Antimicrobial combinations

Abstract

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

Description

Field of the Invention

[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 for killing proliferating (i.e., logarithmic phase) microorganisms associated with bacterial infections, such as Gram-negative bacterial infections.

[0002] Background

[0003] Before the introduction of antibiotics, patients suffering from acute microbial infections, such as tuberculosis or pneumonia, had a very low chance of survival. For example, the mortality rate of tuberculosis was approximately 50%. The introduction of antimicrobial agents in the 1940s and 1950s rapidly changed this scenario, and today approximately 100 antibiotics are used to treat different bacterial infections. This contributed to the birth of modern medicine, as bacterial infections in millions of patients suffering from, for example, cancer, organ transplantation, renal dialysis, immunosuppression, and surgery can be effectively prevented and treated.

[0004] However, bacteria have responded to the widespread use of antibiotics by gradually acquiring resistance. Today, antibiotic-resistant bacteria are present in every country in the world, and this resistance is increasing year by year, thereby reducing the effectiveness of all antibiotics. By 2040 - 2050, deaths from antimicrobial-resistant infections are expected to exceed 10 million per year (The Review on Antimicrobial Resistance, chaired by Jim O’Neill, May 2016).

[0005] The increase in antimicrobial resistance is expected to occur in poorer countries by 2040 - 2050 and this has become a real and practical medical problem in Europe and the United States. In fact, in the United States, more than 70% of the bacteria that cause hospital - acquired infections are resistant to at least one of the major antimicrobial agents commonly used to combat infections (Nature Reviews, Drug Discovery, 1, 895 - 910 (2002)). Thus, the World Health Organization has classified antibiotic resistance as a "serious threat, [which] is no longer a prediction for the future, it is happening in every region of the world and has the potential to affect anyone, of any age, in any country" ("Antimicrobial resistance: global report on surveillance", World Health Organization, April 2014). If not addressed, life expectancy could return to pre - antibiotic levels, i.e., about 20 years less than now.

[0006] Therefore, there is an urgent need for solutions to the growing problem of drug - resistant bacteria. In essence, the medical community needs to replace approximately 100 antibiotics with products that are effective against microbial - resistant infections while avoiding the development of future antimicrobial resistance.

[0007] Current efforts to solve this problem have mainly focused on the development of new chemical entities (NCEs). Each NCE requires more than 10 years of development and costs over $600 million to complete the necessary safety and clinical testing. A large number will fail and thus it typically takes about $3.8 billion to deliver one NCE antibiotic. Ironically, with the increase in antibiotic use, the development of antimicrobial resistance is also faster, meaning that all NCEs have a limited lifespan, usually less than 10 years. Thus, replacing the currently used antibiotics with NCEs over a 10 - year period would require approximately $3.8 trillion, and even if successful, these products would then need ongoing programs to replace them as each antimicrobial resistance emerges during their 10 - year lifespan. This is clearly unsustainable, even for high - income countries.

[0008] The applicant has identified a solution to this major, global problem. Specifically, combinations of four or more known antibiotics have surprisingly been shown to have a synergistic effect against Gram - negative bacteria and / or Gram - positive bacteria. Such combinations have been found to kill antimicrobial - resistant bacteria (also known as drug - resistant bacteria) and avoid the development of antimicrobial resistance. These four or more known antibiotics are defined in the appended claims and described herein.

[0009] The synergistic effect demonstrated means that the composition has higher biological activity at the said dose levels than the additive effect of the individual agents would be expected to have. This means that less amounts (e.g., 5 to 20 times less) of the antibiotics are more effective in combination therapy than any of the individual antibiotics.

[0010] In addition, by combining approved drugs (CADs), the approval time and the costs involved are much lower than for NCEs. For example, the time is about 5 years and it takes no more than $50 million to produce a new product. Since there are far fewer failures in development, the cumulative success / failure cost for a CAD is about $130 million, compared to $3.8 billion for each NCE. To replace the approximately 100 individual antibiotics currently in use, the number of combinations required can be reduced since the CAD has activity against a wide range of different infectious species, and single antibiotics that are unavailable due to antimicrobial resistance in the CAD can be revitalized, the CAD having higher activity against these antimicrobial-resistant strains and avoiding the development of future antimicrobial resistance. As a percentage of GDP, the CAD technology moves the 4% of GDP represented by NCEs in high-income countries to about 0.005%, enabling all high, middle and even low-income countries to contribute and benefit.

[0011] Currently, the global focus is only on repetitive NCE projects, which have a higher cost / time / failure probability and a shorter remaining useful life, and it is expected that effective antibiotics will be exhausted within twenty years and antimicrobial resistance deaths will set back 200 years. Even investing 0.1% of the NCE development costs in the CAD approach would provide a practical, affordable and more durable solution, which offers a viable alternative for continuing to provide available antibiotic products - effective and affordable globally - and brings hope for "antibiotics for all, always and affordably".

[0012] 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 the present application, the applicant has developed a new approach for replacing all antibiotics by discovering a synergistic combination of three antimicrobial agents. The combination is effective against at least extended-spectrum β-lactamase (ESBL), carbapenemase-producing (CPE) and carbapenem-resistant Gram-negative bacteria. Even more surprisingly, the applicant has revealed that the active concentrations of each antimicrobial agent in the combination are at very low concentrations, e.g., as low as 1 / 16 of the MIC of the agent. mono Therefore, compared to, for example, monotherapy, it is possible to advantageously reduce any potential toxicity burden by using less amounts of each antimicrobial agent in the combination.

[0013] WO2015 / 114340 describes the use of a combination of zidovudine with a polymyxin selected from colistin or polymyxin B, an anti-tuberculosis antibiotic selected from rifampicin, rifapentine or rifabutin, and optionally piperine for the treatment of microbial infections. WO2018 / 011562 describes a combination comprising zidovudine and a carbapenem, and optionally a polymyxin selected from polymyxin B and polymyxin E. Accordingly, the present invention does not cover the combinations previously identified by these applicants.

[0014] When two or more active agents are used in combination, the synergistic effect is unpredictable or unexpected. Synergism in the context of antimicrobial agents is measured in a variety of ways, which is in line with the generally accepted view that "synergism is an effect greater than additivity". One of the methods for assessing whether synergism has been observed is to use the "checkerboard" technique. This is a widely accepted method that results in the generation of a value known as the fractional inhibitory concentration index (FICI). Orhan et al. J. Clin. Microbiol. 2005, 43(1):140 describe the checkerboard method and analysis in the bridging paragraph on pages 140 - 141, and explain that the FICI value is the ratio of the sum of the MIC (minimum inhibitory concentration) levels of each individual component in isolation and in the mixture.

[0015] The FICI (or fractional inhibitory concentration index) refers to the sum of the FICs (or fractional inhibitory concentrations) of each antimicrobial agent when used in combination. The FIC (or fractional inhibitory concentration) of an antimicrobial agent in a combination is equal to the MIC of the antimicrobial agent in the combination divided by the MIC of the same antimicrobial agent when used alone. The minimum inhibitory concentration in the art is defined as the lowest concentration of an antimicrobial agent that will inhibit visible growth of a microorganism after overnight incubation.

[0016] The combination is active against drug - resistant bacteria (see the examples herein). In many cases, the individual antimicrobial agents in these combinations have significant activity at concentrations significantly lower than their MICs when used alone. However, there does not appear to be a method in the art for defining the synergism of a 4 - mer (a combination of four antimicrobial agents) expressed in terms of fractional inhibitory concentration. In fact, very little prior work has been done in this area. Accordingly, the present inventors have devised such a method. The method is applicable to any combination of "n" antimicrobial agents.

[0017] In this method, two antibiotics are "fixed" as part of the backbone, while the other two antibiotics start at the MIC (×1) of the effective dose of monotherapy against the test microorganism and vary on a doubling concentration scale. Then, ΣFIC is calculated as shown below.

[0018] Divide ΣFIC by "0.5 / n", where n = the number of antimicrobial agents in the combination to provide FIC. The inventors chose this representation because it is closer to the level of synergy in pairs. Apply the same FIC scale used for pairs: Synergy is observed when FIC < 0.5. "Additive" effect is observed when FIC is 0.5 to <1. Indifference is observed when FIC is 1 to <2. Antagonism is observed when FIC is 2 to 4.

[0019] Synergy can be expressed as "ΣFIC ≤ 0.25×n".

[0020] Examples of Synergy

[0021]

[0022] The above method was employed in the examples herein. Summary of the Invention

[0024] In one aspect, the present invention provides a combination as defined in the appended claims. The combination comprises at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and their pharmaceutically acceptable derivatives. In various embodiments, the combination includes ceftazidime, doxycycline, or their pharmaceutically acceptable derivatives. This means that the remaining antimicrobial agents in the combination are selected from polymyxin E, polymyxin B, levofloxacin, and rifampicin, or their pharmaceutically acceptable derivatives. Pharmaceutically acceptable derivatives of polymyxin E can be defined as colistin sulfate, colistin methanesulfonate, or colistin methane sulfonate sodium.

[0025] In another aspect, the present invention provides the use of the combination defined herein for the treatment of bacterial infections.

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising the combination defined herein and a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition can be used for the treatment of bacterial infections.

[0027] On the other hand, the present invention provides a product comprising at least four antimicrobial agents selected from rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and their pharmaceutically acceptable derivatives, as a combined preparation for simultaneous, separate, or sequential use in the treatment of bacterial infections. In various embodiments, the product comprises ceftazidime, doxycycline, or their pharmaceutically acceptable derivatives. This means that the remaining antimicrobial agents in the product are selected from polymyxin E, polymyxin B, levofloxacin, and rifampicin, or their pharmaceutically acceptable derivatives. Pharmaceutically acceptable derivatives of polymyxin E can be defined as colistin sulfate, colistin mesylate, or colistin mesylate sodium.

[0028] On the other hand, 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 Gram-negative or Gram-positive bacterial infections. The first antimicrobial agent is rifampicin or its pharmaceutically acceptable derivative. The second, third, and fourth antimicrobial agents are selected from levofloxacin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and their pharmaceutically acceptable derivatives.

