METHOD FOR RESTORING EFFICACY OF AN ANTIBACTERIAL AGENT

Field of the Invention

The present invention relates to a method for restoring the efficacy or prolonging the life of an antibacterial agent by combining the antibacterial agent with at least two antibiotic resistance breakers, wherein each antibiotic resistance breaker is used independently with the antibacterial agent so as to at least partially restore the efficacy thereof and wherein each antibiotic resistance breaker has a different mechanism of action. The invention also provides the use of at least two antibiotic resistance breakers to restore efficacy or prolong the life of an antibacterial agent. Background

Before the introduction of antibiotics, patients suffering from acute microbial infections (e.g. tuberculosis or pneumonia) had a low chance of survival. For example, mortality from tuberculosis was around 50%. Although the introduction of antibacterial agents in the 1940s and 1950s rapidly changed this picture, bacteria have responded by progressively gaining resistance to commonly used antibiotics. Now, every country in the world has antibiotic- resistant bacteria. Indeed, more than 70% of bacteria that give rise to hospital acquired infections in the USA resist at least one of the main antibacterial agents that are typically used to fight infection {Nature Reviews, Drug Discovery, 1 , 895-910 (2002)), and in its 2014 report of global antibacterial resistance, the World Health Organization focused on the high levels of antibiotic resistance in the bacteria that cause common infections.

One way of tackling the growing problem of resistant bacteria is the development of new classes of antibacterial agents. However, until the introduction of linezolid in 2000, there had been no new class of antibiotic marketed for over 37 years. Moreover, even the development of new classes of antibiotic provides only a temporary solution, and indeed there are already reports of resistance of certain bacteria to linezolid (Lancet 357, 1179 (2001) and Lancet 358, 207-208 (2001)).

In order to develop more long-term solutions to the problem of bacterial resistance, it is clear that alternative approaches are required.

One approach is to co-administer another drug with the failing antibiotic so as to restore sufficient antibacterial activity. These compounds are known in the art as "antibiotic resistance breakers" (ARBs), and the use of such compounds to restore antibiotics is exemplified by the successful co-administration of β-lactamase inhibitors, such as clavulanic acid, with β-lactam antibiotics, such as amoxicillin (White, A. R, el al. _: J. Aniimicrob. Chemother. 53 (Suppl. 1), i3-i20 (2004); Prabhudesai, P. P. et al., J. Indian Med. Assoc. 109, 124-127 (2011)). Existing β-ΐ3θί3Γη/β-ΐ3θί3Γη33β inhibitor combinations include Tazocin® (piperacillin/tazobactam), Avycaz® (ceftazidime/avibactam) and Carbavance® (meropenem/vaborbactam). It would, however, be preferable to identify ARBs which restore one or more key members of each mechanistic antibiotic class, for example those antibiotics used against Gram-negative bacteria. According to Nature Reviews, Drug Discovery, 14, 821-832 (2015), the antibiotics that most need ARBs are:

cephalosporins and carbapenems;

- polymyxins;

fluoroquinolones;

tetracyclines and aminoglycosides; and

macrolides.

Of these, polymyxins are of particular interest because polymyxin E (colistin) is one of the agents currently used to combat bacteria that are resistant to the strongest antibiotics.

"Colistin" is commercially available in Europe under the trade name Colomycin® in intravenous form. Intravenous Colomycin® includes colistimethate sodium which undergoes hydrolysis to the active substance colistin in aqueous solution and is indicated for the treatment of serious infections due to selected aerobic Gram-negative pathogens in patients with limited treatment options.

There have, however, been reports which show that colistin may be losing its effectiveness in antibacterial therapy. The U.S. Military HIV Research Program has for instance reported colistin resistance in a human E. coli infection

International Patent Application, Publication Number WO2012032360 discloses a combination comprising phenoxybenzamine or a pharmaceutically acceptable derivative thereof and a polymyxin selected from polymyxin E and polymyxin B or a pharmaceutically acceptable derivative thereof, and its use in treating a microbial infection.

International Patent Application, Publication Number WO2014147405 discloses the use of colistin (polymyxin E) in combination with zidovudine for treating a microbial infection. International Patent Application, Publication Number WO2016097754 discloses a combination comprising suloctidil or a pharmaceutically acceptable derivative or prodrug thereof, and a polymyxin selected from polymyxin E and polymyxin B or a pharmaceutically acceptable derivative thereof, and its use in treating a microbial infection. Antibiotic resistance to these combinations of an antibacterial agent (e.g. colistin) and antibiotic resistance breaker will also, however, inevitably occur.

The present invention is therefore based on the unexpected finding that the efficacy of an antibacterial agent can be prolonged and/or restored by using at least two antibiotic resistance breakers with different mechanisms of action for at least two time periods: the first antibiotic resistance breaker being combined with the antibacterial agent for a first time period, and the second antibiotic resistance breaker being combined with the antibacterial agent for a second time period.

"Efficacy" in the context of the present invention refers to the antibacterial activity of the antibacterial agent against a particular bacterium, preferably a Gram-negative bacterium. For example, the antibacterial activity of colistin against a Gram-negative bacterium such as E.coli or K. pneumoniae, preferably a drug-resistant strain of E.coli or K.pneumoniae.

In one embodiment, the antibacterial activity of the antibacterial agent and the antibiotic resistance breaker against the bacterium of interest is additive or synergistic. Preferably the antibacterial activity is synergistic. Synergy in the context of antibacterial drugs is measured in a number of ways that conform to the generally accepted opinion that "synergy is an effect greater than additive".

One of the ways to assess whether synergy has been observed is to use the "chequerboard" technique. This is a well-accepted method that leads to the generation of a value called the fractional inhibitory concentration index (FICI).

Orhan et al J. Clin. Microbiol. 2005, 43(1): 140 describes the chequerboard method and analysis in the paragraph bridging pages 140-141 , and explains that the FICI value is a ratio of the sum of the MIC (Minimum Inhibitory Concentration) level of each individual component alone and in the mixture. The combination is considered synergistic when the∑FIC is≤0.5, indifferent when the∑FIC is >0.5 but <2, and antagonistic when the∑FIC is≥2.

Another accepted test for ascertaining the presence or absence of synergy is to use time-kill methods. This involves the dynamic effect of a drug combination being compared to each drug alone when assessing the effect on bacterial log or stationary-growth over time. Again, the possible results are synergy, indifference or antagonism. Summary of invention

A first aspect of the invention is a method for restoring efficacy of an antibacterial agent. The method comprises (i) combining the antibacterial agent with a first antibiotic resistance breaker after a first time period; and (ii) combining the antibacterial agent with a second antibiotic resistance breaker after a second time period. During each time period the efficacy of the antibacterial agent decreases due to increasing antibiotic resistance, and each antibiotic resistance breaker at least partially restores the efficacy of the antibacterial agent relative to the end of the preceding time period. The first and second antibiotic resistance breakers have different mechanisms of action.

In a second aspect the invention provides the use of at least two antibiotic resistance breakers to prolong the efficacy of an antibacterial agent, where the at least two antibiotic resistance breakers have different mechanisms of action. Figures

Figure 1 is a graph of relative efficacy of antibiotic therapy against an approximate historic and anticipated timeline. This graph portrays the invention in a schematic manner showing how antibiotic resistance breakers (ARBs) can be combined with an antibacterial agent after successive time periods so as to at least partially restore the efficacy of the antibacterial agent.

Figure 2 contains images of E.coli tolC cells showing the characteristic phenotype produced by treatment with the compounds in Example 3. Each image is entitled with the compound tested and the concentration used (in μg/ml). In Figure 2A cell membranes are shown in red, DNA is shown in blue and green staining indicates permeabilization of the cell membrane. White scale bar is 1 μηι. Figure 2B is the greyscale version of Figure 2A.

Figure 3 contains images of E.coli ATCC 25922 cells showing the predominant phenotypes produced by treatment in Example 3 with colistin and/or mefloquine for 2 hours at the concentrations indicated ^g/ml). In Figure 3A cell membranes are shown in red, DNA in blue and green staining indicates permeabilization of the cell membrane. White scale bar is 1 μηι. Figure 3B is the greyscale version of Figure 3A.

Figure 4 is a chart which shows the viable cell counts for E.coli ATCC 25922 treated with DMSO, colistin, mefloquine or a combination of colistin and mefloquine at 0, 2 and 4 hours. Concentrations are in μg/ml.

Figure 5 contains images of E.coli ATCC 25922 cells showing the predominant phenotypes produced by treatment in Example 3 with colistin and/or suloctidil for 2 hours at the concentrations indicated ^g/ml). In Figure 5A cell membranes are shown in red, DNA in blue and green staining indicates permeabilization of the cell membrane. White scale bar is 1 μηι. Figure 5B is the greyscale version of Figure 5A. Figure 6 is a chart which shows the viable cell counts for E.coli ATCC 25922 treated with DMSO, colistin, suloctidil or a combination of colistin and suloctidil at 0, 2 and 4 hours. Concentrations are in μςΛηΙ.

Figure 7 contains images of E.coli ATCC 25922 cells showing the predominant phenotypes produced by treatment in Example 3 with colistin and/or zidovudine for 2 hours at the concentrations indicated ^g/ml). In Figure 7A cell membranes are shown in red, DNA in blue and green staining indicates permeabilization of the cell membrane. White scale bar is 1 μηι. Figure 7B is the greyscale version of Figure 7A.

Figure 8 is a chart which shows the viable cell counts for E.coli ATCC 25922 treated with DMSO, colistin, zidovudine or a combination of colistin and zidovudine at 0, 2 and 4 hours. Concentrations are in μg/ml.

Figure 9 contains images of E.coli ATCC 25922 cells showing the predominant phenotypes produced by treatment in Example 3 with colistin and/or phenoxybenzamine for 2 hours at the concentrations indicated ^g/ml). In Figure 9A cell membranes are shown in red, DNA in blue and green staining indicates permeabilization of the cell membrane. White scale bar is 1 μηι. Figure 9B is the greyscale version of Figure 9A.

Figure 10 is a chart which shows the viable cell counts for E.coli ATCC 25922 treated with DMSO, colistin, phenoxybenzamine or a combination of colistin and phenoxybenzamine at 0, 2 and 4 hours. Concentrations are in μg/ml. Figure 11 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 0.5% DMSO for 30 minutes (left) and 120 minutes (right). In Figure 11A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) did not produce any visible results. White scale bar is 1 μηι. Figure 1 1 B is the greyscale version of Figure 1 1A. Figure 12 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 0.06 μg/ml azidothymidine (zidovudine) for 30 minutes (left) and 120 minutes (right). In Figure 12A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) did not produce any visible results. White scale bar is 1 μηι. Figure 12B is the greyscale version of Figure 12A.

Figures 13A and 13B are the same as Figures 12A and 12B except treatment is with 0.3 μg/ml azidothymidine (zidovudine). Figure 14 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 0.002 μg/ml ciprofloxacin for 30 minutes (left) and 120 minutes (right). In Figure 14A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) did not produce any visible results. White scale bar is 1 μηι. Figure 14B is the greyscale version of Figure 14A.

Figures 15A and 15B are the same as Figures 14A and 14B except treatment is with 0.01 μg/ml ciprofloxacin.

