USO09549965B2

(12) United States Patent (10) Patent No.: US 9,549,965 B2 Hu et al. (45) Date of Patent: Jan. 24, 2017

(54) PHARMACEUTICAL COMPOSITION FOR (52) U.S. Cl. TREATING AMCROBAL INFECTION CPC ...... A61K 38/12 (2013.01); A61 K3I/05 (2013.01); A61K 31/10 (2013.01); A61 K (71) Applicant: HELPERBY THERAPEUTICS 3 1/138 (2013.01); A61K 31/145 (2013.01); LIMITED, London (GB) A61K 31/167 (2013.01); A61K 31/18 (2013.01); A61K 3L/216 (2013.01); A61 K (72) Inventors: Yanmin Hu, London (GB); Anthony R 3 I/282 (2013.01); A61 K3I/305 (2013.01); M Coates, London (GB) A61 K3I/382 (2013.01); A61 K3I/395 (2013.01); A61K 31/407 (2013.01); A61 K (73) Assignee: Helperby Therapeutics Limited, 31/417 (2013.01); A61K 31/4178 (2013.01); London (GB) A61K 31/445 (2013.01); A61K 31/473 (*) Notice: Subject to any disclaimer, the term of this (2013.01); A61 K3I/475 (2013.01); A61 K patent is extended or adjusted under 35 31/4709 (2013.01); A61 K3I/4741 (2013.01); U.S.C. 154(b) by 0 days. A6 IK3I/517 (2013.01); A61K 3.1/5415 (2013.01); A61 K3I/704 (2013.01); A61 K (21) Appl. No.: 14/862,108 3 1/7052 (2013.01); A61K 33/24 (2013.01); A61K 38/14 (2013.01); A61K 45/06 (2013.01) (22) Filed: Sep. 22, 2015 (58) Field of Classification Search O O CPC ... A61K 38/12: A61K 31/138: A61K 2300/00 (65) Prior Publication Data See application file for complete search history. US 2016/OO82O73 A1 Mar. 24, 2016 (56) References Cited Related U.S. Application Data U.S. PATENT DOCUMENTS (63) Continuation of application No. 13/821,448, filed as 5,290,774. A * 3/1994 Morita ...... A61K9/0048 application No. PCT/GB2011/051695 on Sep. 9, 514,218 2011. 2003/0229082 A1 12/2003 James et al. 2008/0113031 A1* 5/2008 Moodley ...... A61K9/5073 O O 424/490 (30) Foreign Application Priority Data 2009, O131342 A1 ck 5/2009 Ellis ...... A61 K 31,04 514, 29 Sep. 10, 2010 (GB) ...... 10 15079.5 FOREIGN PATENT DOCUMENTS (51) Int. Cl. A6 IK 38/12 (2006.01) EP O527101 2, 1993 A6 IK3I/38 (2006.01) TR 2001 01991 2, 2003 A6 IK 3/8 (2006.01) WO WO 99.433.54 9, 1999 A 6LX 3L/395 (2006.01) A6 IK 3/47 (2006.01) OTHER PUBLICATIONS A6 IK 3/475 (2006.01) Chodosh, S. et al. “Prazosin in hypertensive patients with chronic A 6LX 3/57 (2006.01) bronchitis and asthma: A brief report.” American Journal of Medi A6 IK 45/06 (2006.01) cine, vol. 86, No. 1, Jan. 23, 1989, pp. 91-93. A6 IK3I/05 (2006.01) (Continued) A6 IK3I/It (2006.01) A6 IK3I/I67 (2006.01) A6 IK 3L/26 (2006.01) Primary Examiner — Sreeni Padmanabhan A6 IK3I/282 (2006.01) Assistant Examiner — Jody Karol A6 IK3I/305 (2006.01) (74) Attorney, Agent, or Firm — Steptoe & Johnson LLP A6 IK3I/382 (2006.01) A6 IK 3/407 (2006.01) A6 IK 3/478 (2006.01) (57) ABSTRACT A6 IK 3/445 (2006.01) The present invention relates to a combination comprising at AA6 6LX IK 3L/247093/473 (2006.01)2006.O1 least one compound selected from the group consisting of an ( .01) C.-adrenergic antagonist, an anthelmintic agent, an antifun A6 IK 3/474 (2006.01) gal agent, an antimalarial agent, an antineoplastic agent, an A6 IK3I/545 (2006.01) antipsychotic agent, an antioxidant, a vasodilator, a vitamin, A6 IK3I/704 (2006.01) or a pharmaceutically acceptable derivative thereof, and an A 6LX 3L/7052 (2006.01) antimicrobial compound. A6 IK 33/24 (2006.01) A6 IK 38/14 (2006.01) A6 IK 3L/45 (2006.01) 6 Claims, 15 Drawing Sheets US 9,549,965 B2 Page 2

(56) References Cited

OTHER PUBLICATIONS Szreder, Z. et al. "Inhibition of pyrogen Escherichia coli fever with intracerebral administration of prazosin, dihydrobenzperidol and nifedipin in the rabbits.” General Pharmacology, vol. 22, No. 2, Jan. 1, 1991, pp. 381-388. Bermudez et al., “Mefloquine, Moxifloxacin, and Ethanbutol Area Triple-Drug Alternative to Macrollide-Containing Regimens for Treatment of Mycobacterium avium Disease.” Brief Report. J. of Infectious Diseases, 2003: 187; 1977-1980. * cited by examiner U.S. Patent Jan. 24, 2017 Sheet 1 of 15 US 9,549,965 B2