[0029] On the other hand, 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 Gram-negative or Gram-positive bacterial infections. The first antimicrobial agent is levofloxacin or its pharmaceutically acceptable derivative. The second, third, and fourth antimicrobial agents are selected from rifampicin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and their pharmaceutically acceptable derivatives.

[0030] On the other hand, 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 Gram-negative or Gram-positive bacterial infections. The first antimicrobial agent is polymyxin E or B, or their pharmaceutically acceptable derivatives. The second, third, and fourth antimicrobial agents are selected from levofloxacin, rifampicin, doxycycline, ceftazidime, and their pharmaceutically acceptable derivatives.

[0031] On the other hand, 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 Gram-negative or Gram-positive bacterial infections. The first antimicrobial agent is doxycycline or its pharmaceutically acceptable derivative. The second, third, and fourth antimicrobial agents are selected from levofloxacin, polymyxin E, polymyxin B, rifampicin, ceftazidime, and their pharmaceutically acceptable derivatives.

[0032] On the other hand, 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 Gram-negative or Gram-positive bacterial infections. The first antimicrobial agent is ceftazidime or a pharmaceutically acceptable derivative thereof. The second, third, and fourth antimicrobial agents are selected from levofloxacin, polymyxin E, polymyxin B, doxycycline, rifampicin, and pharmaceutically acceptable derivatives thereof.

[0033] On the other hand, the present invention provides a method for treating Gram-negative or Gram-positive bacterial infections, which method comprises 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.

[0034] These aspects and their embodiments are set forth in the appended independent and dependent claims. It will be understood that the features of the dependent claims may be combined with each other and with the features of the independent claims in combinations other than those explicitly set forth in the claims. Furthermore, the present disclosure is not limited to the specific embodiments set forth below, but includes and encompasses any combination of the features presented herein.

[0035] The foregoing and other aspects, embodiments, features, and advantages of the present disclosure will be apparent from the following detailed description. In this regard, specific portions of the specification should not be read in isolation from the other portions.

[0036] Detailed Description

[0037] Although various exemplary embodiments are described or presented herein, other exemplary embodiments employing various methods and materials similar or equivalent to those described or presented herein are also encompassed within the general inventive concept. For the sake of brevity, those features or embodiments that are implemented in a conventional manner may not be discussed or described in detail. Accordingly, it will be understood that features not described in detail in the devices, products, or methods described herein may be implemented according to any conventional techniques for implementing such features in the respective circumstances.

[0038] As used herein, the expressions "combination of... " and "in combination with... " encompass separate, sequential, or simultaneous administration of the medicaments. Unless otherwise specified, these expressions are also intended to exclude any additional active ingredients. For example, "a combination comprising at least four antimicrobial agents" means that the defined agents are administered alone, sequentially, or simultaneously, but no other active ingredients are administered.

[0039] When the agents are administered sequentially, rifampicin, levofloxacin, polymyxin E, polymyxin B, doxycycline or ceftazidime can be administered first. When the 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.

[0040] The combinations of the present invention can be used for treating Gram-positive bacterial or Gram-negative bacterial infections. Specifically, they can be used to kill proliferating and / or clinically latent bacteria associated with such infections, preferably proliferating bacteria associated with such infections, such as proliferating bacteria associated with Gram-negative bacterial infections. Thus, the treatment of bacterial infections as referred to herein includes killing proliferating and / or clinically latent microorganisms associated with such infections.

[0041] As used herein, "kill" refers to the loss of viability as evaluated by the lack of metabolic activity.

[0042] As used herein, "clinically latent bacteria" refers to bacteria that are metabolically active but have a growth rate lower than the threshold for the manifestation of an infectious disease. The threshold for the manifestation of an infectious disease refers to the growth rate threshold below which no symptoms of an infectious disease appear in the host.

[0043] The metabolic activity of clinically latent bacteria can be determined by several methods known to those skilled in the art; for example, by measuring the mRNA levels in the bacteria or by determining their uridine uptake rate. In this regard, clinically latent bacteria have reduced but still significant levels of the following, compared to bacteria under logarithmic growth conditions (in vitro or in vivo):

[0044] (I) mRNA (e.g., 0.0001% to 50%, such as 1% to 30%, 5% to 25% or 10% to 20% of the mRNA level); and / or

[0045] (II) uridine (e.g., [3H]uridine) uptake (e.g., 0.0005% to 50%, such as 1% to 40%, 15% to 35% or 20% to 30% of the [3H]uridine uptake level).

[0046] Clinically latent bacteria typically have a number of recognizable characteristics. For example, they may be viable but non-culturable; that is, they generally cannot be detected by standard culturing techniques, but can be detected and quantified by, for example, broth dilution counting, microscopy, or molecular techniques (such as polymerase chain reaction). In addition, clinically latent bacteria are phenotypically drug-resistant and are thus sensitive (in the logarithmic phase) to the biostatic effect of conventional antimicrobial agents (i.e., bacteria for which the minimum inhibitory concentration (MIC) of a conventional antimicrobial agent remains essentially constant); but have a sharply decreased susceptibility to drug-induced killing (e.g., for any given conventional antimicrobial agent, the ratio of the minimum bactericidal concentration (e.g., minimum bactericidal concentration, MBC) to the MIC is 10 or higher).

[0047] In various embodiments of the present invention, one or more of the foregoing combinations are used to treat bacterial infections, and in particular, the combination can be used to kill proliferating and / or clinically latent bacteria associated with a bacterial infection. As used herein, the term "bacteria" (and its derivatives, such as "bacterial infection") includes, but is not limited to, reference to the following categories and specific types of microorganisms (or infections due to microorganisms):

[0048] Gram-positive cocci, such as Staphylococcus spp. (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. equorum, Staph. gallinarum, Staph. haemolyticus, Staph. hominis hominis, Staph. h. novobiosepticius, Staph. hyicus, Staph. intermedius, Staph. lugdunensis, Staph. pasteuri, Staph. saccharolyticus, Staph. schleiferi schleiferi, Staph. s. coagulans, Staph. sciuri, Staph. simulans, Staph. warneri, and Staph. xylosus)); Streptococcus spp. (e.g., β-hemolytic Streptococcus pyogenes (e.g., Strept. agalactiae, Strept. canis, Strept. dysgalactiae dysgalactiae, Strept. dysgalactiae equisimilis, Strept. equi equi, Strept. equi zooepidemicus, Strept. iniae, Strept. porcinus, and Strept. pyogenespyogenes), microaerophilic Streptococcus pyogenes (“Streptococcus “milleri”), such as Streptococcus anginosus, Streptococcus constellatus subsp. constellatus, Streptococcus constellatus subsp. pharyngidis, and Streptococcus intermedius), “mitis” oral streptococci (α-hemolytic “viridans” streptococci, such as Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus cristatus, Streptococcus gordonii, and Streptococcus parasanguinis), “salivarius” (non-hemolytic, such as Streptococcus salivarius and Streptococcus vestibularis), and “mutans” (tooth-surface streptococci, such as Streptococcus criceti, Streptococcus mutans, Streptococcus ratti, and Streptococcus sobrinus) groups, Streptococcus acidominimus, Streptococcus bovis, Streptococcus faecalis, Streptococcus equinus, Streptococcus pneumoniae, and Streptococcus suisStreptococci of the suis species, or alternatively streptococci that can be classified into groups A, B, C, D, E, G, L, P, U or V; Enterococcus spp. (e.g., Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus 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;.