Figure 16 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 20 μg/ml cephalexin for 30 minutes (left) and 120 minutes (right). In Figure 16A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) did not produce any visible results. White scale bar is 1 μηι. Figure 16B is the greyscale version of Figure 16A.

Figure 17 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 25 μg/ml cerulenin for 120 minutes (left) and 240 minutes (right). In Figure 17A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) indicates permeabilization of the cell membrane. White scale bar is 1 μηι. Figure 17B is the greyscale version of Figure 17A.

Figure 18 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 7.5 μg/ml rifampicin for 30 minutes (left) and 120 minutes (right). In Figure 18A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) did not produce any visible results. White scale bar is 1 μηι. Figure 18B is the greyscale version of Figure 18A.

Figure 19 is a clustergram of azidothymine (zidovudine) and the different classes of antibiotics with E.coli tolC treated with 5X MIC for 120 minutes (or 240 minutes for cerulenin). Figure 20 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 1.5 μg/ml daunorubicin for 30 minutes (left) and 120 minutes (right). In Figure 20A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) did not produce any visible results. White scale bar is 1 μηι. Figure 20B is the greyscale version of Figure 20A. Figures 21 A and 21 B are the same as Figures 20A and 20B except treatment is with 7.5 μg/ml daunorubicin. The green staining (SYTOX Green) indicates permeabilization of the cell membrane. Figure 22 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 1 μg/ml novobiocin for 30 minutes (left) and 120 minutes (right). In Figure 22A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) did not produce any visible results. White scale bar is 1 μηι. Figure 22B is the greyscale version of Figure 22A.

Figures 23A and 23B are the same as Figures 22A and 22B except treatment is with 5 μg/ml novobiocin.

Figure 24 contains images of E.coli tolC showing the predominant phenotypes produced by treatment with 0.03 μg/ml mitomycin C for 30 minutes (left) and 120 minutes (right). In Figure 24A cell membranes are shown in red and DNA in blue. The green staining (SYTOX Green) did not produce any visible results. White scale bar is 1 μηι. Figure 24B is the greyscale version of Figure 24A.

Figures 25A and 25B are the same as Figures 24A and 24B except treatment is with 0.15 μg/ml mitomycin C. Figure 26 is a clustergram of azidothymine (zidovudine) and the DNA replication inhibitors with E.coli tolC treated with 5X MIC for 120 minutes.

Detailed description of invention

Abbreviations used herein: cy or cyclo refers to the cyclic part of the peptide, enclosed within brackets; Dab or Dbu refers to α,γ-diamino-n-butyryl (i.e. 2,4-diaminobutyryl); Abu refers to 2-aminobutyryl; Thr refers to L-threonine; DThr refers to D-threonine; DPhe refers to D- phenylamine; Leu refers to L-leucine; DSer refers to D-serine and OA refers to octanoyl.

As mentioned above, a first aspect of the invention relates to a method for restoring efficacy of an antibacterial agent as defined herein.

"Efficacy" in the context of the present invention refers to the antibacterial activity of the antibacterial agent against a particular bacterium, preferably a Gram-negative bacterium. In one embodiment, the antibacterial activity of the antibacterial agent and the antibiotic resistance breaker is additive (indifferent) or synergistic. Preferably the antibacterial activity is synergistic.

In one embodiment the antibacterial activity is against a drug-resistant Gram-negative bacterium such as E.coli or K.pneumoniae.

1 In one embodiment, the method further comprises (iii) combining the antibacterial agent with a third antibiotic resistance breaker after a third time period, where during the third time period the efficacy of the antibacterial agent decreases due to increasing antibiotic resistance, and the third antibiotic resistance breaker at least partially restores the efficacy of the antibacterial agent relative to the end of the second time period. The third antibiotic resistance breaker preferably has a different mechanism of action to both the first and second antibiotic resistance breakers.

In an additional embodiment, the method comprises (iv) combining the antibacterial agent with a fourth antibiotic resistance breaker after a fourth time period, where during the fourth time period, the efficacy of the antibacterial agent decreases due to increasing antibiotic resistance, and the fourth antibiotic resistance breaker at least partially restores the efficacy of the antibacterial agent relative to the end of the third time period. The fourth antibiotic resistance breaker preferably has a different mechanism of action to each of the first, second and third antibiotic resistance breakers. As used herein, "antibiotic resistance" refers to the ability of bacteria and other microorganisms to resist the effects of an antibiotic to which they were once sensitive. The antibiotic becomes ineffective against the bacteria. When the bacteria become resistant to most antibacterial agents, they are often referred to in the art as "superbugs".

There are various mechanisms by which bacteria become resistant to antibiotics. These mechanisms can chemically modify the antibiotic, render it inactive through physical removal from the cell, or modify the target site so that it is not recognized by the antibiotic. Efflux pumps for example are high-affinity reverse transport systems located in the membrane that transport the antibiotic out of the cell. Alternatively there may be a specific enzyme which modifies the antibiotic in a way that it loses its activity or degrades the antibiotic so it becomes inactive.

Bacteria may alternatively be inherently resistant to an antibiotic. For example, an organism may lack a transport system for an antibiotic, or the target for the antibiotic molecule, or as in the case of Gram-negative bacteria, the cell wall may be covered with an outer membrane that establishes a permeability barrier against the antibiotic. As used herein, the term "antibiotic resistance breaker" refers to non-antibiotic compounds that, when combined with existing antibiotics, act to block resistance or enhance antibacterial activity. These compounds are also known in the art as "antibiotic adjuvants", "resistance breakers" and "antibiotic potentiators". They may be known compounds (repurposed existing drugs) or new chemical entities. In one embodiment at least one of the antibiotic resistance breakers of the present invention is a repurposed existing drug. Preferably each of the antibiotic resistance breakers is a repurposed existing drug.

One advantage of using a repurposed drug is that these drugs have known toxicology and pharmacology profiles, and therefore can lead to considerable cost savings by eliminating much of the toxicological and pharmacokinetic assessment that would normally be required for approval of a new drug.

The antibiotic resistance breakers in the method or use of the present invention may, for example, be selected from the group consisting of zidovudine, suloctidil, mefloquine, phenoxybenzamine or pharmaceutically acceptable derivatives thereof. All of these compounds are known non-antibiotic drugs and in the context of the present invention are repurposed existing drugs.

Zidovudine is also known as azidothymidine (AZT) and is an anti retroviral medication used to prevent and treat HIV/AIDs. It is generally recommended for use with other antiretrovirals and has the following chemical structure:

Its lUPAC name is 1-[(2/ ^: ?,4S,5S)-4-Azido-5-(hydroxymethyl)oxolan-2-yl]-5-methy lpyrimidine- 2,4-dione, and it is available by prescription only under the trade name Retrovir®.

Suloctidil is a sulfur-containing amino alcohol that was brought to market in the early 1970s as a vasodilator. It has the following chemical structure:

Mefloquine is an orally administered medication used in the prevention and treatment of malaria. It is commercially available in Europe under the trade name Lariam® in tablet form. Lariam® tablets include the hydrochloride salt of mefloquine and are indicated for the therapy and prophylaxis of malaria. Phenoxybenzamine is a non-selective, irreversible alpha blocker which is marketed under the trade name Dibenzyline®. It is used in the treatment of hypertension and has the following chemical structure:

In one embodiment the first antibiotic resistance breaker is zidovudine or a pharmaceutically acceptable derivative thereof.

In one embodiment the second, third and/or fourth antibiotic resistance breakers are selected from the group consisting of suloctidil, mefloquine and phenoxybenzamine or pharmaceutically acceptable derivatives thereof, provided that each of the antibiotic resistance breakers is different.

The antibiotic resistance breakers used in the present invention have different mechanisms of action. The term "mechanism of action" describes the specific biochemical interaction through which the ARB produces its pharmacological effect. In other words, how the ARB restores the efficacy (i.e. antibacterial activity) of the antibacterial agent against the bacterium of interest. The mechanism of actions of the ARBs mefloquine, suloctidil, zidovudine and phenoxybenzamine are discussed in more detail in the Examples.

In some literature articles, the term mechanism of action and mode of action (MoA) are used interchangeably; typically referring to the way in which the drug interacts and produces a medical effect. However, in actuality, a mode of action describes functional or anatomical changes, at the cellular level, resulting from the exposure of a living organism to a substance. This differs from a mechanism of action, as it is a more specific term that focuses on the interaction between the drug itself and an enzyme or receptor and its particular form of interaction, whether through inhibition, activation, agonism, or antagonism.

It is known in the art how to determine a mechanism of action for a particular compound. For example, the skilled person may use microscopy-based methods, direct biochemical methods or computation inference methods.

Without wishing to be bound by theory, it is believed that antibiotic resistance breakers may work by disrupting the bacterial cell membrane, by destroying the bacterial cell wall, inhibiting resistance mechanisms such as drug efflux from the cell, inhibiting bacterial enzymes which destroy antibiotics, including enhancing access of antibiotics to drug targets, and preventing the direct modification or inactivation of antibiotics.

By combining the antibacterial agent with at least two antibiotic resistance breakers each having a different mechanism of action, the present invention advantageously allows the lifetime of an antibacterial agent to be extended, despite the inevitable build-up of antibiotic resistance against the antibacterial agent alone and in combination with the antibiotic resistance breakers. The antibacterial agent effectively becomes renewable.

As used herein, the term "combining" or "in combination with" covers both separate and sequential use of the antibacterial agent and each antibiotic resistance breaker. When the compounds are used or administered sequentially, either the antibacterial agent or the antibiotic resistance breaker may be administered first. When administration is simultaneous, the compounds may be administered either in the same or a different pharmaceutical composition.

As will be appreciated and readily understood by the skilled person, the time periods of the present invention will depend on the rate at which antibiotic resistance develops to the antibacterial agent, used alone or in combination with an antibiotic resistance breaker. The time periods can for instance be identical or different in length, for example, the first time period could be longer, shorter or the same length as the second time period. The second time period could be longer, shorter or the same length as the third time period, etc. In one embodiment each of the time periods is between 5 and 50 years, preferably between 10 and 40 years, more preferably between 20 and 30 years.

As used herein, the term "antibacterial agent" refers to compounds which kill bacteria or inhibit their growth.