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x Caistin & ragi. x riftioperaxi & hydrochiorida + Cristin US 9,549,965 B2 1. 2 PHARMACEUTICAL COMPOSITION FOR duction of the target enzyme and bypass of the inhibited TREATING AMCROBAL INFECTION steps. Nevertheless, the rate of resistance emerges to a particular agent has been observed to vary widely, depend This invention relates to the use of certain classes of ing upon factors such as the agents mechanism of action, known compounds for the treatment of microbial infections. whether the agent's mode of killing is time- or concentra In particular, it relates to the use of Such compounds to kill tion-dependent, the potency against the population of bac multiplying, non-multiplying and/or clinically latent micro teria and the magnitude and duration of the available serum organisms associated with microbial infections. concentration. Before the introduction of antibiotics, patients suffering It has been proposed (Science 264,388-393 (1994)) that from acute microbial infections (e.g. tuberculosis or pneu 10 agents that target single enzymes (e.g. rifampicin) are the monia) had a low chance of Survival. For example, mortality from tuberculosis was around 50%. Although the introduc most prone to the development of resistance. Further, the tion of antimicrobial agents in the 1940s and 1950s rapidly longer that Suboptimal levels of antimicrobial agent are in changed this picture, bacteria have responded by progres contact with the bacteria, the more likely the emergence of resistance. sively gaining resistance to commonly used antibiotics. 15 Now, every country in the world has antibiotic-resistant Moreover, it is now known that many microbial infections bacteria. Indeed, more than 70% of bacteria that give rise to include Sub-populations of bacteria that are phenotypically hospital acquired infections in the USA resist at least one of resistant to antimicrobials (J. Antimicrob. Chemother: 4. the main antimicrobial agents that are typically used to fight 395-404 (1988); J. Med. Microbiol. 38, 197-202 (1993); J. infection (Nature Reviews, Drug Discovery 1, 895-910 Bacteriol. 182, 1794-1801 (2000): ibid. 182, 6358-6365 (2002)). (2000): ibid. 183, 6746-6751 (2001); FEMS Microbiol. Lett. One way of tackling the growing problem of resistant 202, 59-65 (2001); and Trends in Microbiology 13, 34-40 bacteria is the development of new classes of antimicrobial (2005)). There appear to be several types of such pheno agents. However, until the introduction of linezolid in 2000, typically resistant bacteria, including persisters, stationary there had been no new class of antibiotic marketed for over 25 phase bacteria, as well as those in the depths of biofilms. 37 years. Moreover, even the development of new classes of However, each of these types is characterised by its low rate antibiotic provides only a temporary Solution, and indeed of growth compared to log-phase bacteria under the same there are already reports of resistance of certain bacteria to conditions. Nutritional starvation and high cell densities are linezolid (Lancet 357, 1179 (2001) and Lancet 358, 207-208 also common characteristics of Such bacteria. (2001)). 30 Although resistant to antimicrobial agents in their slow In order to develop more long-term solutions to the growing state, phenotypically resistant bacteria differ from problem of bacterial resistance, it is clear that alternative those that are genotypically resistant in that they regain their approaches are required. One Such alternative approach is to susceptibility to antimicrobials when they return to a fast minimise, as much as is possible, the opportunities that growing state (e.g. when nutrients become more readily bacteria are given for developing resistance to important 35 available to them). antibiotics. Thus, strategies that can be adopted include The presence of phenotypically resistant bacteria in an limiting the use of antibiotics for the treatment of non-acute infection leads to the need for prolonged courses of antimi infections, as well as controlling which antibiotics are fed to crobial agents, comprising multiple doses. This is because animals in order to promote growth. the resistant, slowly multiplying bacteria provide a pool of However, in order to tackle the problem more effectively, 40 “latent organisms that can convert to a fast-growing state it is necessary to gain an understanding of the actual when the conditions allow (thereby effectively re-initiating mechanisms by which bacteria generate resistance to anti the infection). Multiple doses over