[0049] Gram-negative cocci, such as Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria sicca, Neisseria subflava, and Neisseria weaveri; Enterobacteriaceae such as Escherichia coli; Enterobacter spp. (e.g., Enterobacter aerogenes, Enterobacter agglomerans, and Enterobacter cloacae); Citrobacter spp. (such as Citrob. freundii and Citrob. 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, Shigella boydii, and Shigella sonnei); Klebsiella spp. (e.g., Klebs. Pneumoniae, Klebs. oxytoca, Klebs. ornitholytica, Klebs. planticola, Klebs. ozaenae, Klebs. terrigena, Klebs. granulomatis (Calymmatobacteriumgranulomatis)) and Klebsiella rhinoscleromatis)); Proteus spp. (e.g., Proteus mirabilis, Proteus rettgeri, and Proteus vulgaris), Providencia spp. (e.g., Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii)); Serratia spp. (e.g., Serratia marcescens and Serratia liquifaciens) and Yersinia spp. (e.g., Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis)); Helicobacter spp. (e.g., Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fennelliae)); Acinetobacter spp. (e.g., Acinetobacter baumanii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter lwoffii, and Acinetobacter radioresistens)); Pseudomonas spp. (e.g., Pseudomonas aeruginosa, Pseudomonas maltophilia (Stenotrophomonas maltophiliaPseudomonas maltophilia), Pseudomonas alcaligenes, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas luteola, Pseudomonas mendocina, Pseudomonas monteilii, Pseudomonas oryzihabitans, Pseudomonas pertocinogena, Pseudomonas pseudalcaligenes, Pseudomonas putida, and Pseudomonas stutzeri); Bacteroides fragilis; Peptococcus spp. (e.g., Peptococcus nigerniger)); Peptostreptococcus; Clostridium (e.g., Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tetani, Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bifermentans, Clostridium beijerinckii, Clostridium butyricum, Clostridium cadaveris, Clostridium carnis, Clostridium celatum, Clostridioforme clostridium, 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, 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)); Mycoplasma (e.g., Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, and Mycoplasma urealyticum)); Mycobacterium (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium fortuitumfortuitum), Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium leprae, Mycobacterium smegmitis, Mycobacterium africanum, Mycobacterium alvei, Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium bohemicum, Mycobacterium bovis, Mycobacterium branderi, Mycobacterium brumae, Mycobacterium celatum, Mycobacterium chubense, Mycobacterium confluentis, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium flavescens, Mycobacterium gadium, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium hassicum, Mycobacterium intracellulare, Mycobacterium interjectum, Mycobacterium heidelberense, Mycobacteriumlentiflavum), Mycobacterium malmoense, Mycobacterium microgenicum, Mycobacterium microti, Mycobacterium mucogenicum, Mycobacterium neoaurum, Mycobacterium nonchromogenicum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium thermoresistabile, Mycobacterium triplex, Mycobacterium triviale, Mycobacterium tusciae, Mycobacterium ulcerans, Mycobacterium vaccae, Mycobacterium wolinskyi, and Mycobacterium xenopi)); Haemophilus spp. (e.g., Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus)); Actinobacillus spp. (e.g., Actinobacillus actinomycetemcomitans, Actinobacillus equuli, ActinobacillusActinobacillus (e.g., Actinobacillus hominis, Actinobacillus lignieresii, Actinobacillus suis, and Actinobacillus ureae); Actinomyces (e.g., Actinomyces israelii); Brucella (e.g., Brucella abortus, Brucella canis, Brucella melintensis, and Brucella suis); Campylobacter (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus); Listeria monocytogenes; Vibrio (e.g., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus, and Vibrio vulnificus); Erysipelothrix rhusopathiae; Corynebacteriaceae (e.g., Corynebacterium diphtheriae, Corynebacterium jeikeum, and Corynebacterium urealyticum); Spirochaetaceae, such as Borrelia (e.g., Borrelia recurrentis, Borrelia burgdorferi, Borrelia afzelii, Borrelia andersonii, Borrelia bissettii, Borrelia garinii,garinii), Borrelia japonica, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, Borrelia valaisiana, 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 the genus Treponema (Treponema pallidum ssp. pallidum, Treponema pallidum ssp. endemicum, Treponema pallidum ssp. pertenue, and Treponema carateum); the genus Pasteurella (e.g., Pasteurella aerogenes, Pasteurella bettyae, Pasteurella canis, Pasteurella dagmatis, Pasteurella gallinarum, Pasteurella haemolytica, Pasteurella multocida multocida, Pasteurella multocida gallicida, Pasteurella multocida septicaseptica), Pasteurella pneumotropica, and Pasteurella stomatis); Bordetella spp. (such as Bordetella bronchiseptica, Bordetella hinzii, Bordetella holmseii, Bordetella parapertussis, Bordetella pertussis, and Bordetella trematum); Nocardiaceae, such as Nocardia spp. (e.g., Nocardia asteroides and Nocardia brasiliensis); Rickettsia (e.g., Ricksettsii or Coxiella burnetii); Legionella spp. (such as Legionella anisa, 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 micdadei, Legionella oakridgensis, Legionella pneumophila, Legionella saintelensi, Legionella tucsonensis, and Legionella wadsworthii)); Moraxellacatarrhalis); Cyclospora cayetanensis; Entamoeba histolytica; Giardia lamblia; Trichomonas vaginalis; Toxoplasma gondii; Stenotrophomonas maltophilia; Burkholderia stenotrophomonas; Burkholderia cepacia; Burkholderia mallei and Burkholderia pseudomallei; Francisella tularensis; Gardnerella spp. (e.g., Gardnerella vaginalis and Gardnerella mobiluncus); Streptobacillus moniliformis; Flavobacteriaceae, such as Capnocytophaga spp. (e.g., Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica, Capnocytophaga ochracea, and Capnocytophaga putigena)); Bartonella spp. (Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella elizabethae, Bartonella henselae, Bartonella quintana, and Bartonella vinsonii arupensis)); Leptospira spp. (e.g., LeptospiraLeptospira biflexa, Leptospira borgpetersenii, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira santarosai, and Leptospira weilii); Spirillum (e.g., Spirillum minus); Bacteroides (e.g., Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides merdae, Bacteroides ovatus, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides splanchinicus, Bacteroides stercoris, Bacteroides tectus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, and Bacteroides vulgatus); Prevotella (e.g., Prevotella bivia, Prevotella buccae, Prevotella corporis, Prevotella dentalis (Mitsuokella dentalis), Prevotella denticola, Prevotella peptolytica, PrevotellaPrevotella (e.g., Prevotella enoeca, Prevotella heparinolytica, Prevotella intermedia, Prevotella loeschii, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulora, Prevotella tannerae, Prevotella venoralis, and Prevotella zoogleoformans)); Porphyromonas (e.g., Porphyromonas asaccharolytica, Porphyromonas cangingivalis, Porphyromonas canoris, Porphyromonas cansulci, Porphyromonas catoniae, Porphyromonas circumdentaria, Porphyromonascrevioricanis, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonasgingivicanis, Porphyromonas levii, and Porphyromonas macacae)); Fusobacterium (e.g., F. gonadiaformans, F. mortiferum, F. naviforme, F. necrogenes, F. necrophorum necrophorum, F. necrophorum fundiliforme, F. nucleatum nucleatumF. nucleatum), Fusobacterium nucleatum fusiforme, Fusobacterium nucleatum polymorphum, Fusobacterium nucleatum vincentii, Fusobacterium periodonticum, Fusobacterium russii, Fusobacterium ulcerans, and Fusobacterium varium); Chlamydia (e.g., Chlamydia trachomatis); Cryptosporidium (e.g., Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium meleagridis, and Cryptosporidium muris); Chlamydophila (e.g., Chlamydophila abortus (Chlamydia psittaci), Chlamydophila pneumoniae (Chlamydia pneumoniae), and Chlamydophila psittaci (Chlamydia psittaci)); Leuconostoc (e.g., Leuconostoc citreum, Leuconostoc cremoris, Leuconostoc dextranicum, Leuconostoc lactis, Leuconostoc mesenteroides, and Leuconostoc pseudomesenteroides); Gemella (e.g., Gemella bergeri, Gemella haemolysans, Gemella morbillorum, and Gemella sanguinis); Aeromonas (Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii biovar sobria) and Ureaplasma (e.g., Ureaplasma parvum and Ureaplasma urealyticum).

[0050] Preferably, the combination of the present invention has a synergistic effect on Gram-positive bacteria or Gram-negative bacteria selected from the following:

[0051] Gram-negative bacteria: Enterobacteriaceae, Enterobacter spp, Pseudomonas spp, Acinetobacter spp, Shigella spp, Salmonella spp, Burkholderia stenotrophomonas, Citrobacter spp, Serratia spp, Proteus spp, Morganella spp, Providencia spp, Haemophilus spp, Aeromonas spp, Pasteurella spp, Brucella spp, Helicobacter spp, Campylobacter spp, Francisella tularensis, Legionella spp, Vibrio spp, Neisseria spp, Mycobacterium spp, Yersinia pestis, Rickettsia spp.

[0052] Gram-positive bacteria: Staphylococcus spp, Enterococcus spp, Streptococcus spp, Bacillus anthracis.

[0053] Gram-negative bacteria can be, for example: Enterobacteriaceae bacteria, such as Escherichia coli; Enterobacter spp. (such as Enterobacter aerogenes, Enterobacter agglomerans, and Enterobacter cloacae); Citrobacter spp. (such as Citrob. freundii and Citrob. divernis); Pseudomonas spp. (such as Ps. aeruginosa, Ps. maltophilia (Stenotrophomonas maltophilia), Ps. alcaligenes, Ps. chlororaphis, Ps. fluorescens, Ps. luteola, Ps. mendocina, Ps. monteilii, Ps. oryzihabitans, Ps. pertocinogena, Ps. pseudalcaligenes, Ps. putida, and Ps. stutzeri); Yersinia spp. (such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis); Helicobacter spp. (such as Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fennelliae); Acinetobacter spp. (such as A. baumanii, A. calcoaceticus, A. haemolyticus, A. johnsonii, A. junii, A. lwoffi, and A. radioresistens); Morganella spp. (such as Morganella morganii), Salmonella spp. (Salmonella enterica and SalmonellaSalmonella typhi)); Shigella spp. (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei)); (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella ornitholytica, Klebsiella planticola, Klebsiella ozaenae, Klebsiella terrigena, Klebsiella granulomatis (Calymmatobacterium granulomatis), and Klebsiella rhinoscleromatis)); Stenotrophomonas maltophilia; Francisella tularensis; Serratia spp. (e.g., Serratia marcescens and Serratia liquifaciens)); Proteus spp. (e.g., Proteus mirabilis, Proteus rettgeri, and Proteus vulgaris)); Providencia spp. (e.g., Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii)); Haemophilus spp. (e.g., Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus)); Aeromonas spp. (Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii biovarsobria)); Pasteurella (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)); 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 birminghamensis, Legionella bozemanii, Legionella cincinnatiensis, Legionella dumoffii, Legionella feeleii, Legionella gormanii, Legionella hackeliae, Legionella israelensis, Legionella jordanis, Legionella lansingensis, Legionella longbeachae, Legionella maceacherniimaceachernii), Legionella micdadei, Legionella oakridgensis, Legionella pneumophila, Legionella sainthelensi, Legionella tucsonensis, and Legionella wadsworthii); Vibrio spp. (e.g., Vibrio cholerae and Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus, and Vibrio vulnificus)); Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria sicca, Neisseria subflava, and Neisseria weaveri; Mycobacterium spp. (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium leprae, Mycobacterium smegmatisMycobacterium smegmitis), Mycobacterium africanum, Mycobacterium alvei, Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium bohemicum, Mycobacterium bovis, Mycobacterium branderi, Mycobacterium brumae, Mycobacterium celatum, Mycobacterium chubense, Mycobacterium confluentis, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium flavescens, Mycobacterium gadium, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium hassicum, Mycobacterium intracellulare, Mycobacterium interjectum, Mycobacterium heidelberense, Mycobacterium lentiflavum, Mycobacterium malmoense, Mycobacterium microgenicum, Mycobacterium microti, Mycobacterium mucogenicum, Mycobacterium neoaurum, Mycobacterium nonchromogenicumnonchromogenicum), Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium thermoresistabile, Mycobacterium triplex, Mycobacterium triviale, Mycobacterium tusciae, Mycobacterium ulcerans, Mycobacterium vaccae, Mycobacterium wolinskyi, and Mycobacterium xenopi)); Rickettsia (e.g., Ricksettsii or Coxiella burnetii).