Antibacterial agents are used in the treatment and prevention of bacterial infections and are commonly classified based on their mechanism of action, chemical structure or spectrum of activity. For example, penicillins and cephalosporins target the bacterial cell wall, polymyxins target the cell membrane, rifamycins, lipiarmycins, quinolones and sulphonamides interfere with essential bacterial enzymes, and macrolides, lincosamides and tetracyclines target protein synthesis. Suitable antibacterial agents for the present invention include one or more compounds selected from the following:

(1) β-Lactams, including:

(i) penicillins, such as (I) benzylpenicillin, procaine benzylpenicillin, phenoxy-methylpenicillin, methicillin, propicillin, epicillin, cyclacillin, hetacillin, 6-aminopenicillanic acid, penicillic acid, penicillanic acid sulphone (sulbactam), penicillin G, penicillin V, phenethicillin, phenoxymethylpenicillinic acid, azlocillin, carbenicillin, cloxacillin, D-(-)-penicillamine, dicloxacillin, nafcillin and oxacillin,

(II) penicillinase-resistant penicillins (e.g. flucloxacillin),

(III) broad-spectrum penicillins (e.g. ampicillin, amoxicillin, metampicillin and bacampicillin),

(IV) antipseudomonal penicillins (e.g. carboxypenicillins such as ticarcillin or ureidopenicillins such as piperacillin),

(V) mecillinams (e.g. pivmecillinam), or

(VI) combinations of any two or more of the agents mentioned at (I) to (V) above, or combinations of any of the agents mentioned at (I) to (V) above with a β-lactamase inhibitor such as tazobactam or, particularly, clavulanic acid (which acid is optionally in metal salt form, e.g. in salt form with an alkali metal such as sodium or, particularly, potassium);

(ii) cephalosporins, such as cefaclor, cefadroxil, cefalexin (cephalexin), cefcapene, cefcapene pivoxil, cefdinir, cefditoren, cefditoren pivoxil, cefixime, cefotaxime, cefpirome, cefpodoxime, cefpodoxime proxetil, cefprozil, cefradine, ceftazidime, cefteram, cefteram pivoxil, ceftriaxone, cefuroxime, cefuroxime axetil, cephaloridine, cephacetrile, cephamandole, cephaloglycine, ceftobiprole, PPI-0903 (TAK-599), 7-aminocephalosporanic acid, 7-aminodes- acetoxycephalosporanic acid, cefamandole, cefazolin, cefmetazole, cefoperazone, cefsulodin, cephalosporin C zinc salt, cephalothin, cephapirin; and

(iii) other β-lactams, such as monobactams (e.g. aztreonam), carbapenems (e.g. imipenem (optionally in combination with a renal enzyme inhibitor such as cilastatin), meropenem, ertapenem, doripenem (S-4661) and RO4908463 (CS-023)), penems (e.g. faropenem) and 1-oxa^-lactams (e.g. moxalactam).

(2) Tetracyclines, such as tetracycline, demeclocycline, doxycycline, lymecycline, minocycline, oxytetracycline, chlortetracycline, meclocycline and methacycline, as well as glycylcyclines (e.g. tigecycline).

(3) Aminoglycosides, such as amikacin, gentamicin, netilmicin, neomycin, streptomycin, tobramycin, amastatin, butirosin, butirosin A, daunorubicin, dibekacin, dihydrostreptomycin, G 418, hygromycin B, kanamycin B, kanamycin, kirromycin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptozocin and thiostrepton. (4) (i) Macro I ides, such as azithromycin, clarithromycin, erythromycin, roxithromycin, spiramycin, amphotericins B (e.g. amphotericin B), bafilomycins (e.g. bafilomycin A1), brefeldins (e.g. brefeldin A), concanamycins (e.g. concanamycin A), filipin complex, josamycin, mepartricin, midecamycin, nonactin, nystatin, oleandomycin, oligomycins (e.g. oligomycin A, oligomycin

B and oligomycin C), pimaricin, rifampicin, rifamycin, rosamicin, tylosin, virginiamycin and fosfomycin.

(ii) Ketolides such as telithromycin and cethromycin (ABT-773).

(iii) Lincosamines, such as lincomycin.

(5) Clindamycin and clindamycin 2-phosphate.

(6) Phenicols, such as chloramphenicol and thiamphenicol.

(7) Steroids, such as fusidic acid (optionally in metal salt form, e.g. in salt form with an alkali metal such as sodium).

(8) Glycopeptides such as vancomycin, teicoplanin, bleomycin, phleomycin, ristomycin, telavancin, dalbavancin and oritavancin.

(9) Oxazolidinones, such as linezolid and AZD2563.

(10) Streptogramins, such as quinupristin and dalfopristin, or a combination thereof.

(11) (i) Peptides, such as polymyxins (e.g. polymyxin E (colistin) and polymyxin B), lysostaphin, duramycin, actinomycins (e.g. actinomycin C and actinomycin D), actinonin, 7-aminoactinomycin D, antimycin A, antipain, bacitracin, cyclosporin

A, echinomycin, gramicidins (e.g. gramicidin A and gramicidin C), myxothiazol, nisin, paracelsin, valinomycin and viomycin.

(ii) Lipopeptides, such as daptomycin.

(iii) Lipoglycopeptides, such as ramoplanin.

(12) Sulfonamides, such as sulfamethoxazole, sulfadiazine, sulfaquinoxaline, sulfathiazole (which latter two agents are optionally in metal salt form, e.g. in salt form with an alkali metal such as sodium), succinylsulfathiazole, sulfadimethoxine, sulfaguanidine, sulfamethazine, sulfamonomethoxine, sulfanilamide and sulfasalazine.

(13) Trimethoprim, optionally in combination with a sulfonamide, such as sulfamethoxazole (e.g. the combination co-trimoxazole).

(14) Antituberculous drugs, such as isoniazid, rifampicin, rifabutin, pyrazinamide, ethambutol, streptomycin, amikacin, capreomycin, kanamycin, quinolones (e.g. those at (q) below), para-aminosalicylic acid, cycloserine and ethionamide.

(15) Antileprotic drugs, such as dapsone, rifampicin and clofazimine.

(16) (i) Nitroimidazoles, such as metronidazole and tinidazole.

(ii) Nitrofurans, such as nitrofurantoin. (17) Quinolones, such as nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, garenoxacin, DX-619, WCK 771 (the arginine salt of S-(-)-nadifloxacin), 8-quinolinol, cinoxacin, enrofloxacin, flumequine, lomefloxacin, oxolinic acid and pipemidic acid.

(18) Amino acid derivatives, such as azaserine, bestatin, D-cycloserine, 1 ,10- phenanthroline, 6-diazo-5-oxo-L-norleucine and L-alanyl-L-1-aminoethyl-phosphonic acid.

(19) Aureolic acids, such as chromomycin A3, mithramycin A and mitomycin C C.

(20) Benzochinoides, such as herbimycin A.

(21) Coumarin-glycosides, such as novobiocin.

(22) Diphenyl ether derivatives, such as irgasan.

(23) Epipolythiodixopiperazines, such as gliotoxin from Gliocladium fimbriatum.

(24) Fatty acid derivatives, such as cerulenin.

(25) Glucosamines, such as 1-deoxymannojirimycin, 1-deoxynojirimycin and A/-methyl-1- deoxynojirimycin.

(26) Indole derivatives, such as staurosporine.

(27) Diaminopyrimidines, such as iclaprim (AR-100).

(28) Macrolactams, such as ascomycin.

(29) Taxoids, such as paclitaxel.

(30) Statins, such as mevastatin.

(31) Polyphenolic acids, such as (+)-usnic acid.

(32) Polyethers, such as lasalocid A, lonomycin A, monensin, nigericin and salinomycin.

(33) Picolinic acid derivatives, such as fusaric acid.

(34) Peptidyl nucleosides, such as blasticidine S, nikkomycin, nourseothricin and puromycin.

(35) Nucleosides, such as adenine 9^-D-arabinofuranoside, 5-azacytidine, cordycepin, formycin A, tubercidin and tunicamycin.

(36) Pleuromutilins, such as GSK-565154, GSK-275833 and tiamulin.

(37) Peptide deformylase inhibitors, such as LBM415 (NVP PDF-713) and BB 83698. (38) Antibacterial agents for the skin, such as fucidin, benzamycin, clindamycin, erythromycin, tetracycline, silver sulfadiazine, chlortetracycline, metronidazole, mupirocin, framycitin, gramicidin, neomycin sulfate, polymyxins (e.g. polymyxin B or polymyxin E) and gentamycin.

(39) Miscellaneous agents, such as methenamine (hexamine), doxorubicin, piericidin A, stigmatellin, actidione, anisomycin, apramycin, coumermycin A1 , L(+)-lactic acid, cytochalasins (e.g. cytochalasin B and cytochalasin D), emetine and ionomycin. (40) Antiseptic agents, such as chlorhexidine, phenol derivatives (e.g. thymol and triclosan), quarternary ammonium compounds (e.g. benzalkonium chloride, cetylpyridinium chloride, benzethonium chloride, cetrimonium bromide, cetrimonium chloride and cetrimonium stearate), octenidine dihydrochloride, and terpenes (e.g. terpinen-4-ol). or a pharmaceutically acceptable derivative thereof.

Preferably the antibacterial agent is a polymyxin selected from colistin (polymyxin E), polymyxin B, or pharmaceutically acceptable derivatives thereof. More preferably the antibacterial agent is colistin or a pharmaceutically acceptable derivative thereof.

In one embodiment the antibacterial agent is a polymyxin derivative of formula (I):

¾R5 „ _s ,*18

wherein: R1 is Dab;

R2 is Thr;

R3 is DThr;

R4 is Dab;

R5 is Dab;

R6 is DPhe;

R7 is Leu;

R8 is Abu;

R9 is Dab;

R10 is Thr; and

R(FA) is octanoyl;

or wherein: R1 is absent;

R2 is Thr;

R3 is DSer;

R4 is Dab;

R5 is Dab;

R6 is DPhe;

R7 is Leu;

R8 is Dab;

R9 is Dab;

R10 is Thr; and R(FA) is octanoyl;

or a pharmaceutically acceptable salt thereof.

The polymyxin derivatives of formula (I) are known compounds developed by Northern Antibiotics Oy. The polymyxin derivative of formula (I) wherein R1 is Dab; R2 is Thr; R3 is DThr; R4 is Dab; R5 is Dab; R6 is DPhe; R7 is Leu; R8 is Abu; R9 is Dab; R10 is Thr; and R(FA) is octanoyl; was first disclosed in WO20161 13470 (A1) as NAB815. Example 1 of WO20161 13470 (A1) discloses the synthesis of NAB815. The disclosure of this reference insofar as it relates to NAB815 is incorporated herein by reference. The polymyxin derivative of formula (I) wherein R1 is absent (i.e. replaced by a covalent bond); R2 is Thr; R3 is DSer; R4 is Dab; R5 is Dab; R6 is DPhe; R7 is Leu; R8 is Dab; R9 is Dab; R10 is Thr; and R(FA) is octanoyl; was first disclosed in WO2008017734 (A1) as NAB739. Example 1 of WO2008017734 (A1) discloses the synthesis of NAB739. The disclosure of this reference insofar as it relates to NAB739 is incorporated herein by reference.

Preferably the polymyxin derivative of formula (I) is NAB 815, wherein R1 is Dab; R2 is Thr; R3 is DThr; R4 is Dab; R5 is Dab; R6 is DPhe; R7 is Leu; R8 is Abu; R9 is Dab; R10 is Thr; and R(FA) is octanoyl.

In one embodiment the polymyxin derivative of formula (I) has R1-R10 representing an amino acid sequence Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-]. In one embodiment the polymyxin derivative of formula (I) has R1-R10 representing SEQ ID NO. 2 defined herein.

SEQ ID NO.2 is Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-]. SEQ ID NO.2 can also be written as Thr-DSer-[cyclo-Dbu-Dbu-DPhe-Leu-Dbu-Dbu-Thr] where Thr refers to Threonine; DSer refers to D-Serine; Dbu refers to 2,4-diaminobutyric acid; DPhe refers to D- Phenylalanine; Leu refers to Leucine and residues 3-9 form the cyclic heptapeptide portion.