time deal with this issue biotic agents. To do this requires first a consideration of how by gradually killing off the “latent bacteria that convert to current antibiotic agents work to kill bacteria. “active’ form. Antimicrobial agents target essential components of bac 45 However, dealing with “latent bacteria by administering terial metabolism. For example, the B-lactams (e.g. penicil prolonged courses of antimicrobials poses its own problems. lins and cephalosporins) inhibit cell wall synthesis, whereas That is, prolonged exposure of bacteria to Suboptimal con other agents inhibit a diverse range of targets, such as DNA centrations of antimicrobial agent can lead to the emergence gyrase (quinolones) and protein synthesis (e.g. macrollides, of genotypically resistant bacteria, which can then multiply aminoglycosides, tetracyclines and oxazolidinones). The 50 rapidly in the presence of even high concentrations of the range of organisms against which the antimicrobial agents antimicrobial. are effective varies, depending upon which organisms are Long courses of antimicrobials are more likely to encour heavily reliant upon the metabolic step(s) that is/are inhib age the emergence of genotypic resistance than shorter ited. Further, the effect upon bacteria can vary from a mere courses on the grounds that non-multiplying bacterial will inhibition of growth (i.e. a bacteriostatic effect, as seen with 55 tend to Survive and, interestingly, probably have an agents such as the tetracyclines) to full killing (i.e. a bac enhanced ability to mutate to resistance (Proc. Natl. Acad. tericidal effect, as seen, e.g. with penicillin). Sci. USA 92, 11736-11740 (1995); J. Bacteriol. 179, 6688 Bacteria have been growing on Earth for more than 3 6691 (1997); and Antimicrob. Agents Chemother: 44, 1771 billion years and, in that time, have needed to respond to vast 1777 (2000)). numbers of environmental stresses. It is therefore perhaps 60 In the light of the above, a new approach to combating the not surprising that bacteria have developed a seemingly problem of bacterial resistance might be to select and inexhaustible variety of mechanisms by which they can develop antimicrobial agents on the basis of their ability to respond to the metabolic stresses imposed upon them by kill "latent microorganisms. The production of Such agents antibiotic agents. Indeed, mechanisms by which the bacteria would allow, amongst other things, for the shortening of can generate resistance include strategies as diverse as 65 chemotherapy regimes in the treatment of microbial infec inactivation of the drug, modification of the site of action, tions, thus reducing the frequency with which genotypical modification of the permeability of the cell wall, overpro resistance arises in microorganisms. US 9,549,965 B2 3 4 International Patent Application, Publication Number infection therefore include killing multiplying, non-multi WO2000028074 describes a method of screening com plying and/or clinically latent microorganisms associated pounds to determine their ability to kill clinically latent with such infections. In a preferred embodiment, the afore microorganisms. Using this method, the Applicant has mentioned compounds are used to kill non-multiplying observed that many conventional antimicrobial agents. Such and/or clinically latent microorganisms, most preferably as augmentin, azithromycin, levofloxacin, lineZolid and non-multiplying microorganisms. mupirocin, which otherwise exhibit excellent biological As used herein, “kill means a loss of viability as assessed activity against log phase (i.e. multiplying) bacteria, exhibit by a lack of metabolic activity. little or no activity against clinically latent microorganisms. As used herein, "clinically latent microorganism' means This observation has necessitated the development of novel 10 a microorganism that is metabolically active but has a antimicrobials which may be used to kill clinically latent growth rate that is below the threshold of infectious disease microorganisms. expression. The threshold of infectious disease expression International Patent Application, Publication Numbers refers to the growth rate threshold below which symptoms of WO2007054693, WO2008117079 and WO2008142384 