[0054] Gram-positive bacteria can be, for example, Staphylococcus (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. equorum, Staph. gallinarum, Staph. haemolyticus, Staph. hominis hominis, Staph. h. novobiosepticius, Staph. hyicus, Staph. intermedius, Staph. lugdunensis, Staph. pasteuri, Staph. saccharolyticus, Staph. schleiferi schleiferi, Staph. s. coagulans, Staph. sciuri, Staph. simulans, Staph. warneri, and Staph. xylosus)); Streptococcus (e.g., β-hemolytic Streptococcus pyogenes (e.g., Strept. agalactiae, Strept. canis, Strept. dysgalactiae dysgalactiae, Strept. dysgalactiae equisimilis, Strept. equi equi, Strept. equi zooepidemicus, Strept. iniae, Strept. porcinus, and Strept.pyogenes), microaerophilic Streptococcus pyogenes (“Streptococcus “milleri”), such as Streptococcus anginosus, Streptococcus constellatus constellatus, Streptococcus constellatus pharyngidis, and Streptococcus intermedius), “mitis” oral streptococci (alpha-haemolytic “viridans” streptococci, such as Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus cristatus, Streptococcus gordonii, and Streptococcus parasanguinis), “salivarius” (non-haemolytic, such as Streptococcus salivarius and Streptococcus vestibularis) and “mutan” (tooth surface streptococci, such as Streptococcus criceti, Streptococcus mutans, Streptococcus ratti, and Streptococcus sobrinus) groups, Streptococcus acidominimus, Streptococcus bovis, Streptococcus faecalis, Streptococcus equinus, Streptococcus pneumoniae, and Streptococcus suisStreptococcus suis), or alternatively streptococci that can be classified into groups A, B, C, D, E, G, L, P, U or V; Enterococcus spp. (e.g., Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, and Enterococcus solitarius); Bacillus anthracis).

[0055] Preferably, the bacterial infection treated by the combination therapy described herein is a Gram-negative bacterial infection. Specific Gram-negative bacteria that can be treated using the combination of the present invention include:

[0056] Enterobacteriaceae, such as Escherichia coli, Klebsiella spp. (e.g., Klebsiella pneumoniae and Klebsiella oxytoca), and Proteus spp. (e.g., Proteus mirabilis, Proteus rettgeri, and Proteus vulgaris); Haemophilus influenzae; Mycobacterium spp. (e.g., Mycobacterium tuberculosis); and Enterobacter spp. (e.g., Enterobacter cloacae). Preferably, the bacteria are Enterobacteriaceae, such as Escherichia coli, Klebsiella spp. (e.g., Klebsiella pneumoniae and Klebsiella oxytoca). Particularly preferred are Escherichia coli and Klebsiella pneumoniae (e.g., Klebsiella pneumoniae subsp. pneumoniae).

[0057] The combination of the present invention is particularly beneficial in treating (multi)-drug resistant ((M)DR) bacteria. For Enterobacteriaceae, resistance most often progresses to carbapenemases, i.e., carbapenemase-resistant strains and "extended-spectrum beta-lactamase" (ESBL) strains, such as New Delhi Metallo-beta-lactamase-1 (NDM-1)-resistant Klebsiella pneumoniae and NDM-1 Escherichia coli. The combination therapy of the present invention is also particularly effective against carbapenemase-producing Enterobacteriaceae (CPE). Other resistant strains can also be used, such as colistin-resistant strains, as well as carbapenemase-resistant strains outside Enterobacteriaceae, which include carbapenem-resistant Acinetobacter and carbapenem-resistant Pseudomonas spp. microorganisms carrying the blaKPC gene.

[0058] In various embodiments, the combinations of the present invention are beneficial against ESKAPE pathogens. These six highly virulent and typically antibiotic-resistant bacterial pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. This group of Gram-positive and Gram-negative bacteria can evade or "escape" commonly used antibiotics due to their enhanced multi-drug resistance. Thus, the combinations of the present invention are beneficial against (M)DR strains of ESKAPE pathogens.

[0059] Advantageously, in various embodiments, the combinations of the present invention have a broader range of activity than monotherapy or combinations of only two active agents. Specifically, the various combinations can be effective against at least Acinetobacter, Pseudomonas, and Enterobacteriaceae, which bacteria have been identified by the World Health Organization as including multi-drug resistant bacteria for which there is an urgent need for new antibiotics.

[0060] It should be remembered that although combinations (such as the claimed combinations) may initially be shown to be effective in treating (M)DR strains, they can additionally be used to treat non-resistant strains. This is extremely valuable in the context of the currently claimed combinations, where the primary therapies for Enterobacteriaceae such as Escherichia coli and Klebsiella spp. (such as Klebsiella pneumoniae and Klebsiella oxytoca) are expensive antimicrobial drugs due to existing patent protection. From a therapeutic perspective as well as from a financial / economic perspective (at a time when governments are seeking to reduce healthcare costs), the substitution of such "prescription" drugs by combinations of "generic" antibiotics is considered beneficial.

[0061] The combinations of the present invention can be used to treat infections associated with any of the bacterial microorganisms mentioned above, and in particular they can be used to kill proliferating and / or clinically latent microorganisms associated with such infections (such as ESKAPE pathogen bacterial infections).

[0062] In various embodiments, the combinations of the present invention are effective in treating infections caused by: (1) carbapenem-resistant Escherichia coli, Klebsiella, Acinetobacter, Pseudomonas aeruginosa, Serratia, or Proteus; (2) MRSA, vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococcus faecium (VRE), clarithromycin-resistant Helicobacter pylori, or quinolone-resistant Salmonella; or (3) penicillin-resistant Streptococcus pneumoniae, ampicillin-resistant Haemophilus influenzae, or quinolone-resistant Shigella. In various embodiments, the combinations of the present invention are effective in treating infections caused by Acinetobacter baumannii, Pseudomonas aeruginosa, or MRSA.

[0063] Specific medical conditions for which the combination therapy of the present invention can be used include tuberculosis (such as pulmonary tuberculosis, non-pulmonary tuberculosis (such as scrofula, genitourinary tuberculosis, osteoarticular tuberculosis, tuberculous meningitis), and miliary tuberculosis), anthrax, abscess, acne vulgaris, actinomycosis, asthma, bacillary dysentery, bacterial conjunctivitis, bacterial keratitis, bacterial vaginosis, botulism, Buruli ulcer, bone and joint infections, bronchitis (acute or chronic), brucellosis, burn wounds, cat scratch fever, cellulitis, chancroid, cholangitis, cholecystitis, cutaneous diphtheria, cystic fibrosis, cystitis, diffuse panbronchiolitis, diphtheria, dental caries, upper respiratory diseases, eczema, empyema, endocarditis, endometritis, enteric fever, enteritis, epididymitis, epiglottitis, erysipelas, erysipeloid, erythrasma, eye infections, furuncle, gardnerella vaginitis, gastrointestinal infections (gastroenteritis), genital infections, gingivitis, gonorrhea, granuloma inguinale, Haverhill fever, burn infections, dental post-operative infections, oral cavity infections, prosthesis-related infections, intra-abdominal abscesses, Legionnaires' disease, leprosy, leptospirosis, listeriosis, liver abscesses, Lyme disease, lymphogranuloma venereum, mastitis, mastoiditis, meningitis and nervous system infections, mycetoma, nocardiosis (such as Madura foot), non-specific urethritis, ophthalmia (such as neonatal ophthalmia), osteomyelitis, otitis (such as otitis externa and otitis media), orchitis, pancreatitis, paronychia, pelvic peritonitis, peritonitis, peritonitis with appendicitis, pharyngitis, cellulitis, pinta, plague, pleural effusion, pneumonia, post-operative wound infections, post-operative gas gangrene, prostatitis, pseudomembranous colitis, psittacosis, pulmonary emphysema, pyelonephritis, pyoderma (such as impetigo), Q fever, rat bite fever, reticulocytosis, ricin poisoning, Ritter's disease, salmonellosis, salpingitis, septic arthritis, septic infections, septicemia, sinusitis, skin infections (such as skin granulomas, impetigo, folliculitis, and furunculosis), syphilis, systemic infections, tonsillitis, toxic shock syndrome, trachoma, tularemia, typhoid fever, typhus (such as epidemic typhus, murine typhus, scrub typhus, and spotted fever), urethritis, wound infections, yaws, aspergillosis, candidiasis (such as oropharyngeal candidiasis, vaginal candidiasis, or balanitis), cryptococcosis, favus, histoplasmosis, intertrigo, mucormycosis, tinea (such as tinea corporis, tinea capitis, tinea cruris, tinea pedis, and onychomycosis), onychomycosis, pityriasis versicolor, trichomycosis, and sporotrichosis;or infections by MSSA, MRSA, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Haemophilus influenzae, Enterococcus faecalis, and Enterococcus faecium;

[0064] Specific medical conditions that can be treated using the combinations of the present invention also include those caused by Gram-negative bacteria, such as abscess, asthma, bacillary dysentery, bacterial conjunctivitis, bacterial keratitis, bacterial vaginosis, bone and joint infections, bronchitis (acute or chronic), brucellosis, burn wounds, cat scratch fever, cellulitis, chancroid, cholangitis, cholecystitis, cystic fibrosis, cystitis, nephritis, diffuse panbronchiolitis, dental caries, upper respiratory tract diseases, empyema, endocarditis, endometritis, enteric fever, enteritis, epididymitis, epiglottitis, eye infections, furuncle, gardnerella vaginitis, gastrointestinal infections (gastroenteritis), genital infections, gingivitis, gonorrhea, granuloma inguinale, Haverhill fever, burn infections, post-dental infections, oropharyngeal infections, prosthesis-related infections, intra-abdominal abscesses, Legionnaires' disease, leptospirosis, listeriosis, liver abscesses, Lyme disease, lymphogranuloma venereum, mastitis, otitis media, meningitis and nervous system infections, non-specific urethritis, ophthalmia (e.g., ophthalmia neonatorum), osteomyelitis, otitis (such as otitis externa and otitis media), orchitis, pancreatitis, paronychia, pelvic peritonitis, peritonitis, peritonitis with appendicitis, pharyngitis, pleural effusion, pneumonia, post-operative wound infections, post-operative gas gangrene, prostatitis, pseudomembranous colitis, psittacosis, pyelonephritis, Q fever, Ritter's disease, salmonellosis, salpingitis, septic arthritis, septic infections, sepsis, systemic infections, tonsillitis, trachoma, typhoid, urethritis, urinary tract infections, wound infections; or infections by Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Haemophilus influenzae, Enterococcus faecalis, Enterococcus faecium, and Enterobacter cloacae.