In another embodiment the polymyxin derivative of formula (I) has R1-R10 representing the amino acid sequence Dab-Thr-DThr-cy[Dab-Dab-DPhe-Leu-Abu-Dab-Thr-]. In one embodiment the polymyxin derivative of formula (I) has R1-R10 representing SEQ ID NO. 1 defined herein. Preferably the polymyxin derivative of formula (I) has R1-R10 representing SEQ ID N0.1 defined herein.

SEQ ID N0.1 is Dab-Thr-DThr-cy[Dab-Dab-DPhe-Leu-Abu-Dab-Thr-]. SEQ ID N0.1 can also be written as Dbu-Thr-DThr-[cyclo -Dbu-Dbu-DPhe-Leu-Abu-Dbu-Thr] where Thr refers to Threonine; Dbu refers to 2,4-diaminobutyric acid; DThr refers to D- Threonine; DPhe refers to D-Phenylalanine; Leu refers to Leucine; Abu refers to 2- aminobutyric acid; and residues 4-10 form the cyclic heptapeptide portion. The polymyxin derivative of formula (I) can also be represented as OA-Thr-DSer-cy[Dab- Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-SEQ ID NO. 2 or as OA-Dab-Thr-DThr-cy[Dab-Dab- DPhe-Leu-Abu-Dab-Thr-], i.e. OA-SEQ ID NO. 1. OA-SEQ ID NO. 1 is preferred.

Both NAB 815 and NAB 739 have a total number of positive charges at physiological pH of 3. For NAB 739 these positive charges are located at positions R5, R8 and R9 of formula (I), i.e. within the heptapeptide ring. For NAB 815 two of these positive charges are located within the heptapeptide ring at positions R5 and R9, and one is located on the hydrophobic tail at position R1.

As used herein, "physiological pH" refers to a pH value of more than 7.0 and below 7.6, such as a pH value in the range of from 7.1 to 7.5, for example in the range of from 7.2 to 7.4. In one embodiment the antibacterial agent is a compound capable of killing log-phase or fast growing bacteria.

In one embodiment the antibacterial agent is a compound capable of killing clinically latent microorganisms, preferably a compound capable of killing clinically latent bacteria.

As used herein, "kill" means a loss of viability as assessed by a lack of metabolic activity and "clinically latent microorganism" means a microorganism that is metabolically active but has a growth rate that is below the threshold of infectious disease expression. The threshold of infectious disease expression refers to the growth rate threshold below which symptoms of infectious disease in a host are absent.

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

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

(II) uridine (e.g. [ ³ H]uridine) uptake (e.g. from 0.0005 to 50%, such as from 1 to 40, 15 to 35 or 20 to 30% of the level of [ ³ H]uridine uptake). Clinically latent microorganisms typically possess a number of identifiable characteristics. For example, they may be viable but non-culturable; i.e. they cannot typically be detected by standard culture techniques, but are detectable and quantifiable by techniques such as broth dilution counting, microscopy, or molecular techniques such as polymerase chain reaction. In addition, clinically latent microorganisms are phenotypically tolerant, and as such are sensitive (in log phase) to the biostatic effects of conventional antibacterial agents (i.e. microorganisms for which the minimum inhibitory concentration (MIC) of a conventional antibacterial is substantially unchanged); but possess drastically decreased susceptibility to drug-induced killing (e.g. microorganisms for which, with any given conventional antibacterial agent, the ratio of minimum microbiocidal concentration (e.g. minimum bactericidal concentration, MBC) to MIC is 10 or more).

Test procedures that may be employed to determine the biological (e.g. bactericidal or antibacterial) activity of the active ingredients include those known to persons skilled in the art for determining:

(a) bactericidal activity against clinically latent bacteria; and

(b) antibacterial activity against log phase bacteria.

In relation to (a) above, methods for determining activity against clinically latent bacteria include a determination, under conditions known to those skilled in the art (such as those described in Nature Reviews, Drug Discovery 1 , 895-910 (2002), the disclosures of which are hereby incorporated by reference), of Minimum Stationary-cidal Concentration ("MSC") or Minimum Dormicidal Concentration ("MDC") for a test compound.

By way of example, WO2000028074 describes a suitable method of screening compounds to determine their ability to kill clinically latent microorganisms. A typical method may include the following steps:

(1) growing a bacterial culture to stationary phase;

(2) treating the stationary phase culture with one or more antibacterial agents at a concentration and or time sufficient to kill growing bacteria, thereby selecting a phenotypically resistant sub-population;

(3) incubating a sample of the phenotypically resistant subpopulation with one or more test compounds or agents; and

(4) assessing any antibacterial effects against the phenotypically resistant subpopulation.

According to this method, the phenotypically resistant sub-population may be seen as representative of clinically latent bacteria which remain metabolically active in vivo and which can result in relapse or onset of disease. In relation to (b) above, methods for determining activity against log phase bacteria include a determination, under standard conditions (i.e. conditions known to those skilled in the art, such as those described in WO 2005014585, the disclosures of which document are hereby incorporated by reference), of MIC or MBC for a test compound. As used herein, the term "microorganisms" means fungi and bacteria. Preferably "microorganisms" means bacteria.

Examples of compounds capable of killing clinically latent bacteria include those compounds disclosed in International Patent Application, Publication Numbers WO2007054693, WO2008117079 and WO2008142384. These applications describe suitable methods for the preparation of such compounds and doses for their administration.

Preferred examples of an antibacterial agent include a compound selected from the group consisting of:

6,8-dimethoxy-4-methyl-1-(3-phenoxyphenyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

6,8-dimethoxy-4-methyl-1-(2-phenoxyethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

1-cyclopropyl-6,8-dimethoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

8-methoxy-4-methyl-1-(4-phenoxyphenyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

{2-[4-(8-methoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinolin-1-yl)- phenyoxy]ethyl}dimethylamine;

8-methoxy-4-methyl-1-[4-(pyridin-3-yloxy)phenyl]-2,3-dihydro -1 H-pyrrolo[3,2-c]quinoline; 4-methyl-8-phenoxy-1-phenyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

1-benzyl-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline

1-(indan-2-yl)-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline

4-methyl-6-phenoxy-1-phenyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

1-benzyl-4-methyl-6-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

1-(indan-2-yl)-4-methyl-6-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-1-(2-phenylethyl)-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

8-methoxy-4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinolin-6-ol;

1-(1-benzyl-piperidin-4-yl)-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

1-(indan-1-yl)-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

1-(benzodioxan-2-ylmethyl)-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-8-phenoxy-1-(1 ,2,3,4-tetrahydronaphthalen-1-yl)-2,3-dihydro-1 H-pyrrolo[3,2- c]quinoline;

1-cyclohexyl-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

8-ethoxy-4-methyl-1-(4-phenoxyphenyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

1-(4-methoxyphenyl)-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

4-methyl-1-(4-phenoxyphenyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline; 4-methyl-1-(2-methylphenyl)methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-8-phenoxy-1-(4-/ ^' so-propylphenyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

4-methyl-8-phenoxy-1-(1-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

8-methoxy-4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

6,8-dimethoxy-1-(4-hydroxyphenyl)-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-1-(3-hydroxyphenyl)-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-1-(3-hydroxy-5-methylphenyl)-4-methyl-2,3-d ihydro-1 H-pyrrolo[3,2- c]quinoline;

8-methoxy-1-(4-methoxyphenyl)-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

8-trifluoromethoxy-1-(4-phenoxyphenyl)-4-methyl-2,3-dihyd ro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-4-methyl-1-[4-(pyridin-3-yloxy)phenyl]-2,3- dihydro-1 H-pyrrolo[3,2-c]quinoline;

1-benzyl-6,8-dimethoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-1-(2-phenylethyl)-8-trifluoromethoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-1-(indan-1-yl)-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

6,8-dimethoxy-4-methyl-1-[(6-phenoxy)pyridin-3-yl]-2,3-dihyd ro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-1-[(6-methoxy)pyridin-3-yl]-4-methyl-2,3-di hydro-1 H-pyrrolo[3,2-c]quinoline;

1-(benzodioxol-5-ylmethyl)-6,8-dimethoxy-4-methyl-2,3-dih ydro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-4-methyl-1-(3-methylbutyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

1-cyclopropylmethyl-6,8-dimethoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-8-(morpholin-4-yl)-1-(4-phenoxyphenyl)-2,3-dihydro- 1 H-pyrrolo[3,2-c]quinoline;

8-methoxy-4-methyl-1-(1 ,2,3,4-tetrahydronaphthalen-1-yl)-2,3-dihydro-1 H-pyrrolo[3,2- c]quinoline;

4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4,6-dimethyl-1-(2-methylphenyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4,6-dimethyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-8-(piperidin-1-yl)-1-[4-(piperidin-1-yl)phenyl]-2,3 -dihydro-1 H-pyrrolo[3,2-c]quinolin

4-methyl-8-(piperidin-1-yl)-1-(3-phenoxyphenyl)-2,3-dihyd ro-1 H-pyrrolo[3,2-c]quinoline;

1-{4-[2-(A/,A/-dimethylamino)ethoxy]phenyl}-4-methyl-8-ph enoxy-2,3-dihydro-1 H-pyrrolo[3,^ c]quinoline;

1-[4-(4-fluorophenoxy)phenyl]-8-methoxy-4-methyl-2,3-dihydro -1 H-pyrrolo[3,2-c]quinoline; 1-(benzodioxan-2-ylmethyl)-8-methoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline; 1-cyclohexyl-8-methoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

8-methoxy-4-methyl-1-phenyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-8-phenoxy-1-[4-(3-pyridyl)phenyl]-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

4-methyl-8-phenoxy-1-[2-(3-pyridyl)ethyl]-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

4-methyl-8-phenoxy-1-(2-pyridylmethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline; 4-methyl-1-(5-methylpyrazin-2-ylmethyl)-8-phenoxy-2,3-dihydr o-1 H-pyrrolo[3,2-c]quinolin

8-chloro-4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline-8-carboxylate;

4-methyl-8-(morpholin-1-yl)-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

ethyl [4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline-8-yl]acetate;

1-[3-(4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinolin-1-yl)propyl]-pyrrolidin-2-one;

4-methyl-8-phenoxy-1-[2-(2-pyridyl)ethyl]-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

ethyl 3-(8-methoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline-1-yl)propionate;

ethyl 4-(4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline-1-yl)butanoate;

methyl 4-(4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline-1-yl)butanoate;

ethyl (4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline-1-yl)acetate;

4-methyl-1-(1-methylpiperidin-4-yl)-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

1-(1-benzylpyrrolidin-3-yl)-8-methoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

methyl 3-(4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline-1-yl)propionate;

1-((S)-indan-1-yl)-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

^((/^-indan-l-y ^-methyl-S-phenoxy^.S-dihydro-I H-pyrrolofS^-cl-quinoline;

1-(3-methoxypropyl)-4-methyl-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

4-methyl-8-phenoxy-1-(tetrahydrofuran-2-ylmethyl)-2,3-dihydr o-1 H-pyrrolo[3,2-c]quinoline;

1-[2-(4-chlorophenyl)ethyl]-4-methyl-8-phenoxy-2,3-dihydr o-1 H-pyrrolo[3,2-c]quinoline;

1-[2-(4-methoxyphenyl)ethyl]-4-methyl-8-phenoxy-2,3-dihyd ro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-8-phenoxy-1-(2-phenylpropyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