infectious disease in a host are absent. describe compounds which exhibit biological activity 15 against clinically latent microorganisms. Examples of Such The metabolic activity of clinically latent microorganisms compounds include 4-methyl-1-(2-phenylethyl)-8-phenoxy can be determined by several methods known to those 2,3-dihydro-1H-pyrrolo3.2-c-quinoline, 4-(3-benzylpyrro skilled in the art; for example, by measuring mRNA levels lidin-1-yl)-2-methyl-6-phenoxyquinoline, N-4-(3-ben in the microorganisms or by determining their rate of uridine Zylpyrrolidin-1-yl)-2-methylduinolin-6-ylbenzamide and uptake. In this respect, clinically latent microorganisms, pharmaceutically acceptable derivatives thereof. when compared to microorganisms under logarithmic The present invention is based upon the unexpected growth conditions (in vitro or in vivo), possess reduced but finding that certain classes of known biologically active still significant levels of: compounds have been found to exhibit bactericidal activity (I) mRNA (e.g. from 0.0001 to 50%, such as from 1 to 30, against a variety of microorganisms. 25 5 to 25 or 10 to 20%, of the level of mRNA); and/or Thus, in one embodiment the present invention provides (II) uridine (e.g. Huridine) uptake (e.g. from 0.0005 to the use of one or more compounds selected from the 50%, such as from 1 to 40, 15 to 35 or 20 to 30% of the following: an O-adrenergic antagonist, an anthelmintic level of Huridine uptake). agent, an antifungal agent, an antimalarial agent, an antine Clinically latent microorganisms typically possess a num oplastic agent, an antipsychotic agent, an antioxidant, a 30 ber of identifiable characteristics. For example, they may be vasodilator and/or a vitamin, or a pharmaceutically accept viable but non-culturable; i.e. they cannot typically be able derivative thereof, for the treatment of a microbial detected by standard culture techniques, but are detectable infection; with the proviso that when the biologically active and quantifiable by techniques such as broth dilution count agent is an antifungal agent, the microbial infection is a ing, microscopy, or molecular techniques such as poly bacterial infection. 35 merase chain reaction. In addition, clinically latent micro In a further embodiment, the invention provides a method organisms are phenotypically tolerant, and as Such are of treating a microbial infection which comprises adminis sensitive (in log phase) to the biostatic effects of conven tering to a mammal, including man, one or more compounds tional antimicrobial agents (i.e. microorganisms for which selected from the following: an O-adrenergic antagonist, an the minimum inhibitory concentration (MIC) of a conven anthelmintic agent, an antifungal agent, an antimalarial 40 tional antimicrobial is Substantially unchanged); but possess agent, an antineoplastic agent, an antipsychotic agent, an drastically decreased Susceptibility to drug-induced killing antioxidant, a vasodilator and/or a vitamin, or a pharmaceu (e.g. microorganisms for which, with any given conven tically acceptable derivative thereof; with the proviso when tional antimicrobial agent, the ratio of minimum microbio the biologically active agent is an antifungal agent, the cidal concentration (e.g. minimum bactericidal concentra microbial infection is a bacterial infection. 45 tion, MBC) to MIC is 10 or more). There is also provided a pharmaceutical composition As used herein, the term “microorganisms' means fungi comprising one or more compounds selected from the and bacteria. References herein to “microbial”, “antimicro following: an O-adrenergic antagonist, an anthelmintic bial and “antimicrobially shall be interpreted accordingly. agent, an antifungal agent, an antimalarial agent, an antine For example, the term “microbial” means fungal or bacte oplastic agent, an antipsychotic agent, an antioxidant, a 50 rial, and “microbial infection” means any fungal or bacterial vasodilator and/or a vitamin, or a pharmaceutically accept infection. able derivative thereof, and a pharmaceutically acceptable In one embodiment of the invention, one or more of the adjuvant, diluent or carrier, for use in the treatment of a aforementioned classes of biologically active compounds is microbial infection; with the proviso when the biologically used to treat a bacterial infection; in particular, the com active agent is an antifungal agent, the microbial infection is 55 pounds may be used to kill clinically latent microorganisms a bacterial infection. associated with a bacterial infection. As used herein, the term “bacteria” (and derivatives thereof, such as “microbial BRIEF DESCRIPTION OF THE DRAWINGS infection') includes, but is not limited to, references to organisms (or infections due to organisms) of the following FIG. 1 is a graph showing bacterialcidal activity. 