[0065] Preferably, the combinations of the present invention are used for treating 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.

[0066] It will be understood that the term "treatment" as used herein extends to prophylaxis as well as the treatment of established diseases or symptoms.

[0067] As used herein, the term "pharmaceutically acceptable derivative" means: (a) pharmaceutically acceptable salts; (b) solvates (including hydrates) and / or (c) prodrugs (where appropriate).

[0068] The pharmaceutically acceptable salts of the compounds included in the combination of the present invention include their suitable acid addition salts or base salts. A review of suitable pharmaceutical salts can be found in Berge et al., J Pharm Sci, 66, 1-19 (1977).

[0069] Suitable acid addition salts include carboxylates (e.g., formate, acetate, trifluoroacetate, propionate, isobutyrate, heptanoate, decanoate, caprate, octanoate, stearate, acrylate, hexanoate, propiolate, 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, octanedioate, decanedioate, fumarate, malate, maleate, hydroxymaleate, hippurate, phthalate or terephthalate), 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, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, or nitrate. Suitable base salts include metal salts, such as sodium salts, calcium salts and amine salts.

[0070] For example, ceftazidime pentahydrate, colistin sulfate, polymyxin B sulfate, doxycycline hyclate (doxycycline semi-ethanol semi-hydrate), doxycycline hydrochloride, doxycycline monohydrate, levofloxacin hemihydrate and rifampicin N-oxide are commercially available from Sigma Aldrich. Other suppliers are also known in the art.

[0071] As used herein, the term "prodrug" refers to an antimicrobial compound in which one or more groups have been modified such that the modification can be reversed after administration to a human or mammalian subject. This reversal is typically carried out by enzymes naturally present in the subject, but a second agent can also be administered together with such prodrug in order to effect the reversal in vivo. Examples of such modifications include the formation of esters (e.g., any of those described above), where the reversal can be effected by esterases and the like. Other such systems will be known to those skilled in the art.

[0072] Polymyxin E or colistin is commercially available as a mesylate derivative: colistimethate sodium or colistin sodium methanesulfonate (CMS). Colistimethate sodium is a prodrug. It is produced by the reaction of colistin with formaldehyde and sodium bisulfite, which results in the addition of a sulfomethyl group to the primary amine of colistin. In aqueous solution, it undergoes hydrolysis to form a complex mixture of partially sulfomethylated derivatives and colistin.

[0073] This invention includes the use of these pharmaceutically acceptable derivatives and prodrugs. Specifically, this invention includes the use of colistin and its pharmaceutically acceptable derivatives (including colistin sulfate, colistimethate sodium, and colistin sodium methanesulfonate).

[0074] This invention also includes all enantiomers and tautomers (where appropriate) of the compounds. Those skilled in the art will recognize compounds having optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and / or tautomers can be separated or prepared by methods known in the art.

[0075] Certain compounds included in the combinations of this invention may exist as stereoisomers and / or geometric isomers - for example, they may have one or more asymmetric centers and / or geometric centers and thus may exist in two or more stereoisomeric and / or geometric isomeric forms. This invention contemplates the use of all individual stereoisomers and geometric isomers of those inhibitors and their mixtures. The terms used in the claims cover these forms, provided that said forms retain the appropriate functional activity (although not necessarily to the same degree).

[0076] The present invention also includes all suitable isotopic variants of the compound or its pharmaceutically acceptable salts. Isotopic variants or their pharmaceutically acceptable salts are defined as those in which at least one atom is replaced by an atom having the same atomic number but a different atomic mass from that typically found in nature. Examples of isotopes that can be incorporated include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F, and 36Cl, respectively. Certain isotopic variants, such as those incorporating radioactive isotopes (e.g., 3H or 14C), can be used for drug and / or substrate tissue distribution studies. Tritium (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred due to their ease of preparation and detection. In addition, replacement with an isotope such as deuterium (i.e., 2H) can provide certain therapeutic advantages, such as increased in vivo half-life or reduced dose requirements, due to higher metabolic stability and may thus be preferred in certain cases. Isotopic variants can generally be prepared by conventional procedures using appropriate isotopic variants of suitable reagents.

[0077] The compounds for use in the combinations of the present invention, including their pharmaceutically acceptable derivatives or prodrugs, 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 methyl sulfate, polymyxin B sulfate, doxycycline hyclate (doxycycline hyclate semi-ethanolate hemihydrate), doxycycline, doxycycline monohydrate, levofloxacin hemihydrate, and rifampicin N-oxide are commercially available from Sigma Aldrich, for example, from Sigma- obtained. Other commercial suppliers are known in the art.

[0078] Ceftazidime is sold under the trade name Fortaz, etc. and is a third-generation cephalosporin antibiotic used to treat a variety of bacterial infections. It has the following chemical structure:

[0079]

[0080] Polymyxin E is also known as colistin. It is an antibiotic drug used as a last resort for treating infections including multi-drug resistant Gram-negative bacteria infections (including pneumonia). These infections may involve bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter. It has two forms: colistimethate sodium can be administered intravenously, intramuscularly, or by inhalation; and colistin sulfate is mainly administered topically or orally. It has the following chemical structure:

[0081]

[0082] Polymyxin B, sold under the trade name Poly-Rx, etc., is an antibiotic used to treat meningitis, pneumonia, sepsis, and urinary tract infections. It can be administered by intravenous injection, intramuscular injection, or cerebrospinal fluid injection, or by inhalation. It has the following chemical structure:

[0083]

[0084] Doxycycline is a broad-spectrum tetracycline antibiotic used to treat infections caused by bacteria and certain parasites. It is used to treat bacterial pneumonia, acne, chlamydia infections, Lyme disease, cholera, typhus, and syphilis. Doxycycline can be taken orally or by intravenous injection. It has the following chemical structure:

[0085]

[0086] Levofloxacin, sold under the trade name Levaquin, etc., is an antibiotic drug used to treat a variety of bacterial infections, including acute bacterial sinusitis, pneumonia, urinary tract infections, chronic prostatitis, and certain types of gastroenteritis. It is available by oral, intravenous injection, and eye drops. It is the (S)-isomer of ofloxacin and has the following chemical structure:

[0087]

[0088] Rifampicin, also known as rifampin, is an ansamycin antibiotic used to treat several types of bacterial infections, including tuberculosis, Mycobacterium avium complex disease, leprosy, and Legionnaires' disease. Rifampicin can be administered orally or by intravenous injection and has the following chemical structure:

[0089]

[0090] The synergistic combination of the present invention includes four antimicrobial agents. These agents are grouped in the appended claims so as to cover the exemplified combinations in the most effective manner.

[0091] In various embodiments, at least four antimicrobial agents are selected from rifampicin, polymyxin E, polymyxin B, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof. Alternatively, at least four antimicrobial agents are selected from levofloxacin, polymyxin B, polymyxin E, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof. Another option is where at least four antimicrobial agents are selected from rifampicin, levofloxacin, doxycycline, ceftazidime, and pharmaceutically acceptable derivatives thereof.

[0092] In various embodiments, at least four antimicrobial agents are rifampicin, polymyxin E, doxycycline, and ceftazidime, or pharmaceutically acceptable derivatives thereof.

[0093] In various embodiments, at least four antimicrobial agents are levofloxacin, polymyxin E, doxycycline, and ceftazidime, or pharmaceutically acceptable derivatives thereof.

[0094] In various embodiments, at least four antimicrobial agents are rifampicin, levofloxacin, doxycycline, ceftazidime, or pharmaceutically acceptable derivatives thereof.

[0095] Preferably, at least four antimicrobial agents include at least ceftazidime, doxycycline, or pharmaceutically acceptable derivatives thereof.

[0096] More preferably, 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, provided that (iii) and (iv) are different.

[0097] The compounds for use according to the invention can be administered in raw material form, but are preferably provided in the form of a pharmaceutical composition. The compounds can be used in separate formulations or in a single combined formulation. When combined in the same formulation, it will be understood that the two compounds must be stable and compatible with each other and with the other ingredients in the formulation.

[0098] The formulations of the invention include those suitable for administration by oral, parenteral (including subcutaneous such as by injection or depot tablets, intrathecal, intramuscular such as by depot formulations, and intravenous) and rectal, or suitable for administration by inhalation or insufflation. The most suitable route of administration may depend on the condition and disorder of the patient. Preferably, the compositions of the invention are formulated for oral administration.

[0099] The pharmaceutical compositions can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy, for example as described in "Remington: The Science and Practice of Pharmacy", Lippincott Williams and Wilkins, 21st Edition, (2005). Suitable methods include the step of bringing the active ingredient into association with a carrier which constitutes one or more excipients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. It will be understood that when two active ingredients are administered independently, each can be administered by a different route.

[0100] When formulated with excipients, the active ingredient can be present in a concentration of from 0.1% to 99.5% (e.g., 0.5% to 95%) by weight of the total mixture; suitably from 30% to 95% for tablets and capsules, and from 0.01% to 50% (e.g., 3% to 50%) for liquid formulations.

[0101] The concentration of each antimicrobial agent in the synergistic combination is equal to or lower than the minimum inhibitory concentration (i.e., MIC mono ) in monotherapy with that agent against the bacteria for which the combination is used. Thus, "MIC" as referred to herein should be understood as MIC mono , unless otherwise stated. Preferably, the concentration of at least one antimicrobial agent in the synergistic combination is lower than MIC mono , more preferably, the concentrations of at least two antimicrobial agents in the synergistic combination are lower than MIC mono . The use of such concentrations is advantageous because it avoids toxicity problems and reduces the likelihood of development of antimicrobial resistance as compared to one or more of the agents in the combination.