8-cyano-4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

8-hydroxy-4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

8-phenoxy-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-1-(4-hydroxyphenyl)-4-methylpyrrolo[3,2-c]q uinoline;

8-methoxy-4-methyl-1-[4-(4-methylpiperazin-1-yl)-3-fluorophe nyl]-2,3-dihydro-1 H-pyrrolo[3,2- c]quinoline;

4-methyl-8-phenylamino-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline;

[4-methyl-1-(2-phenylethyl)-2,3-dihydro-1 H-pyrrolo[2,3-c]quinoline-8-oyl]-piperidine;

6,8-dimethoxy-1-(4-/ ^' so-propylphenyl)-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

6-methoxy-1-(4-phenoxyphenyl)-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

6-methoxy-1-(4-/ ^' so-propylphenyl)-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

6,8-dimethoxy-1-(4-phenoxyphenyl)-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

4-methyl-8-phenoxy-1-(4-phenoxyphenyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

1-(4-/ ^' so-propylphenyl)-6-phenoxy-4-methyl-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline; and

4,6-dimethyl-1-(4-methylphenyl)-2,3-dihydro-1 H-pyrrolo[3,2-c]quinoline;

or a pharmaceutically acceptable derivative thereof. Further preferred examples of the antibacterial agent include a compound selected from the group consisting of:

(1-methyl-1 H-benzimidazol-2-yl)-(6-hydroxy-2-methylquinolin-4-yl)amine;

(1-methyl-1 H-benzimidazol-2-yl)-(2-methyl-6-phenoxyquinolin-4-yl)amine;

(1-methyl-1 H-benzimidazol-2-yl)-(6-chloro-2-methylquinolin-4-yl)amine;

(1-methyl-1 H-benzimidazol-2-yl)-(6-cyano-2-methylquinolin-4-yl)amine;

(1-methyl-1 H-benzimidazol-2-yl)-(6-benzyloxy-2-methylquinolin-4-yl)amin e;

(1-methyl-1 H-benzimidazol-2-yl)-(5,6-dichloro-2-methylquinolin-4-yl)ami ne;

(1-methyl-1 H-benzimidazol-2-yl)-(7-chloro-2-methylquinolin-4-yl)amine hydrochloride;

(1-methyl-1 H-benzimidazol-2-yl)-(6,8-dichloro-2-methylquinolin-4-yl)ami ne;

[6-(4-fluorophenoxy)-2-methylquinolin-4-yl]-(1-methyl-1 H-benzimidazol-2-yl)amine;

(2-methyl-6-phenylaminoquinolin-4-yl)-(1-methyl-1 H-benzimidazol-2-yl)amine;

(1 H-benzimidazol-2-yl)-(2-methyl-6-phenoxyquinolin-4-yl)amine;

(benzoxazol-2-yl)-(2-methyl-6-phenoxyquinolin-4-yl)amine;

(1 H-benzimidazol-2-yl)-(6-chloro-2-methylquinazolin-4-yl)amine ;

[2-methyl-6-(pyrimidin-2-yloxy)quinolin-4-yl]-(1-methyl-1 H-benzimidazol-2-yl)amine;

(1-methyl-1 H-benzimidazol-2-yl)-[2-methyl-6-(4-methylpiperazin-1-yl)-qu inolin-4-yl]amine; and

(1-methyl-1 H-benzimidazol-2-yl)-(2-morpholin-4-yl-6-phenoxyquinolin-4-y l)amine;

or a pharmaceutically acceptable derivative thereof.

Still further preferred examples of the antibacterial agent include a compound selected from the group consisting of:

6-chloro-2-methyl-4-(3-phenylpyrrolidin-1-yl)quinoline;

6-benzyloxy-2-methyl-4-(3-phenylpyrrolidin-1-yl)quinoline;

2-methyl-4-(3-phenylpyrrolidin-1-yl)-6-(pyridin-3-ylmetho xy)quinoline;

6-(4-methanesulfonylbenzyloxy)-2-methyl-4-(3-phenylpyrrolidi n-1-yl)quinoline;

6-(4-methoxybenzyloxy)-2-methyl-4-(3-phenylpyrrolidin-1-yl)q uinoline

2-methyl-6-phenethyloxy-4-(3-phenylpyrrolidin-1-yl)quinoline ;

2-methyl-6-(5-methylisoxazol-3-ylmethoxy)-4-(3-phenylpyrroli din-1-yl)quinoline;

4-(3-benzylpyrrolidin-1-yl)-2-methyl-6-phenoxyquinoline;

4-[3-(4-methoxyphenyl)pyrrolidin-1-yl]-2-methyl-6-phenoxyqui noline;

4-[3-(4-chlorophenyl)pyrrolidin-1-yl]-2-methyl-6-phenoxyquin oline;

[1-(2-methyl-6-phenoxyquinolin-4-yl)-pyrrolidin-3-yl]phenyla mine;

N-[2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6-yl]benzami de;

N-[2-methyl-4-(3-phenylpyrrolidin-1-yl)-quinolin-6-yl]-2- phenylacetamide;

4-chloro-N-[2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6-y l]benzamide;

4-methoxy-N-[2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6- yl]benzamide; 2- methyl-N-[2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6-yl] benzamide;

pyrazine-2-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]amide;

1 H-pyrazole-4-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]amide;

furan-2-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]amide;

N-[2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6-yl]nico tinamide;

3- methyl-3H-imidazole-4-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6- yl]amide;

5-methyl-1 H-pyrazole-3-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1-yl)-quinolin-6- yl]amide;

pyridazine-4-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1-yl)-quinolin-6-yl]amide;

2-(4-methoxyphenyl)-N-[2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]acetami

2-(4-chlorophenyl)-N-[2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]acetamide;

3,5-dimethyl-isoxazole-4-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6- yl]amide;

2-(3-methyl-isoxazol-5-yl)-N-[2-methyl-4-(3-phenyl-pyrrol idin-1 -yl)-quinolin-6-yl]-acetam

N-[2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6-yl]benz enesulfonamide;

benzyl-[2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6-yl ]amine;

(R- or S-)Benzyl-[2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]arnine;

(S- or /?-)Benzyl-[2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]arnine;

(4-methoxybenzyl)-[2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]amine;

4- {[2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-ylamino]methyl}benzonitri

1- [2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]pyrrolidin-2-one;

N-[2-methyl-4-(3-phenylpyrrolidin-1-yl)quinolin-6-yl]-3-p henyl propionamide;

5- methyl-isoxazole-3-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1-yl)-quinolin-6- yl]amide;

pyridine-2-carboxylic acid [2-methyl-4-(3-phenylpyrrolidin-1 -yl)quinolin-6-yl]amide;

N-[4-(3-benzylpyrrolidin-1 -yl)-2-methylquinolin-6-yl]benzamide; and

2- methyl-6-phenoxy-4-(3-phenylpyrrolidin-1-yl)quinoline; or

or a pharmaceutically acceptable derivative thereof. Particularly preferred antibacterial agents are 4-methyl-1 -(2-phenylethyl)-8-phenoxy-2,3- dihydro-1 H-pyrrolo[3,2-c]-quinoline (Example 9, WO2007054693), 4-(3-benzylpyrrolidin-1 -yl)- 2-methyl-6-phenoxyquinoline (Example 8, WO2008142384), and N-[4-(3-benzylpyrrolidin-1 - yl)-2-methylquinolin-6-yl]benzamide (Example 38, WO2008142384), and pharmaceutically acceptable derivatives thereof. In one embodiment of the invention the antibacterial agent is 4-(3-benzylpyrrolidin-1-yl)-2- methyl-6-phenoxyquinoline, N-[4-(3-benzylpyrrolidin-1 -yl)-2-methylquinolin-6-yl]benzamide or a pharmaceutically acceptable derivative thereof. A more preferred antibacterial agent is 4- methyl-1-(2-phenylethyl)-8-phenoxy-2,3-dihydro-1 H-pyrrolo[3,2-c]-quinoline or a pharmaceutically acceptable derivative thereof such as the hydrochloride salt thereof.

As used herein, the term "bacteria" includes, but is not limited to, references to organisms (or infections due to organisms) of the following classes and specific types:

Gram-positive cocci, such as Staphylococci (e.g. Staph, aureus, Staph, epidermidis, Staph. saprophyticus, Staph, auricularis, Staph, capitis capitis, Staph, c. ureolyticus, Staph, caprae,

Staph, cohnii cohnii, Staph, c. urealyticus, Staph, 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); Streptococci (e.g.beta-haemolytic, pyogenic streptococci (such as Strept. agalactiae, Strept. canis, Strept. dysgalactiae dysgalactiae, Strept. dysgalactiae equisimilis,

Strept. equi equi, Strept. equi zooepidemicus, Strept. iniae, Strept. porcinus and Strept. pyogenes), microaerophilic, pyogenic streptococci (Streptococcus "milleri", such as Strept. anginosus, Strept. constellatus constellatus, Strept. constellatus pharyngidis and Strept. intermedius), oral streptococci of the "mitis" (alpha-haemolytic - Streptococcus "viridans", such as Strept. mitis, Strept. oralis, Strept. sanguinis, Strept. cristatus, Strept. gordonii and

Strept. parasanguinis), "salivarius" (non-haemolytic, such as Strept. salivarius and Strept. vestibularis) and "mutans" (tooth-surface streptococci, such as Strept. criceti, Strept. mutans,

Strept. ratti and Strept. sobrinus) groups, Strept. acidominimus, Strept. bovis, Strept. faecalis, Strept. equinus, Strept. pneumoniae and Strept. suis, or Streptococci alternatively classified as Group A, B, C, D, E, G, L, P, U or V Streptococcus);