60 classes and specific types: FIGS. 2-15 are graphs depicting time kill curves. Gram-positive cocci. Such as Staphylococci (e.g. Staph. The aforementioned classes of biologically active com aureus, Staph. epidermidis, Staph. Saprophyticus, Staph. pounds may be used to treat microbial infections. In par auricularis, Staph. Capitis capitis, Staph. c. ureolyticus, ticular they may be used to kill multiplying (log phase), Staph. Caprae, Staph. cohnii Cohnii, Staph. c. urealyticus, non-multiplying (stationary phase) and/or clinically latent 65 Staph. equorum, Staph. gallinarum, Staph. haemolyticus, (persistent) microorganisms associated with microbial infec Staph. hominis hominis, Staph. h. novobiosepticius, Staph. tions. References herein to the treatment of a microbial hyicus, Staph. intermedius, Staph. lugdunensis, Staph, pas US 9,549,965 B2 5 6 teuri, Staph. Saccharolyticus, Staph. Schleiferi Schleiferi, Clostridium (e.g. C. perfingens, C. difficile, C. botuli Staph. S. coagulans, Staph. Sciuri, Staph. Simulans, Staph. num, C. tetani, C. absonium, C. argentinense, C. baratii, C. warneri and Staph. xylosus); bifermentans, C. beijerinckii, C. butyricum, C. cadaveris, C. Streptococci (e.g. beta-haemolytic, pyogenic streptococci camis, C. cellatum, C. clostridioforme, C. cochlearium, C. (such as Strept. agalactiae, Strept. Canis, Strept dysgalactiae cocleatum, C. fallax, C. ghonii, C. glycolicum, C. haemo dysgalactiae, Strept. dysgalactiae equisimilis, Strept equi lyticum, C. hastiforme, C. histolyticum, C. indolis, C. equi, Strept. equi ZOOepidemicus, Strept. iniae, Strept. porci innocuum, C. irregulare, C. leptum, C. limosum, C. male nus and Strept. pyogenes), microaerophilic, pyogenic strep nominatum, C. novyi, C. Oroticum, C. paraputrificum, C. tococci (Streptococcus “milleri”. Such as Strept. anginosus, piliforme, C. putrefasciens, C. ramosum, C. septicum, C. 10 sordelii, C. sphenoides, C. Sporogenes, C. subterminale, C. Strept. Constellatus constellatus, Strept. constellatus phar symbiosum and C. tedium); yngidis and Strept. intermedius), oral Streptococci of the Mycoplasma (e.g. M. pneumoniae, M. hominis, M. geni “mitis” (alpha-haemolytic-Streptococcus “viridans, such as talium and M. urealyticum); Strept. mitis, Strept. Oralis, Strept. Sanguinis, Strept. Crista Mycobacteria (e.g. Mycobacterium tuberculosis, Myco tus, Strept.gordonii and Strept. parasanguinis), "salivarius' 15 bacterium avium, Mycobacterium fortuitum, Mycobacte (non-haemolytic, such as Strept salivarius and Strept. ves rium marinum, Mycobacterium kansasii, Mycobacterium tibularis) and "mutans' (tooth-Surface Streptococci. Such as chelonae, Mycobacterium abscessus, Mycobacterium lep Strept. Criceti, Strept. mutans, Strept. ratti and Strept. Sobri rae, Mycobacterium Smegmitis, Mycobacterium africanum, nus) groups, Strept. acidominimus, Strept. bovis, Strept. Mycobacterium alvei, Mycobacterium asiaticum, Mycobac faecalis, Strept. equinus, Strept. pneumoniae and Strept. terium aurum, Mycobacterium bohemicum, Mycobacterium suis, or Streptococci alternatively classified as Group A, B, bovis, Mycobacterium branderi, Mycobacterium brumae, C, D, E, G, L. P. U or V Streptococcus); Mycobacterium cellatum, Mycobacterium chubense, Myco Gram-negative cocci. Such as Neisseria gonorrhoeae, bacterium confluentis, Mycobacterium conspicuum, Myco Neisseria meningitidis, Neisseria cinerea, Neisseria elon bacterium cookii, Mycobacterium flavescens, Mycobacte gata, Neisseria flavescens, Neisseria lactanica, Neisseria 25 rium gadium, Mycobacterium gastri, Mycobacterium mucosa, Neisseria sicca, Neisseria subflava and Neisseria genavense, Mycobacterium gordonae, Mycobacterium