[0102] In various embodiments, the concentration of ceftazidime against the bacteria for which the combination is used is 1xMIC or lower, where MIC is the minimum inhibitory concentration of ceftazidime alone against said bacteria, i.e., MIC mono . Preferably, the concentration of ceftazidime against the bacteria for which the combination is used can be at 0.5xMIC mono or lower. More preferably, the concentration of ceftazidime against the bacteria for which the combination is used is 0.25xMIC mono or lower. Most preferably, the concentration of ceftazidime against the bacteria for which the combination is used is 0.125xMIC mono or lower. In some embodiments, the concentration of ceftazidime against the bacteria for which the combination is used is as low as 0.0625xMIC mono - equivalent to 1 / 16MICmono 。In other embodiments, the concentration of ceftazidime against the bacteria being targeted by the combination is as low as 0.0039xMIC mono - equivalent to 1 / 32MIC mono 。

[0103] As an example, in a combination showing synergy against ESBL E. coli, the concentration of ceftazidime can be about 32 mg / L or lower. In a preferred embodiment, in a combination showing synergy against ESBL E. coli, the concentration of ceftazidime can be from about 1 to about 32 mg / L.

[0104] As another example, in a combination showing synergy against KPC Klebsiella pneumoniae, the concentration of ceftazidime can be about 32 mg / L or lower. In a preferred embodiment, in a combination showing synergy against KPC Klebsiella pneumoniae, the concentration of ceftazidime can be from about 0.125 mg / L to about 32 mg / L.

[0105] In various embodiments, the concentration of doxycycline against the bacteria being targeted by the combination is 1xMIC mono or lower. Preferably, the concentration of doxycycline against the bacteria being targeted by the combination can be 0.5xMIC mono or lower. More preferably, the concentration of doxycycline against the bacteria being targeted by the combination can be 0.25xMIC mono or lower. Most preferably, the concentration of doxycycline against the bacteria being targeted by the combination can be 0.125xMIC mono or lower.

[0106] As an example, in a combination showing synergy against ESBL E. coli, the concentration of doxycycline can be about 1 mg / L or lower, preferably from about 0.03 to about 1 mg / L.

[0107] As another example, in a combination showing synergy against KPC Klebsiella pneumoniae, the concentration of doxycycline can be about 1 mg / L or lower, preferably from about 0.03125 to about 1 mg / L.

[0108] The concentration range of doxycycline can be combined with the concentration range of ceftazidime. In various embodiments, in a combination showing synergy against ESBL E. coli, ceftazidime is used at a concentration of from about 1 to about 32 mg / L and doxycycline is used at a concentration of from about 0.03 to about 1 mg / L. In various embodiments, in a combination showing synergy against KPC Klebsiella pneumoniae, ceftazidime is used at a concentration of from about 0.125 to about 32 mg / L and doxycycline is used at a concentration of from about 0.03125 to about 1 mg / L, more preferably from 0.25 to about 32 mg / L.

[0109] In various embodiments, the concentration of rifampicin against the bacteria targeted by the combination can be 1xMIC mono or lower. Preferably, the concentration of rifampicin against the bacteria targeted by the combination can be 0.5xMIC mono or lower. More preferably, the concentration of rifampicin against the bacteria targeted by the combination can be 0.25xMIC mono or lower. Most preferably, the concentration of rifampicin against the bacteria targeted by the combination can be 0.125xMIC mono or lower. For example, in various embodiments, the concentration of rifampicin against the bacteria targeted by the combination can be 0.0625xMIC mono .

[0110] As an example, in a combination showing synergy against ESBL Escherichia coli, the rifampicin concentration can be about 4 mg / L or lower, preferably about 0.125 to about 4 mg / L. In another example, in a combination showing synergy against KPC Klebsiella pneumoniae, the rifampicin concentration can be about 4 mg / L or lower, preferably about 0.125 mg / L to about 4 mg / L.

[0111] The concentration range of rifampicin can be combined with the respective concentration ranges of ceftazidime and doxycycline mentioned above.

[0112] In various embodiments, in a combination showing synergy against ESBL Escherichia 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 showing synergy against ESBL Escherichia 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.

[0113] In various embodiments, in a combination showing synergy against ESBL Escherichia 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.

[0114] In various embodiments, in a combination showing synergy against ESBL Escherichia 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.

[0115] In various embodiments, 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 rifampin is used at a concentration of about 0.125 to about 4 mg / L in combinations that exhibit synergy against ESBL Escherichia coli. Preferably, in combinations that exhibit synergy against ESBL Escherichia 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, and rifampin is used at a concentration of about 0.125 to about 4 mg / L.

[0116] In various embodiments, 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 rifampin is used at a concentration of about 0.125 mg / L to about 4 mg / L in combinations that exhibit synergy against KPC Klebsiella pneumoniae. Preferably, in combinations that exhibit synergy against KPC Klebsiella 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 rifampin is used at a concentration of about 0.125 mg / L to about 4 mg / L. More preferably, in combinations that exhibit synergy against KPC Klebsiella 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 rifampin is used at a concentration of about 0.25 mg / L to about 4 mg / L.

[0117] The concentrations of ceftazidime, doxycycline, and rifampin can also be expressed as multiples of the MIC mono For example, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, and rifampin can be used at a concentration of 1xMIC mono or lower. Preferably, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, and rifampin can be used at a concentration of 0.125 to 1xMIC mono For example, ceftazidime can be used at a concentration of 0.5xMIC

[0118] In various embodiments, ceftazidime can be used at a concentration of 0.5xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, and rifampin at a concentration of 1xMIC mono or lower. Alternatively, ceftazidime can be used at a concentration of 1xMIC monoUsed at a concentration of 0.5xMIC or lower, doxycycline mono Used at a concentration of 1xMIC or lower, and rifampicin mono Used at a concentration of 1xMIC or lower.

[0119] In various embodiments, for the bacteria targeted by the use of the combination, the concentration of polymyxin E or polymyxin B can be 1xMIC mono Or lower. Preferably, for the bacteria targeted by the use of the combination, the concentration of polymyxin E or polymyxin B can be 0.5xMIC mono Or lower. More preferably, for the bacteria targeted by the use of the combination, the concentration of polymyxin E or polymyxin B can be 0.25xMIC mono Or lower. Most preferably, for the bacteria targeted by the use of the combination, the concentration of polymyxin E or polymyxin B can be 0.125xMIC mono Or lower.

[0120] As an example, in a combination showing synergy against ESBL Escherichia coli, the concentration of polymyxin E or polymyxin B can be about 2 mg / L or lower, preferably about 0.06 to about 2 mg / L.

[0121] In another example, in a combination showing synergy against KPC Klebsiella pneumoniae, the concentration of polymyxin E or polymyxin B can be about 1 mg / L or lower, preferably about 0.125 mg / L to about 1 mg / L.

[0122] The concentration range of polymyxin E / B can be combined with the respective concentration ranges of ceftazidime, doxycycline and rifampicin as described above.

[0123] In various embodiments, in a combination showing synergy against ESBL Escherichia 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 showing synergy against ESBL Escherichia coli, the concentration of ceftazidime can be about 1 to about 32 mg / L, the concentration of doxycycline can be about 0.125 to about 1 mg / L, the concentration of rifampicin can be about 0.125 to about 4 mg / L, and the concentration of polymyxin E / B can be about 0.125 to about 2 mg / L.

[0124] In various embodiments, in combinations showing synergy 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 showing synergy 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 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.

[0125] In various embodiments, in combinations showing synergy 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 1 to 4 mg / L, and polymyxin E / B is used at a concentration of about 0.06 to about 2 mg / L.

[0126] In various embodiments, in combinations showing synergy 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 combinations showing synergy 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.

[0127] In various embodiments, in combinations showing synergy against Klebsiella pneumoniae carbapenemase (KPC) - producing Klebsiella 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 combinations showing synergy against KPC - producing Klebsiella 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 combinations showing synergy against KPC - producing Klebsiella 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 combinations showing synergy against KPC - producing Klebsiella 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.

[0128] The concentrations of ceftazidime, doxycycline, rifampicin, and polymyxin E / B can also be expressed as multiples of the MIC mono For example, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, rifampicin can be used at 1xMIC mono or lower, and polymyxin E / B can be used at a concentration of 1xMIC mono or lower. Preferably, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, rifampicin can be used at a concentration of 0.0625 to 1xMIC mono and polymyxin E / B can be used at a concentration of 0.0625 to 1xMIC mono More preferably, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC monoUsed at a concentration of 0.0625 to 1xMIC or lower, rifampicin can be used at a concentration of 0.0625 to 1xMIC mono and polymyxin E / B can be used at a concentration of 0.125 to 1xMIC mono Used at a concentration of 0.125 to 1xMIC

[0129] In various embodiments, ceftazidime can be used at a concentration of 0.5xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, and polymyxin E / B and rifampicin can each be used at a concentration of 1xMIC mono or lower. Alternatively, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 0.5xMIC mono or lower, and polymyxin E / B and rifampicin can each be used at a concentration of 1xMIC mono or lower. Used at a concentration of 1xMIC

[0130] In various embodiments, ceftazidime can be used at a concentration of 0.5xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, rifampicin can be used at a concentration of 1xMIC mono or lower, and polymyxin E / B can be used at a concentration of 0.125 to 1xMIC mono Used at a concentration of 0.125 to 1xMIC

[0131] In various embodiments, for the bacteria targeted by the combination, the concentration of levofloxacin is 1xMIC mono or lower. Preferably, for the bacteria targeted by the combination, the concentration of levofloxacin can be 0.5xMIC mono or lower. More preferably, for the bacteria targeted by the combination, the concentration of levofloxacin can be 0.25xMIC mono or lower. Most preferably, for the bacteria targeted by the combination, the concentration of levofloxacin is 0.125xMIC mono or lower. Used at a concentration of 0.125xMIC

[0132] As an example, in a combination showing synergy against ESBL Escherichia coli, the concentration of levofloxacin can be about 8 mg / L or lower, preferably about 0.25 to about 8 mg / L. Used at a concentration of about 0.25 to about 8 mg / L

[0133] The concentration ranges of levofloxacin can be combined with the respective concentration ranges of ceftazidime and doxycycline. In various embodiments, in combinations showing synergy 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 levofloxacin is used at a concentration of about 0.25 to about 8 mg / L. Preferably, in combinations showing synergy against ESBL 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 combinations showing synergy 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.