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; Bacillaceae, such as Bacillus anthracis, Bacillus subtilis, Bacillus thuringiensis, Bacillus stearothermophilus and Bacillus cereus; Enterobacteriaceae, such as Escherichia coli, Enterobacter (e.g. Enterobacter aerogenes, Enterobacter agglomerans and Enterobacter cloacae), Citrobacter (such as Citrob. freundii and Citrob. divernis), Hafnia (e.g. Hafnia alvei), Erwinia (e.g. Erwinia persicinus), Morganella morganii, Salmonella (Salmonella enterica and Salmonella typhi), Shigella (e.g. Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Klebsiella (e.g. Klebs. pneumoniae, Klebs. oxytoca, Klebs. ornitholytica, Klebs. planticola, Klebs. ozaenae, Klebs. terrigena, Klebs. granulomatis (Calymmatobacterium granulomatis) and Klebs. rhinoscleromatis), Proteus (e.g. Pr. mirabilis, Pr. rettgeri and Pr. vulgaris), Providencia (e.g. Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Serratia (e.g. Serratia marcescens and Serratia liquifaciens), and Yersinia (e.g. Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis); Enterococci (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); Helicobacter (e.g. Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae); Acinetobacter (e.g. A. baumanii, A. calcoaceticus, A. haemolyticus, A. johnsonii, A. junii, A. Iwoffi and A. radioresistens); Pseudomonas (e.g. 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); Bacteriodes fragilis; Peptococcus (e.g. Peptococcus niger); Peptostreptococcus; Clostridium (e.g. C. perfringens, C. difficile, C. botulinum, C. tetani, C. absonum, C. argentinense, C. baratii, C. bifermentans, C. beijerinckii, C. butyricum, C. cadaveris, C. carnis, C. celatum, C. clostridioforme, C. cochlearium, C. cocleatum, C. fallax, C. ghonii, C. glycolicum, C. haemolyticum, C. hastiforme, C. histolyticum, C. indolis, C. innocuum, C. irregulare, C. leptum, C. limosum, C. malenominatum, C. novyi, C. oroticum, C. paraputrificum, C. piliforme, C. putrefasciens, C. ramosum, C. septicum, C. sordelii, C. sphenoides, C. sporogenes, C. subterminale, C. symbiosum and C. tertium); Mycoplasma (e.g. M. pneumoniae, M. hominis, M. genitalium and M. urealyticum); Mycobacteria (e.g. Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium fortuitum, 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 intracellular, Mycobacterium interjectum, Mycobacterium heidelberense, Mycobacterium lentiflavum, 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 (e.g. Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus); Actinobacillus (e.g. Actinobacillus actinomycetemcomitans, Actinobacillus equuli, 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 and 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, 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 Treponema (Treponema pallidum ssp. pallidum, Treponema pallidum ssp. endemicum, Treponema pallidum ssp. pertenue and Treponema carateum); 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); Bordetella (e.g. Bordetella bronchiseptica, Bordetella hinzii, Bordetella holmseii, Bordetella parapertussis, Bordetella pertussis and Bordetella trematum); Nocardiaceae, such as Nocardia (e.g. Nocardia asteroides and Nocardia brasiliensis); Rickettsia (e.g. Ricksettsii or Coxiella burnetii); Legionella (e.g. Legionalla anisa, Legionalla birminghamensis, Legionalla bozemanii, Legionalla cincinnatiensis, Legionalla dumoffii, Legionalla feeleii, Legionalla gormanii, Legionalla hackeliae, Legionalla israelensis, Legionalla jordanis, Legionalla lansingensis, Legionalla longbeachae, Legionalla maceachernii, Legionalla micdadei, Legionalla oakridgensis, Legionalla pneumophila, Legionalla sainthelensi, Legionalla tucsonensis and Legionalla wadsworthii); Moraxella catarrhalis; Cyclospora cayetanensis; Entamoeba histolytica; Giardia lamblia; Trichomonas vaginalis; Toxoplasma gondii; Stenotrophomonas maltophilia; Burkholderia cepacia; Burkholderia mallei and Burkholderia pseudomallei; Francisella tularensis; Gardnerella (e.g. Gardneralla vaginalis and Gardneralla mobiluncus); Streptobacillus moniliformis; Flavobacteriaceae, such as Capnocytophaga (e.g. Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica, Capnocytophaga ochracea and Capnocytophaga sputigena); Bartonella {Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella elizabethae, Bartonella henselae, Bartonella quintana and Bartonella vinsonii arupensis); Leptospira (e.g. Leptospira biflexa, Leptospira borgpetersenii, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira santarosai and Leptospira weilii); Spirillium (e.g. Spirillum minus); Baceteroides (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 disiens, 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, Porphyromonas crevioricanis, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gingivicanis, Porphyromonas levii and Porphyromonas macacae); Fusobacterium (e.g. F. gonadiaformans, F. mortiferum, F. naviforme, F. necrogenes, F. necrophorum necrophorum, F. necrophorum fundiliforme, F. nucleatum nucleatum, F. nucleatum fusiforme, F. nucleatum polymorphum, F. nucleatum vincentii, F. periodontium, F. russii, F. ulcerans and F. varium); Chlamydia (e.g. Chlamydia trachomatis); Cryptosporidium (e.g. C. parvum, C. hominis, C. canis, C. felis, C. meleagridis and C. 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); and Ureaplasma (e.g. Ureaplasma parvum and Ureaplasma urealyticum).

Preferably, the bacteria are Gram-negative bacteria, e.g. Enterobacteriaceae, such as Escherichia coli, Klebsiella (e.g. Klebs. pneumoniae and Klebs. oxytoca) and Proteus (e.g. Pr. mirabilis, Pr. rettgeri and Pr. vulgaris); Haemophilis influenzae; Mycobacteria, such as Mycobacterium tuberculosis; and Enterobacter (e.g. Enterobacter cloacae). Preferably, the bacteria are Enterobacteriaceae, such as Escherichia coli and Klebsiella (e.g. Klebs. pneumoniae and Klebs. oxytoca). Also preferred are Acinetobacter spp. Particularly preferred are Escherichia coli, and Klebs. pneumoniae (e.g. Klebs. pneumoniae subsp. pneumoniae). Also particularly preferred are Acinetobacter spp. (e.g. Acinetobacter baumannii).

As used herein the term "pharmaceutically acceptable derivative" means:

(a) pharmaceutically acceptable salts; and/or

(b) solvates (including hydrates).

Suitable acid addition salts include carboxylate salts (e.g. formate, acetate, trifluoroacetate, propionate, isobutyrate, heptanoate, decanoate, caprate, caprylate, stearate, acrylate, caproate, propiolate, ascorbate, citrate, glucuronate, glutamate, glycolate, a-hydroxybutyrate, lactate, tartrate, phenylacetate, mandelate, phenylpropionate, phenylbutyrate, benzoate, chlorobenzoate, methylbenzoate, hydroxy benzoate, methoxybenzoate, dinitrobenzoate, o- acetoxybenzoate, salicylate, nicotinate, isonicotinate, cinnamate, oxalate, malonate, succinate, suberate, sebacate, fumarate, malate, maleate, hydroxymaleate, hippurate, phthalate or terephthalate salts), halide salts (e.g. chloride, bromide or iodide salts), sulfonate salts (e.g. benzenesulfonate, methyl-, bromo- or chloro-benzenesulfonate, xylenesulfonate, methanesulfonate, ethanesulfonate, propanesulfonate, hydroxyethanesulfonate, 1- or 2- naphthalene-sulfonate or 1 ,5-naphthalenedisulfonate salts) or sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate or nitrate salts, and the like. When the antibacterial agent is a pharmaceutically acceptable derivative of colistin, or polymyxin B, the compound may be colistin sulfate, colistimethate sodium, colistin sodium methanesulfonate, or polymyxin B sulfate. Particularly preferred is colistin, colistin sulfate, colistin sodium methane sulfonate or colistimethate sodium, e.g. colistin or colistimethate sodium. A preferred salt of polymyxin B is the sulfate salt thereof, i.e. polymyxin B sulfate. A preferred salt of polymyxin E is the sulfate salt thereof, i.e. polymyxin E sulfate. A typically used acid for formation of the pharmaceutically acceptable salt of the polymyxin derivative of formula (I) is sulfuric acid.

The antibacterial agent for use in the present invention is commercially available or can be prepared by synthesis methods known in the art. For example, polymyxin B, polymyxin B sulfate, polymyxin E sulfate and polymyxin E are commercially available from Sigma Aldrich Limited or Finetech Industry Limited. Suitable dosages and formulations are known in the art. As noted above, at least one of the antibiotic resistance breakers for use in the present invention is preferably a known non-antibiotic drug. Such drugs are typically commercially available or can be prepared by synthesis methods known in the art. For example, mefloquine, mefloquine hydrochloride, zidovudine, suloctidil and phenoxybenzamine hydrochloride are commercially available from Sigma Aldrich Limited. Suitable dosages and formulations of these drugs are also known in the art.

A preferred salt of mefloquine is the hydrochloride salt thereof, i.e. mefloquine hydrochloride.

Mefloquine is a chiral molecule with two asymmetric carbon centres, which means that it has four different stereoisomers. Mefloquine is commercially available as a racemate of the (R,S)- and (S,R)-enantiomers:

In all aspects of the invention, the term "mefloquine" includes all enantiomers, tautomers and stereoisomers thereof. The corresponding enantiomers and/or tautomers may also be isolated and/or prepared by methods known in the art. Suloctidil is preferably used in its non-salt form.

Phenoxybenzamine is preferably used as the hydrochloride salt.

Zidovudine is preferably used in its non-salt form.

The compounds used in the present invention may be as the raw materials, but are preferably provided in the form of pharmaceutical compositions or formulations. The antibacterial agent and at least two antibiotic resistance breakers may be used either as separate formulations or as a single combined formulation. When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. The formulation can be suitable for oral, parenteral (including subcutaneous e.g. by injection or by depot tablet, intradermal, intrathecal, intramuscular e.g. by depot and intravenous), rectal and topical (including dermal, buccal and sublingual) or in a form suitable for administration by inhalation or insufflation administration. Preferably, the compositions of the invention are formulated for systemic intravenous, intramuscular injection or inhaled, oral or topical administration. Examples

Example 1 : Microbiological profiling (FIC assay) of suloctidil, phenoxybenzamine hydrochloride and mefloquine in combination with polymyxin B nonapeptide against E. co// ^" To I C and E. coli D22 mutant strains An FIC assay of suloctidil, phenoxybenzamine hydrochloride and mefloquine each in combination with polymyxin B nonapeptide was carried out to demonstrate that these compounds have different mechanisms of action as an ARB.

Materials & Methods

All compounds were obtained from a commercial source such as Sigma Aldrich. The bacterial strains (wild-type and mutant) were obtained from commercially available bacterial collections such as ATCC.

Broth microdilution chequerboard assay was performed following the procedure described in the Clinical Microbiology Procedures Handbook (Isenberg, H.D. Ed.) American Society for Microbiology, Washington, DC. MIC assays were made using EUCAST & CLSI-approved guidelines using CM0473 ISO-SENSITEST™ BROTH medium.

E. coli wild strains

For comparative purposes, the MIC for colistin and suloctidil were obtained against the wild- type strains: E. coli ATCC 25922 and E. coli ATCC BAA 2471. As would be understood by the skilled person, the MIC is the lowest concentration of a drug that will inhibit visible growth of a microorganism after overnight incubation. The MIC values (in mcg/ml) are shown in Table 1 below.

Table 1

E. coli mutant strains To determine whether each of the ARBs suloctidil, phenoxybenzamine and mefloquine had the same mechanism of action when combined with colistin, each ARB was combined with polymyxin B nonapeptide and tested against E.coli mutant strains. Polymyxin antibiotics are known to bind to lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria and disrupt both the outer and inner membranes. They have a general structure consisting of a cyclic peptide with a hydrophobic tail and disrupt the structure of the bacterial cell membrane by interacting with its phospholipids. The hydrophobic tail is believed to be important in causing the membrane damage. Removal of the hydrophobic tail of polymyxin B produces polymyxin B nonapeptide. This compound still binds to LPS but no longer kills the bacterial cell. Additionally it still detectably increases the permeability of the bacterial cell wall to other compounds. To study the mechanistic differences between suloctidil, phenoxybenzamine and mefloquine towards a Gram-negative bacterium (E.coli), polymyxin B nonapeptide was therefore used instead of colistin.

1) E. coli TolC mutant

The E. coli TolC mutant strain was derived from the wild E.coli strain ATCC 25922 but unlike the wild strain, it did not express the AcrAB-TolC multidrug efflux pump. This pump assembly includes the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is known to transport a wide variety of compounds out of the bacterial cell, and confer resistance to a broad spectrum of antibiotics.