goo weaveri, dii, Mycobacterium haemophilum, Mycobacterium hassi Bacillaceae, such as Bacillus anthracis, Bacillus subtilis, cum, Mycobacterium intracellulare, Mycobacterium inter Bacillus thuringiensis, Bacillus Stearothermophilus and jectum, Mycobacterium heidelberense, Mycobacterium Bacillus cereus, 30 lentiflavum, Mycobacterium malmoense, Mycobacterium Enterobacteriaceae, such as Escherichia coli, Entero mucogenicum, Mycobacterium microti, Mycobacterium bacter (e.g. Enterobacter aerogenes, Enterobacter agglom mucogenicum, Mycobacterium neoaurum, Mycobacterium erans and Enterobacter cloacae), Citrobacter (Such as Cit nonchromogenicum, Mycobacterium peregrinum, Mycobac rob, freundii and Citrob. divernis), Hafnia (e.g. Hafinia terium phlei, Mycobacterium scrofitlaceum, Mycobacterium alvei), Erwinia (e.g. Erwinia persicinus), Morganella mor 35 Shimoidei, Mycobacterium simiae, Mycobacterium Szulgai, ganii, Salmonella (Salmonella enterica and Salmonella Mycobacterium terrae, Mycobacterium the rmoresistabile, typhi), Shigella (e.g. Shigella dysenteriae, Shigella flexneri, Mycobacterium triplex, Mycobacterium triviale, Mycobac Shigella boydii and Shigella sonnei), Klebsiella (e.g. Klebs. terium tusciae, Mycobacterium ulcerans, Mycobacterium pneumoniae, Klebs. Oxytoca, Klebs. Ornitholytica, Klebs. vaccae, Mycobacterium wolinskyi and Mycobacterium planticola, Klebs. OZaenae, Klebs. terrigena, Klebs. gran 40 xenopi); ulomatis (Calymmatobacterium granulomatis) and Klebs. Haemophilus (e.g. Haemophilus influenzae, Haemophilus rhinoscleromatis), Proteus (e.g. Pr: mirabilis, Pr: rettgeri ducreyi, Haemophilus aegyptius, Haemophilus parainfluen and Pr: vulgaris), Providencia (e.g. Providencia alcalifa Zae, Haemophilus haemolyticus and Haemophilus parahae ciens, Providencia rettgeri and Providencia Stuartii), Serra molyticus); tia (e.g. Serratia marcescens and Serratia liquifaciens), and 45 Actinobacillus (e.g. Actinobacillus actinomycetemcomi Yersinia (e.g. Yersinia enterocolitica, Yersinia pestis and tans, Actinobacillus equuli, Actinobacillus hominis, Actino Yersinia pseudotuberculosis); bacillus lignieresii, Actinobacillus suis and Actinobacillus Enterococci (e.g. Enterococcus avium, Enterococcus cas ureae); selliflavus, Enterococcus cecorum, Enterococcus dispar; Actinomyces (e.g. Actinomyces israeli); Enterococcus durans, Enterococcus faecalis, Enterococcus 50 Brucella (e.g. Brucella abortus, Brucella canis, Brucella faecium, Enterococcus flavescens, Enterococcus galli melintensis and Brucella suis); narum, Enterococcus hirae, Enterococcus malodoratus, Campylobacter (e.g. Campylobacter jejuni, Campy Enterococcus mundtii, Enterococcus pseudoavium, Entero lobacter coli, Campylobacter lari and Campylobacter fetus); coccus raffinosus and Enterococcus solitarius); Listeria monocytogenes, Helicobacter (e.g. Helicobacter pylori, Helicobacter 55 Vibrio (e.g. Vibrio cholerae and Vibrio parahaemolyticus, cinaedi and Helicobacter fennelliae): Vibrio alginolyticus, Vibrio Carchariae, Vibrio fluvialis, Acinetobacter (e.g. A. baumanii, A. Calcoaceticus, A. Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio haemolyticus, A. johnsonii, A. junfi, A. Iwoffi and A. radiore mimicus and Vibrio vulnificus); sistens); Erysipelothrix rhusopathiae, Pseudomonas (e.g. PS. aeruginosa, Ps. maltophilia 60 Corynebacteriaceae (e.g. Corynebacterium diphtheriae, (Stenotrophomonas maltophilia), Ps. alcaligenes, Ps. chlo Corynebacterium jeikeum and Corynebacterium urealyti roraphis, Ps, fluorescens, Ps. luteola. Ps. mendocina, Ps. cum); monteilii, Ps. Oryzihabitans, Ps. pertocinogena, Ps. pseu Spirochaetaceae, such as Borrelia (e.g. Borrelia recur dalcaligenes, P.S. putida and Ps. Stutzeri): rentis, Borrelia burgdorferi, Borrelia afzelii, Borrelia ander Bacteroides fragilis, 65 sonii, Borrelia bissettii, Borrelia garinii, Borrelia japonica, Peptococcus (e.g. Peptococcus niger); Borrelia lusitaniae, Borrelia tanuki, Borrelia turdi, Borrelia Peptostreptococcus, valaisiana, Borrelia Caucasica, Borrelia crocidurae, Borre US 9,549,965 B2 7 8 lia duttoni, Borrelia graingeri, Borrelia hermsii, Borrelia PrevOtella oralis, PrevOtella Oris, Prevotella Oulora, Pre hispanica, Borrelia latyschewi, Borrelia mazzottii, Borrelia votella tannerae, PrevOtella