[0134] The concentration ranges of ceftazidime, doxycycline, and levofloxacin can also be combined with the concentration ranges of polymyxin E / B or rifampicin, as set forth below.

[0135] In various embodiments, in combinations showing synergy 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, 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 combinations showing synergy against ESBL 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.

[0136] In various embodiments, in combinations showing synergy against ESBL 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.

[0137] The respective concentrations of ceftazidime, doxycycline, levofloxacin, and polymyxin E / B can also be expressed as multiples of the MIC mono For example, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, levofloxacin can be used at a concentration of 1xMIC monoused at a concentration of or lower, and polymyxin E / B can be used at 1xMIC mono or lower. Preferably, ceftazidime can be used at 1xMIC mono or lower, doxycycline can be used at 1xMIC mono or lower, levofloxacin can be used at a concentration of 0.0625 to 1xMIC mono and polymyxin E / B can be used at a concentration of 0.0625 to 1xMIC mono of concentration.

[0138] In various embodiments, ceftazidime can be used at a concentration of 0.5xMIC mono to 1xMIC mono of concentration, doxycycline can be used at 1xMIC mono or lower, and polymyxin E / B and levofloxacin are each used at 1xMIC mono or lower.

[0139] In various embodiments, in combinations showing synergy 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 combinations showing synergy against ESBL E. coli, the concentration of ceftazidime can be about 2 to about 32 mg / L, the concentration of doxycycline can be about 0.06 to about 1 mg / L, the concentration of levofloxacin can be about 2 to about 8 mg / L, and rifampicin is used at a concentration of about 0.03 to 1 mg / L. More preferably, in combinations showing synergy against ESBL E. coli, the concentration of ceftazidime can be about 16 to 32 mg / L, the concentration of doxycycline can be about 0.06 to about 1 mg / L, the concentration of levofloxacin can be about 4 to about 8 mg / L, and rifampicin is used at a concentration of about 0.03 to 1 mg / L.

[0140] Alternatively, in combinations showing synergy against ESBL E. coli, the ceftazidime concentration can be from about 2 to about 32 mg / L, the doxycycline concentration can be from about 0.06 to about 0.5 mg / L, the levofloxacin concentration can be from about 0.25 to about 8 mg / L, the doxycycline concentration can be from about 0.06 to about 1 mg / L, the levofloxacin concentration can be from about 2 to about 8 mg / L, and rifampicin is used at a concentration of about 0.03 to 1 mg / L. Preferably, in combinations showing synergy against ESBL E. coli, the ceftazidime concentration can be from about 16 to about 32 mg / L, the doxycycline concentration can be from about 0.06 to about 1 mg / L, the levofloxacin concentration can be from about 0.25 to about 8 mg / L, and rifampicin is used at a concentration of about 0.03 to 1 mg / L.

[0141] The concentrations of ceftazidime, doxycycline, levofloxacin, and rifampicin can also be expressed as multiples of the MIC mono . For example, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, levofloxacin can be used at a concentration of 1xMIC mono or lower, and rifampicin can be used at a concentration of 1xMIC mono or lower. Preferably, ceftazidime can be used at a concentration of 1xMIC mono or lower, doxycycline can be used at a concentration of 1xMIC mono or lower, levofloxacin can be used at a concentration of 1xMIC mono or lower, and rifampicin can be used at a concentration of 1xMIC mono or lower. Alternatively, ceftazidime can be used at a concentration of 0.5 to 1×MIC mono , doxycycline can be used at a concentration of 1×MIC mono , levofloxacin at a concentration of 1×MIC mono or lower, and rifampicin can be used at a concentration of 1×MIC mono or lower.

[0142] The lower limit of the MIC mono range defined herein is not limited. In cases where it is not specified, preferably 1 / 512MIC mono , 1 / 256MIC mono , 1 / 128MIC mono , 1 / 64MIC mono , 1 / 32MIC mono , or 0.0625MIC mono . For example, "0.5×MIC mono"or less" is changed to "0.5×MIC" mono to 0.0625 MIC mono ".

[0143] Preparations suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets (such as chewable tablets particularly for paediatric administration), each containing a predetermined amount of the active ingredient; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as water-in-oil or oil-in-water liquid emulsions. The active ingredient may also be presented as boluses, lozenges or pastes.

[0144] Tablets may be made by compression or moulding, optionally with one or more excipients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in free-flowing form such as a powder or granules, optionally mixed with other conventional excipients such as binders (such as syrup, gum acacia, gelatin, sorbitol, tragacanth, starch mucilage, polyvinylpyrrolidone and / or hydroxypropylmethylcellulose), fillers (such as lactose, sugar, microcrystalline cellulose, maize starch, calcium phosphate and / or sorbitol), lubricants (such as magnesium stearate, stearic acid, talc, polyethylene glycol and / or silica), disintegrants (such as potato starch, cross-linked sodium carboxymethylcellulose and / or sodium starch glycolate) and wetting agents (such as sodium lauryl sulphate). Moulded tablets may be made by moulding 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 formulated so as to provide controlled release (such as extended, sustained or pulsatile release, or a combination of immediate release and controlled release) of the active ingredient.

[0145] Alternatively, the active ingredient may be incorporated into oral liquid preparations such as aqueous or oily suspensions, solutions, emulsions, syrups or elixirs. Preparations containing the active ingredient may also be presented as dry products to be constituted with water or another suitable vehicle before use.

[0146] Such liquid preparations may contain conventional additives such as suspending agents (such as sorbitol syrup, methylcellulose, glucose / syrup, gelatin, hydroxypropylmethylcellulose, carboxymethylcellulose, aluminium stearate gel and / or hydrogenated edible fats), emulsifying agents (such as lecithin, sorbitan monooleate and / or gum acacia), non-aqueous carriers (such as edible oils like almond oil, fractionated coconut oil, oily esters, propylene glycol and / or ethanol), and preservatives (such as methyl or propyl hydroxybenzoates and / or sorbic acid).

[0147] The compositions used according to the invention can be presented in a packaging or dispensing device, which can contain one or more unit dosage forms containing the active ingredient. The packaging can for example comprise a metal or plastic foil, such as a blister pack. When the composition is intended to be administered as three separate compositions, these can be presented in a dual pack form.

[0148] The pharmaceutical composition can also be prescribed to the patient in the form of a "patient pack" containing the entire course of treatment in a single package, usually a blister pack. The patient pack has the advantage over traditional prescriptions that the pharmacist separates the patient's supply of the drug from the bulk supplies, since the patient always receives the package insert contained in the patient pack, which is usually missing in traditional prescriptions. It has been shown that including the package insert improves patient compliance with the physician's instructions.

[0149] Administering the combination according to the invention by means of a single patient pack or a patient pack for each composition including a package insert instructing the patient on the correct use of the invention is a desirable feature of the invention.

[0150] A further embodiment according to the invention provides a patient pack comprising at least one active substance according to the combination of the invention and an information insert containing instructions for the use of the combination according to the invention. In another embodiment of the invention, there is provided a dual pack which jointly contains an antimicrobial agent for separate administration (preferably having biological activity against clinically latent microorganisms) and one or more of the compounds disclosed herein (preferably having biological activity against clinically latent microorganisms).

[0151] The amount of the active ingredient required for treatment will vary depending on the nature of the condition to be treated and the age and condition of the patient, and will ultimately be determined by the attending physician. However, generally speaking, the dose for adult treatment will usually be in the range of 0.02 to 5000 mg per day, preferably 1 - 1500 mg per day. The desired dose can conveniently be presented as a single dose or as separate doses administered at suitable intervals, for example two, three or more sub-doses per day.

[0152] Therefore, those skilled in the art will readily obtain and understand this information.

[0153] Biological tests

[0154] Test procedures for determining the biological (e.g., bactericidal or antimicrobial) activity of the active ingredient include those known to those skilled in the art for determining the following:

[0155] (a) Bactericidal activity against clinically latent bacteria; and

[0156] (b) Antimicrobial activity against log-phase bacteria.

[0157] Regarding the above (a), methods for determining clinically latent bacterial activity include determining the Minimum Stationary-cidal Concentration (“MSC”) or Minimum Dormicidal Concentration (“MDC”) of a test compound under conditions known to those skilled in the art (such as those described in Nature Reviews, Drug Discovery 1, 895-910 (2002), the disclosure of which is hereby incorporated by reference).

[0158] For example, WO2000028074 describes suitable methods for screening compounds to determine their ability to kill clinically latent microorganisms. Typical methods can include the following steps:

[0159] (1) Grow a bacterial culture to the stationary phase;

[0160] (2) Treat the stationary-phase culture with one or more antimicrobial agents at a concentration and / or time sufficient to kill the growing bacteria, thereby selecting a phenotypically resistant subpopulation;

[0161] (3) Incubate a sample of the phenotypically resistant subpopulation with one or more test compounds or agents; and

[0162] (4) Evaluate the antimicrobial effect on the phenotypically resistant subpopulation.

[0163] According to this method, the phenotypically resistant subpopulation can be regarded as representative of clinically latent bacteria that retain metabolic activity in vivo and can cause disease recurrence or onset.

[0164] Regarding the above (b), methods for determining the activity against log-phase bacteria include determining the Minimum Inhibitory Concentration (“MIC”) or Minimum Bactericidal Concentration (“MBC”) of a test compound under standard conditions (i.e., conditions known to those skilled in the art, such as those described in WO 2005014585, the disclosure of which is hereby incorporated by reference). Specific examples of such methods are described below. Examples

[0165] Antimicrobial agents were obtained from commercial sources. They were weighed and dissolved in water, PBS, DMSO, or acidified water to achieve a final concentration of 1-10 mg / mL. The antimicrobial agent solution was diluted 10-fold to the highest concentration used in the experiment and then serially diluted 2-fold in no more than 11 consecutive dilutions. This left the operator with up to 12 different, decreasing concentrations of the selected antimicrobial agent.