The MIC values (in mcg/ml) for suloctidil, mefloquine, phenoxybenzamine hydrochloride and polymyxin B nonapeptide with this mutant strain are shown in Table 2 below, along with the MIC values with the wild E.coli strain ATCC 25922 from Table 1 above.

Table 2

From the above data it can be seen that the lack of the AcrAB-TolC multidrug efflux pump in the mutant strain caused the MIC value for the polymyxin to significantly increase (suggesting antibiotic resistance), whilst the MIC value for suloctidil decreased from 64 to 8 mcg/ml. This decrease suggests that the high MIC found with the wild strain was due to the "pumping out" of suloctidil by the AcrAB-TolC pump. In other words, that the action of suloctidil is likely to be inside the bacterial cell. The effect of each of the ARBs suloctidil, mefloquine and phenoxybenzamine hydrochloride on polymyxin B nonapeptide against E. coli TolC mutant was then measured by using the chequerboard method to calculate the FIC indexes for the combinations. These FIC values are shown in Table 3 below, along with an indication of whether the combination had a synergistic effect against the mutant strain.

Table 3 (Bold = Lowest FIC index)

2^ The combination of suloctidil and polymyxin B nonapepitde resulted in synergism with suloctidil at 2 mcg/ml in combination with polymyxin B nonapeptide at 0.5, 1 , 4, 16 or 32 mcg/ml, and with suloctidil at 4 mcg/ml in combination with polymyxin B nonapeptide at 0.25 mcg/ml.

2^ The combination of mefloquine and polymyxin B nonapeptide resulted in synergism with mefloquine at 8 mcg/ml in combination with polymyxin B nonapeptide at 0.5 mcg/ml. ^ The combination of phenoxybenzamine hydrochloride and polymyxin B nonapeptide was not synergistic against the mutant strain.

In summary, a positive synergy result (FIC ≤0.5) was obtained between suloctidil and polymyxin B nonapeptide, using only 2 mcg/ml of suloctidil and a variety of concentrations (0.25-32 mcg/ml) of polymyxin B nonapeptide.

Additionally the lack of efflux pump seems to increase the potentiation effect of suloctidil. 2) E. coli D22 mutant

The E.coli D22 mutant (lpxC101 proA23 lac-28 tsx-81 trp-30 his-51 rpsL173(strR) tufA1 ampCp-1) was derived from the wild E.coli strain ATCC 25922 but unlike the wild strain had a reduced biosynthesis of lipid A. Lipid A is a lipid component held responsible for the toxicity of gram-negative bacteria. It is the innermost of the three regions of the lipopolysaccharide (LPS), and its hydrophobic nature allows it to anchor the LPS to the outer membrane.

The MIC values (in mcg/ml) for suloctidil, mefloquine, phenoxybenzamine hydrochloride and polymyxin B nonapeptide with this mutant strain are shown in Table 4 below, along with the MIC values with the wild E.coli strain ATCC 25922 from Table 1 above. Table 4

From the above data it can be seen that the reduction in biosynthesis of lipid A caused the MIC value for the polymyxin to significantly increase. This suggests antibiotic resistance, which might be attributed to the mutational reduction-elimination of the target molecule, lipid A that has been previously documented in the scientific literature (for example in Delcour,

2009). The MIC of suloctidil is then decreased from 64 in the wild strain to 8 mcg/ml in the mutant by reductions in LPS structure. This suggests that the LPS is a barrier for this compound.

The effect of each of the ARBs suloctidil, mefloquine and phenoxybenzamine hydrochloride on polymyxin B nonapeptide against the E.coli D22 mutant strain was then measured by using the chequerboard method to calculate the FIC indexes for the combinations. These FIC values are shown in Table 5 below, along with an indication of whether the combination had a synergistic effect against the mutant strain.

Table 5

2 4 0.28 Yes

2 8 0.31 Yes

2 16 0.37 Yes

2 32 0.5 Yes

4 0.25 0.5 Yes

4 0.5 0.5 Yes

Phenoxybenzamine 32 32 0.5

Yes hydrochloride

64 0.25 0.5 Yes

64 0.5 0.5 Yes

64 1 0.5 Yes

Mefloquine 4 32 0.42 Yes

8 0.5 0.5 Yes

8 1 0.5 Yes

Bold: lowest FIC index

The combination of suloctidil and polymyxin B nonapeptide resulted in synergism with suloctidil at 2 mcg/ml in combination with polymyxin B nonapeptide at 2, 4, 8, 16 or 32 mcg/ml, and with suloctidil at 4 mcg/ml in combination with polymyxin B nonapeptide at 0.25 or 0.5 mcg/ml.

The combination of phenoxybenzamine hydrochloride and polymyxin B nonapeptide resulted in synergism with phenoxybenzamine hydrochloride at 32 mcg/ml in combination with polymyxin B nonapeptide at 32 mcg/ml, and with phenoxybenzamine hydrochloride at 64 mcg/ml in combination with polymyxin B nonapeptide at 0.25, 0.5 or 1 mcg/ml.

The combination of mefloquine and polymyxin B nonapeptide resulted in synergism with mefloquine at 4 mcg/ml in combination with polymyxin B nonapeptide at 32 mcg/ml, and with mefloquine at 8 mcg/ml in combination with polymyxin B nonapeptide at 0.5 or 1 mcg/ml. Example 2: Effect of suloctidil, phenoxybenzamine hydrochloride and mefloquine in combination with colistin sulfate and polymyxin B nonapeptide hydrochloride

Using the chequerboard technique described in Antimicrob Chemo (2013) 68, 374-384, the MIC and FIC index values were obtained for the combinations against NDM-1 E.coli (BAA2469) and NDM-1 K.pneumoniae (BAA2472).

The compounds were obtained from a commercial source, e.g. Sigma Aldrich. The bacterial strains were obtained from a commercially available bacterial collection such as ATCC.

Log phase growth of the bacterial strains was carried out as known in the art. The log phase bacterial cultures were then incubated with each of the combinations overnight by diluting with nutrient broth (Oxoid) to 10 ⁷ CFU/ml and adding 280 μΙ of each culture to each well to make a final concentration of 300 μΙ. Incubation of the combinations with the bacterial suspensions was carried out for 24 hours.

The MIC and FIC index values from the chequerboard analysis are set out in Table 6 below where: CS = colistin sulfate; PBNH = polymyxin B nonapeptide hydrochloride; S = suloctidil; M = mefloquine; and POBH = phenoxybenzamine hydrochloride.

Table 6

Summary 1. BAA2469 and BAA2472 were susceptible to colistin sulfate with MIC at 0.031 mg/L for BAA2469 and 0.5 -1 mg/L for BAA2472.

2. CS or PBNH combined with suloctidil, phenoxybenzamine and mefloquine showed synergistic activities against strain BAA2469 (wild).

3. CS or PBNH combined with suloctidil and mefloquine showed synergistic activities against strain BAA2472. 4. There was no interaction between CS or PBNH with phenoxybenzamine against strain BAA2472.

Summary of results in Example 1 and Example 2

The results in Example 1 and Example 2 show three different patterns of response. This is important because it suggests different mechanisms of action for suloctidil, phenoxybenzamine hydrochloride and mefloquine. The patterns can be summarized as follows:

1. Colistin/Polymyxin B Nonapeptide + Suloctodil (S)

Table 7

It can be seen from Table 7 above that the combination of colistin + S gives rise to a FIC which is tenfold more synergistic than Nonapeptide + S. As noted above, polymyxin B nonapeptide detectably increases the permeability of the bacterial cell wall to other compounds. This data suggests, however, that increased permeability is a relatively small component of the synergy between a polymyxin and suloctidil against this bacterium.

From the results in Example 1 against the E. coli TolC mutant, it can be seen that the combination with suloctidil has a synergistic effect and that suloctidil may have some antibiotic activity. This compound is also efflux-pump dependent and has ARB activity. From the results in Example 1 against the E. coli D22 mutant, it can be seen that the combination with suloctidil is synergistic and that LPS may be a barrier.

Overall therefore suloctidil as an ARB is not dependent on permeability of the bacterial cell wall, is dependent on efflux and LPS, and may have its own antibiotic activity.

2. Colistin/Polymyxin B nonapeptide + phenoxybenzamine hydrochloride

It can be seen from the table in Example 2 that there was no interaction between colistin or polymyxin B hydrochloride nonapeptide with phenoxybenzamine against strain BAA2472. From the results in Example 1 against the E. coli TolC mutant, it can be seen that the combination with phenoxybenzamine hydrochloride did not have a synergistic effect. Against the E. coli D22 mutant, the combination with phenoxybenzamine hydrochloride was, however, synergistic.

Overall therefore phenoxybenzamine hydrochloride as an ARB does not appear to be dependent on efflux and LPS may have an effect. 3. Colistin/Polymyxin B nonapeptide + mefloquine

It can be seen from the table in Example 2 that the FICs for colistin and polymyxin B hydrochloride nonapeptide with mefloquine were similar. This suggests a role for increased permeabilization. From the results in Example 1 against the E. coli TolC mutant and the E. coli D22 mutant, it can be seen that the combinations with mefloquine were synergistic. Overall therefore mefloquine has a different synergy profile to suloctidil and phenoxybenzamine hydrochloride and consequently must have a different mechanism of action with the polymyxin antibacterial agent.

This data demonstrates that each of the antibiotic resistance breakers (suloctidil, mefloquine and phenoxybenzamine hydrochloride) have a different mechanism of action with a polymyxin antibacterial agent. They are therefore suitable for use in the present invention to restore efficacy/prolong the lifetime of the antibacterial agent.

Example 3: Mechanism of action studies

In this Example the effect of colistin, mefloquine, suloctidil, zidovudine and phenoxybenzamine alone in both wild type E.coli (ATCC 25922) and E.coli tolC was examined at various concentrations and time points. Mefloquine, suloctidil, zidovudine and phenoxybenzamine (the ARBs) were also examined in combination with colistin (0.5 μg/ml). These studies were intended to provide more information on the mechanism of action of each of the ARBs when used in combination with colistin.

Materials and methods All compounds and experimental materials (e.g. SYTOX™ Green Nucleic Acid Stain, DAPI and FM 4-64) were obtained from a commercial source such as Sigma Aldrich or ThermoFisher Scientific. The bacterial strains (wild-type and mutant) were obtained from or derived from commercially available bacterial collections such as ATCC.

Samples of bacteria were grown in Lysogeny Broth at 30°C to OD ₆ oo~0.1. These cultures were then split and each sample treated with the appropriate concentration of the test compound. Samples were collected at two time points: 2 hours and 4 hours. 100 μΙ_ of each sample was stained with 0.4 μΜ SYTOX Green, 1.5 μςΛηί. DAPI (4'-6-diamidino-2- phenylindole; a blue-fluorescent DNA stain), and 0.8 μg/mL of FM 4-64 (N-(3- triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatri enyl) pyridinium dibromide; a red-fluorescent dye). Cells were centrifuged, re-suspended and transferred to an agarose pad (20% LB and 1 % agarose) for imaging. Membranes are shown in red, DNA in blue, and green staining indicates permeabilization of the cell membrane. White scale bar is 1 μηι. The greyscale copy of the images is included as the B version of the relevant Figure.