venoralis and PrevOtella zoog parkeri, Borrelia persica, Borrelia turicatae and Borrelia leoformans); venezue/ensis) and Treponema (Treponema pallidum ssp. Porphyromonas (e.g. Porphyromonas asaccharolytica, pallidum, Treponema pallidum ssp. endemicum, Treponema Porphyromonas cangingivalis, Porphyromonas canoris, pallidum ssp. pertenue and Treponema carateum); Porphyromonas can sulci, Porphyromonas Catoniae, Por phyromonas circumdentaria, Porphyromonas crevioricanis, Pasteurella (e.g. Pasteurella aerogenes, Pasteurella bet Porphyromonas endodontalis, Porphyromonas gingivalis, tyae, Pasteurella canis, Pasteurella dagmatis, Pasteurella Porphyromonas gingivicanis, Porphyromonas levil and Por gallinarum, Pasteurella haemolytica, Pasteurella multocida 10 phyromonas macacae); multocida, Pasteurella multocida gallicida, Pasteurella Fusobacterium (e.g. F. gonadiaformans, F. mortiferum, F. multocida septica, Pasteurella pneumotropica and Pasteur naviforme, F. necrogenes, F. necrophorum necrophorum, F. ella Stomatis); necrophorum findiliforme, F. nucleatum nucleatum, F. Bordetella (e.g. Bordetella bronchiseptica, Bordetella nucleatum fusiforme, F. nucleatum polymorphum, F. nuclea hinzii, Bordetella holmseii, Bordetella parapertussis, Bor 15 tum vincentii, F. periodonticum, F. russi, F. ulcerans and F. detella pertussis and Bordetella trematum); varium); Nocardiaceae. Such as Nocardia (e.g. Nocardia asteroides Chlamydia (e.g. Chlamydia trachomatis); and Nocardia brasiliensis); Cryptosporidium (e.g. C. parvum, C. hominis, C. canis, C. Rickettsia (e.g. Ricksettsii or Coxiella burnetii); felis, C. meleagridis and C. muris); Legionella (e.g. Legionalla anisa, Legionalla birmingha Chlamydophila (e.g. Chlamydophila abortus (Chlamydia mensis, Legionalla bozemanii, Legionalla cincinnatiensis, psittaci), Chlamydophila pneumoniae (Chlamydia pneumo Legionalla dunoffii, Legionalla feeleii, Legionalla gor niae) and Chlamydophila psittaci (Chlamydia psittaci)); manii, Legionalla hackeliae, Legionalla israe/ensis, Legion Leuconostoc (e.g. Leuconostoc citreum, Leuconostoc Cre alla jordanis, Legionalla lansingensis, Legionalla long moris, Leuconostoc dextranicum, Leuconostoc lactis, Leu beachae, Legionalla maceachernii, Legionalla micaladei, 25 conostoc mesenteroides and Leuconostoc pseudomes Legionalla Oakridgensis, Legionalla pneumophila, Legion enteroides); alla Sainthe/ensi, Legionalla tucsonensis and Legionalla Gemella (e.g. Gemella bergeri, Gemella haemolysans, Wadsworthii); Gemella morbillorum and Gemella sanguinis); and Moraxella Catarrhalis, Ureaplasma (e.g. Ureaplasma parvum and Ureaplasma Cyclospora Cayetanensis, 30 urealyticum). Entamoeba histolytica, Preferably the bacteria to be treated are selected from the Giardia lamblia, group consisting of Trichomonas vaginalis, Staphylococci, such as Staph. aureus (either Methicillin Toxoplasma gondii; sensitive (i.e. MSSA) or Methicillin-resistant (i.e. MRSA)) Stenotrophomonas maltophilia, 35 and Staph. epidermidis, Burkholderia cepacia, Burkholderia mallei and Burk Streptococci. Such as Strept. agalactiae and Strept. pyo holderia pseudomalilei, genes, Francisella tularensis, Bacillaceae. Such as Bacillus anthracis, Gardnerella (e.g. Gardneralla vaginalis and Gardneralla Enterobacteriaceae, such as Escherichia coli, Klebsiella mobiluncus); 40 (e.g. Klebs. pneumoniae and Klebs. Oxytoca) and Proteus Streptobacillus moniliformis, (e.g. Pr: mirabilis, Pr: rettgeri and Pr: vulgaris); Haemophi Flavobacteriaceae, such as Capnocytophaga (e.g. Capno lis influenzae, cytophaga Canimorsus, Capnocytophaga Cynodegni, Cap Enterococci. Such as Enterococcus faecalis and Entero nocytophaga gingivalis, Capnocytophaga granulosa, Cap coccus faecium; and nocytophaga haemolytica, Capnocytophaga ochracea and 45 Mycobacteria, Such as Mycobacterium tuberculosis. Capnocytophaga sputigena), More preferably, the bacteria to be treated are selected Bartonella (Bartonella bacilliformis, Bartonella clar from the group consisting of Staphylococcus aureus; either ridgeiae, Bartonella elizabethae, Bartonella hemselae, Bar MSSA or MRSA, Escherichia coli and Pseudomonas tonella quintana and Bartonella vinsonii arupensis); aeruginosa. Leptospira (e.g. Leptospira biflexia, Leptospira borgpe 50 In another