[0166] The 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 the following examples, the bacteria were cultured overnight or until confluent in Mueller Hinton cation-adjusted broth in the presence or absence of supplements. The OD of the bacteria was measured 600 , and bacteria with a reading < 0.25 were returned to the incubator. The bacterial culture was diluted in the medium until the OD 600 register reading = < 0.01, which indicates that the culture was approximately 10 6 CFU / ml.

[0167] All data in this article were generated using the same checkerboard test.

[0168] 20 μL of antibiotic A (backbone) and 20 μL of antibiotic B (backbone) were pipetted as single dilutions into all wells of a 96-well plate for use.

[0169] 20 μL of the lowest concentration of antibiotic C (first variable) was pipetted into column 1 of the 96-well plate. The second concentration (2-fold concentration) was pipetted into column 2. This process was repeated until all concentrations of antibiotic B to be used were added.

[0170] 20 μL of the lowest concentration of antibiotic D (second variable) was pipetted into row A of the 96-well plate. The second concentration (2-fold concentration) was pipetted into row B. This process was repeated until all concentrations of antibiotic C to be used were added.

[0171] 120 μL of sterile medium (BHI / MHB2) was added.

[0172] 20 μL of the prepared bacterial culture was added.

[0173] The culture plate was sealed with a lid and incubated for 16 hours, overnight.

[0174] The culture plate was read in a 96-well plate reader at OD 600 The OD values for each combination are reported below.

[0175] As 4-mer combinations, the concentrations of two antibiotics change while one remains constant, and the latter is the "backbone". The method was explained above, and the calculation of FICI and the indicative values for synergy, indifference, and antagonism were also explained together.

[0176] Example 1: Synergy between ceftazidime, doxycycline, colistin, and rifampicin

[0177] The quadruple combination of ceftazidime, doxycycline, colistin, and rifampicin was tested in the assays outlined above. Colistin at 1 / 16 MIC (0.125 mg / L) and rifampicin at 1 / 16 MIC (0.25 mg / L) were the backbone, and the combination was tested against ESBL Escherichia coli. The highest concentration in each experiment was equal to 1x MIC of the test isolate.

[0178] The isolates of these examples were resistant to cephalosporins and ceftazidime at ≥32 mg / L. Thus, the concentrations used fell within the dosage guidelines recommended by EUCAST (European Committee on Antimicrobial Susceptibility Testing). EUCAST has compiled a breakpoint table for interpreting MICs and zone diameters, see for example version 12.

[0179] The following concentrations (mg / L) of ceftazidime and doxycycline were tested.

[0180]

[0181] The results reported in the above table were OD600 values determined as described herein. The bold values in the above table (and similarly in the corresponding tables of subsequent examples) indicate bacterial growth and are considered to represent ineffective bacterial killing (i.e., lack of synergy).

[0182] The MICs of each drug alone and in combination were calculated according to the method outlined above.

[0183]

[0184] ΣFIC = 0.813 and FIC = 0.406. As explained above, this indicates synergy.

[0185] The quadruple combination of ceftazidime, doxycycline, colistin, and rifampicin was also tested against ESBL Escherichia coli, with 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:

[0186]

[0187] The results reported in the above table were OD600 values determined as described herein.

[0188] The MICs of each drug alone and in combination were calculated according to the method outlined above.

[0189]

[0190] ΣFIC = 0.44 and FIC = 0.221. As explained above, this indicates synergy.

[0191] The quadruple combination of ceftazidime, doxycycline, colistin, and rifampicin was also tested against ESBL E. coli, with 1 / 8 MIC (4 mg / L) of ceftazidime and 1 / 16 MIC (0.25 mg / L) of rifampicin as the backbone. The following concentrations (mg / L) of colistin and doxycycline were tested.

[0192]

[0193] The results reported in the above table are the OD600 values determined as described herein.

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

[0195]

[0196] ΣFIC = 0.34, and FIC = 0.172. As explained above, this indicates synergy.

[0197] Finally, the quadruple combination of ceftazidime, doxycycline, colistin, and rifampicin was tested against ESBL E. coli, with 1 / 8 MIC (4 mg / L) of ceftazidime and 1 / 16 MIC (0.06 mg / L) of doxycycline as the backbone. The following concentrations (mg / L) of colistin and rifampicin were tested:

[0198]

[0199]

[0200] The results reported in the above table are the OD600 values determined as described herein.

[0201] The MICs of each drug alone and in combination were calculated according to the method outlined above.

[0202]

[0203] ΣFIC = 0.375, and FIC = 0.188. As explained above, this indicates synergy.

[0204] As can be seen from the various tests in Example 1, how the quadruple combination of colistin, rifampicin, doxycycline, and ceftazidime synergizes with all compounds that can be included at concentrations below the MIC mono of.

[0205] Synergy is not a predictable outcome. Synergy is particularly difficult to predict at sub-MIC concentrations or against MDR bacteria (ESBL E. coli); this means that these combinations represent an important advance in the fight against antimicrobial resistance. Surprisingly, they were successful against bacteria in the strain found to have the enzyme (ESBL), which is known not to be killed by many of the antibiotics that would be used by a person skilled in the art to treat infections.

[0206] Example 2: Synergy between ceftazidime, doxycycline, colistin, and levofloxacin

[0207] The 4-drug combination of ceftazidime, doxycycline, colistin, and levofloxacin was tested in the assays outlined above. Colistin at 1 / 16 MIC (0.125 mg / L) and doxycycline at 1 / 16 MIC (0.06 mg / L) were the backbone, and the combination was tested against ESBL E. coli. The highest concentration in each experiment was equal to 1x MIC of the test isolate. The following concentrations (mg / L) of ceftazidime and levofloxacin were used.

[0208]

[0209] The results reported in the table above were OD600 values determined as described herein.

[0210] The MIC of each drug alone and in combination was calculated according to the method outlined above.

[0211]

[0212] ΣFIC = 0.719 and FIC = 0.359. As explained above, this indicates synergy.

[0213] Example 3: Synergy between ceftazidime, doxycycline, rifampicin, and levofloxacin

[0214] The 4-drug combination of ceftazidime, doxycycline, rifampicin, and levofloxacin was tested in the assays outlined above. Rifampicin at 1 / 16 MIC (0.06 mg / L) and doxycycline at 1 / 16 MIC (0.06 mg / L) were the backbone, and the combination was tested against ESBL E. coli. The highest concentration in each experiment was equal to 1x MIC of the test isolate. The following concentrations (mg / L) of ceftazidime and levofloxacin were used.

[0215]

[0216] The results reported in the table above were OD600 values determined as described herein.

[0217] The MIC of each drug alone and in combination was calculated according to the method outlined above.

[0218]

[0219] ΣFIC = 0.656 and FIC = 0.328. As explained above, this indicates synergy.

[0220] Example 4: Synergy between ceftazidime (ceft), doxycycline (doxy), rifampicin (rif), and colistin (CSS)

[0221] The 4 - drug combination of ceftazidime, doxycycline, rifampicin, and colistin was tested in the assays outlined above. A backbone of 1 / 256 MIC (0.125 mg / L) ceftazidime and 1 / 8 MIC (0.125 mg / L) colistin was used, and the combination was tested against KPC (Klebsiella pneumoniae carbapenemase) - producing Klebsiella pneumoniae. The highest concentration in each experiment was equal to 1x MIC of the test isolate. The following concentrations (mg / L) of ceftazidime and colistin were used.

[0222]

[0223] The results reported in the table above are reported as the percentage of bacterial killing based on OD600 values relative to a positive control.

[0224]

[0225] The MICs of each drug alone and in combination were calculated according to the method outlined above.

[0226] ΣFIC = 0.441 and FIC = 0.221. As explained above, this indicates synergy.

[0227] The examples support that the combinations of the present invention have synergy. When combining antimicrobial agents, synergy is not an expected result, and especially when four antimicrobial agents are combined and / or against multi - drug - resistant bacteria (such as ESBL strains), synergy is undoubtedly not an expected result. The examples support that the combinations of the present invention have synergy against drug - resistant bacteria and thus provide a solution to the global antimicrobial resistance problem discussed above. This is a significant advancement in the art.

[0228] The various embodiments described herein are presented only to assist in understanding and teaching the claimed features. These embodiments are provided only as representative samples 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 considered as limitations on the scope of the invention defined by the claims or on equivalents of the claims, and that other embodiments may be used and modifications may be made without departing from the scope of the claimed invention. The various embodiments of the invention may suitably comprise, consist of, or consist essentially of suitable combinations of the disclosed elements, components, features, parts, steps, devices, etc., in addition to those specifically described herein. Further, the present disclosure may include other inventions that are not currently claimed but 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 their pharmaceutically acceptable derivatives.

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

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

4. The combination according to any one of the preceding claims, wherein the at least four antimicrobial agents comprise ceftazidime and doxycycline, or their pharmaceutically acceptable derivatives, and at least two of polymyxin E, polymyxin B, rifampicin, levofloxacin, and their pharmaceutically acceptable derivatives.

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

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

7. The combination according to claim 4, wherein the at least four antimicrobial agents are rifampicin, levofloxacin, doxycycline, ceftazidime, or their pharmaceutically acceptable derivatives.

8. The combination according to any one of claims 1 to 7, for use in the treatment of bacterial infections.

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

10. The pharmaceutical composition according to claim 9, for use in the treatment of bacterial infections.

11. The combination for use according to claim 8 or the pharmaceutical composition for use according to claim 10, wherein the use is to kill proliferating microorganisms associated with a bacterial infection.

12. 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. 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 Enterobacteriaceae, preferably wherein the bacterial infection is caused by Escherichia coli or Klebsiella pneumoniae.

14. 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 any one of claims 8 or 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, the product being a combination preparation for use in the simultaneous, separate or sequential treatment of bacterial infections.

17. The product according to 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.

Images