The characteristic phenotype produced by treatment with the indicated compounds for 2 hours is shown in Figure 2 (2A and 2B). The effect of each of the compounds when used alone can be determined by comparing the control (DMSO 1.28%) with the image for the compound in question (e.g. colistin 2.5).

The predominant phenotype produced by treatment of E.coli ATCC 25922 with colistin and/or mefloquine for 2 hours is shown in Figure 3 (3A and 3B). Viable cell counts for E.coli ATCC 25922 treated with DMSO, colistin, mefloquine or a combination of colistin and mefloquine at 0, 2 and 4 hours are shown in Figure 4. It can be seen from Figure 4 that the combination of mefloquine and colistin significantly reduced the viable cell counts at 2 hours and 4 hours compared to colistin alone and mefloquine alone.

The predominant phenotype produced by treatment of E.coli ATCC 25922 with colistin and/or suloctidil for 2 hours is shown in Figure 5 (5A and 5B). Viable cell counts for E.coli ATCC 25922 treated with DMSO, colistin, suloctidil or a combination of colistin and suloctidil at 0, 2 and 4 hours are shown in Figure 6. It can be seen from Figure 6 that the combination of suloctidil (32 μg/ml) and colistin (0.5 μg/ml) significantly reduced the viable cell count at 0 hours, 2 hours and 4 hours compared to colistin alone and suloctidil alone.

The predominant phenotype produced by treatment of E.coli ATCC 25922 with colistin and/or zidovudine for 2 hours is shown in Figure 7 (7A and 7B). Viable cell counts for E.coli ATCC 25922 treated with DMSO, colistin, zidovudine or a combination of colistin and zidovudine at 0, 2 and 4 hours are shown in Figure 8. It can be seen from Figure 8 that the combination of zidovudine (1 μg/ml) and colistin (0.5 μg/ml) produced a similar viable cell count to zidovudine alone and significant reduced the viable cell count of colistin alone. This is consistent with the effect of zidovudine on the phenotype (see comments in Table 8 below).

The predominant phenotype produced by treatment of E.coli ATCC 25922 with colistin and/or phenoxybenzamine for 2 hours is shown in Figure 9 (9A and 9B). Viable cell counts for E.coli ATCC 25922 treated with DMSO, colistin, phenoxybenzamine or a combination of colistin and phenoxybenzamine at 0, 2 and 4 hours are shown in Figure 10. It can be seen from Figure 10 that the combination of phenoxybenzamine and colistin significantly reduces the viable cell amount compared to phenoxybenzamine alone and colistin alone. Some further conclusions which can be drawn from Figures 2 to 10 are set out in Table 8 below.

Table 8

permeabilizing cells with unusual nucleoid (16 μg/ml) and colistin (0.5 μg/ml) structure (appear to not be for 2 hours were permabilized, segregated properly) and similar to colistin alone and variable membrane staining. phenoxybenzamine alone in E.coli In E.coli tolC at "IX and 4X tolC.

MIC, cells were

permeabilized and lysed.

No MIC in ATCC 25922, but

at 128 μg/ml, some cells

were elongated with unusual

nucleoid structure (appeared

to not be segregated

properly).

In summary, it can be concluded from the changes in phenotype and viable cell counts from Figures 2 to 10 and Table 8 that each of the antibiotic resistance breakers mefloquine, suloctidil, zidovudine and phenoxybenzamine has a different mechanism of action when used with colistin against a Gram-negative bacterium. This means that each of these ARBs is suitable for use in the method of the claimed invention. These compounds can effectively "renew" the antibiotic activity of colistin.

Example 4: Additional Mechanism action studies with zidovudine (azidothymidine) and control compounds In this Example, additional studies were carried out to obtain more information on the mechanism by which zidovudine (azidothymidine) targets DNA replication. These studies involved first looking at the effect of zidovudine and other control compounds on E.coli tolC, and second comparing zidovudine to other DNA replication inhibitors. The compounds, their class and their target are shown below in Table 9: Table 9

Firstly the effect of the various compounds on cell growth was examined by determining the minimal inhibitory concentration (see Table 10 below). Secondly the effects of the compounds were examined by performing Bacterial Cytological Profiling (BCP) at a range of exposure times and concentrations. This information allowed a comparison to be made between the profile of zidovudine and the control compounds using cluster analysis.

Materials and Methods All compounds and experimental materials (e.g. SYTOX™ Green Nucleic Acid Stain, DAPI and FM 4-64) were obtained from a commercial source such as Sigma Aldrich or ThermoFisher Scientific. The bacterial strain was obtained from or derived from commercially available bacterial collections such as ATCC.

Cells were grown in Lysogeny Broth (LB) at 30°C to OD ₆ oo~0.4-0.5 then diluted to a final concentration of 5 x 10 ⁵ CFU/mL in 96 well plates containing serial dilutions of the compounds. Serial 1 :2 dilutions of each compound were prepared in LB with a maximum concentration of 128 μg/mL. The plate was incubated on a shaker at 30°C overnight and readings performed after 24 hours.

Bacterial Cytological Profiling (BCP) For BCP, samples of E.coli tolC were grown in LB at 30°C to OD ₆ oo~0.1. These cultures were then split and each sample treated with the appropriate concentration of the test compound. Samples were collected at two time points: 30 and 120 minutes or 120 and 240 minutes. 100 of each sample was stained with 0.4 μΜ SYTOX Green, 1.5 μg/mL DAPI (4'-6-diamidino- 2-phenylindole; a blue-fluorescent DNA stain), and 0.8 μg/mL of FM 4-64 (N-(3- triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatri enyl) pyridinium dibromide; a red-fluorescent dye). Cells were centrifuged, re-suspended and transferred to an agarose pad (20% LB and 1 % agarose) for imaging. Membranes are shown in red, DNA in blue, and green staining indicates permeabilization of the cell membrane. White scale bar is 1 μηι. As above, the greyscale copy of the colour image is included as the B version of the relevant Figure.

Cluster Analysis

Medial focal planes for images were determined and tiffs were created using commercially available image processing software (e.g. (FIJI Is Just) Image J). Cell and DNA objects were identified and their cytological parameters measured using a pipeline created with suitable commercially available software (e.g. CellProfiler). The software and parameters to be measured are known in the art. Dimensionality reduction and dataset variance information was obtained by performing Principal Component Analysis (PCA) with built-in functions in commercially available computer programming software such as MATLAB. The first three Principal Components were used to generate hierarchically clustered heat maps shown in Figures 19 and 26.

Results

The minimum inhibitory concentrations provided an indication of the effect of each compound on cell growth. These concentrations are set out in Table 10:

Table 10

BCP images for the control, zidovudine and a compound of each class of antibiotic tested are contained in Figures 1 1 to 18. Figure 11 (1 1 A and 11 B) includes the images obtained for the DMSO control: E.coli tolC treated with 0.5% DMSO for 30 minutes and 120 minutes. Figure 12 (12A and 12B) includes the images obtained for E.coli tolC treated with 0.06 μg/mL zidovudine for 30 minutes and 120 minutes. Figure 13 (13A and 13B) includes the images obtained for E.coli tolC treated with 0.3 μg/mL zidovudine (azidothymidine) for 30 minutes and 120 minutes. Figure 14 (14A and 14B) includes the images obtained for E.coli tolC treated with 0.002 μg/mL ciprofloxacin for 30 minutes and 120 minutes. Figure 15 (15A and 15B) includes the images obtained for E.coli tolC treated with 0.01 μg/mL ciprofloxacin for 30 minutes and 120 minutes. Figure 16 (16A and 16B) includes the images obtained for E.coli tolC treated with 20 μg/mL cephalexin for 30 minutes and 120 minutes. Figure 17 (17A and 17B) includes the images obtained for E.coli tolC treated with 25 μg/mL cerulenin for 120 minutes and 240 minutes. Figure 18 (18A and 18B) includes the images obtained for E.coli tolC treated with 7.5 μg/mL rifampicin for 30 minutes and 120 minutes.

The effects of the compounds shown in Figures 1 1 to 18 are summarized in Table 11 below: Table 11

Figure 19 contains a clustergram of zidovudine (azidothymidine) and the different classes of antibiotic: transcription (rifampicin), lipid biosynthesis (cerulenin), cell wall biosynthesis (cephalexin), DNA replication (ciprofloxacin) with E.coli tolC treated at 5X MIC for 120 minutes (or for 240 minutes for cerulenin). It can be seen from this figure; along with the information in the table above that zidovudine (azidothymidine) is grouped closer to ciprofloxacin than the other control compounds. This indicates that zidovudine is likely to inhibit DNA replication when acting as an antibiotic. For the other DNA replication inhibitors, profiles were obtained using BCP at two concentrations: 1X MIC and 5X MIC, and two points: 30 minutes and 120 minutes. The images are shown in Figures 20 to 25 (A and B).

Figure 20 (20A and 20B) includes the images obtained for E.coli tolC treated with 1.5 μg/mL daunorubicin for 30 minutes and 120 minutes. Figure 21 (21A and 21 B) includes the images obtained for E.coli tolC treated with 7.5 μg/mL daunorubicin for 30 minutes and 120 minutes. Figure 22 (22A and 22B) includes the images obtained for E.coli tolC treated with 1 μg/mL novobiocin for 30 minutes and 120 minutes. Figure 23 (23A and 23B) includes the images obtained for E.coli tolC treated with 5 μg/mL novobiocin for 30 minutes and 120 minutes. Figure 24 (24A and 24B) includes the images obtained for E.coli tolC treated with 0.03 μg/mL mitomycin C for 30 minutes and 120 minutes. Figure 25 (25A and 25B) includes the images obtained for E.coli tolC treated with 0.15 μg/mL mitomycin C for 30 minutes and 120 minutes.

It can be seen from these figures that condensed, centrally located nucleoids were observed after 30 minutes of treatment with 5X MIC zidovudine, ciprofloxacin, mitomycin C or novobiocin, and that these phenotypes became more pronounced and distinct by 120 minutes. In contrast, cells treated with 5X MIX daunorubicin (Figure 21 (A,B)) were distinct from the other four replication inhibitors, forming only slightly elongated cells with abnormally staining nucleoids.

The effects of the compounds shown in Figures 20 to 25 (A and B) are summarized in Table 12 below:

Table 12

Treatment of E.coli tolC with 5X MIC (5 μς/ηΐ-.) novobiocin resulted in a slight increase in cell length with most cells containing one very condensed nucleoid per cell.

Mitomycin C Mitomycin C crosslinks DNA. Figures 14

Treatment of E.coli tolC with 1X or 5X MIC (0.03 or 0.15 and 15 μg/mL) mitomycin C resulted in long filaments with multiple, (14A, 14B, long nucleoids per cell. 15A, 15B)

Figure 26 contains a clustergram of zidovudine (azidothymidine) and the different types of DNA replication inhibitor: crosslinks DNA (mitomycin C), DNA gyrase A (ciprofloxacin), DNA intercalator (daunorubicin) and DNA gyrase B (novobiocin) with E.coli tolC treated at 5X MIC for 120 minutes. It can be seen from this figure; along with the information in the table above that zidovudine (azidothymidine) is again grouped closer to ciprofloxacin than the other compounds. Both compounds cause the bacteria to form long filaments with most cells containing a single, centrally located, condensed nucleoid. This indicates that zidovudine is likely to target DNA gyrase A when acting as an antibiotic.