wherein the infection is caused by a drug-resistant strain of bacteria.

16. A product comprising at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof as a combination drug for simultaneous, separate or sequential use in the treatment of bacterial infections.

17. 17. The product of claim 16, wherein the at least four antimicrobial agents are (i) rifampicin, polymyxin E, doxycycline, and ceftazidime, or pharmaceutically acceptable derivatives thereof; (ii) levofloxacin, polymyxin E, doxycycline, and ceftazidime, or pharmaceutically acceptable derivatives thereof; or (iii) rifampicin, levofloxacin, doxycycline, and ceftazidime, or pharmaceutically acceptable derivatives thereof.

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