embodiment of the invention, one or more of tersenii, Leptospira inadai, Leptospira interrogans, Lep the aforementioned classes of biologically active com to spira kirschneri, Leptospira noguchii, Leptospira Santa pounds, excluding antifungal agents, is used to treat a fungal rosai and Leptospira weilii); infection; in particular, the compounds may be used to kill Spirillium (e.g. Spirillum minus); clinically latent microorganisms associated with a fungal Baceteroides (e.g. Bacteroides Caccae, Bacteroides cap 55 infection. As used herein, the term “fungi' (and derivatives illosus, Bacteroides coagulans, Bacteroides distasonis, thereof, such as “fungal infection') includes, but is not Bacteroides eggerthii, Bacteroides forsythus, Bacteroides limited to, references to organisms (or infections due to fragilis, Bacteroides merdae, Bacteroides ovatus, Bacte organisms) of the following classes and specific types: roides putredinis, Bacteroides pyogenes, Bacteroides splan Absidia (e.g. Absidia corymbifera); chinicus, Bacteroides Stercoris, Bacteroides tectus, Bacte 60 Ajellomyces (e.g. Ajellomyces capsulatus and Ajellomyces roides thetaiotaomicron, Bacteroides uniformis, Bacteroides dermatitidis); ureolyticus and Bacteroides vulgatus); Arthroderma (e.g. Arthroderma benhamiae, Arthroderma Prevotella (e.g. Prevotella bivia, Prevotella buccae, Pre fiulvum, Arthroderma gypseum, Arthroderma incurvatum, votella corporis, PrevOtella dentalis (Mitsuokella dentalis), Arthroderma Otae and Arthroderma vanbreuseghemii); PrevOtella denticola, PrevOtella disiens, PrevOtella enoeca, 65 Aspergillus (e.g. Aspergillus flavus, Aspergillus filmigatus Prevotella heparinolytica, Prevotella intermedia, PrevOtella and Aspergillus niger); loeschii, PrevOtella melaninogenica, PrevOtella nigrescens, Blastomyces (e.g. Blastomyces dermatitidis); US 9,549,965 B2 10 Candida (e.g. Candida albicans, Candida glabrata, Can abamectin, albendazole, bithionate (such as bithionate dida guilliermondii, Candida krusei, Candida parapsilosis, Sodium), closantel, diethylcarbamazine, emodepside, fen Candida tropicalis and Candida pelliculosa); bendazole, flubendazole, ivermectin, levamisole, mebenda Cladophialophora (e.g. Cladophialophora carrionii); Zole, metrifonate, morantel, monepantel, niclosamide, Coccidioides (e.g. Coccidioides immitis and Coccidioides 5 oxamnaduine, pararosaniline (such as pararosaniline pamo posadasii); ate), piperazine (Such as piperazine citrate and piperazine Cryptococcus (e.g. Cryptococcus neoformans); hydrate), praziquantel, pyrvinium (such as pyrvinium pamo Cunninghamella (e.g. Cunninghamella sp.) ate), pyrantel (such as pyrantel pamoate), quinacrine (Such Epidermophyton (e.g. Epidermophyton floccosum); as quinacrine hydrochloride), Suramin, thiabendazole and/or Exophiala (e.g. Exophiala dermatitidis); 10 triclabendazole, and pharmaceutically acceptable derives Filobasidiella (e.g. Filobasidiella neoformans); thereof. Preferably, the anthelmintic agent is selected from Fonsecaea (e.g. Fonsecaea pedrosoi); one or more of the following compounds: bithionate Sodium, Fusarium (e.g. Fusarium Solani); pararosaniline pamoate, pyrvinium pamoate, and/or quina Geotrichum (e.g. Geotrichum candidum); crine hydrochloride. Histoplasma (e.g. Histoplasma capsulatum); 15 An antifungal agent is a compound used to treat fungal Hortaea (e.g. Hortaea werneckii); infections. Examples of Suitable antifungal agents include Issatschenkia (e.g. Issatschenkia Orientalis); one or more compounds selected from the group consisting Madurella (e.g. Madurella grisae); of abafungin, acrisorcin, anidulafungin, amorolfine, ampho Malassezia (e.g. Malassezia firfiur; Malassezia globosa, tericin B, butenafine, candicin, caspofungin, filipin, hamy Malassezia obtusa, Malassezia pachydermatis, Malassezia cin, natamycin, nystatin, rimocidin, bifonazole, butocon restricta, Malassezia sloofiae and Malassezia sympodialis); azole, clotrimazole, econazole, fenticonazole, fluconazole, Microsporum (e.g. Microsporum canis, Microsporum full isoconazole, itraconazole, isavuconazole, ketoconazole, naf vum and Microsporum gypseum); tifine, micafungin miconazole, oxiconazole, phenylmercuric Microsporidia, acetate, posaconazole, ravuconazole, Sertraconazole, Sul Mucor (e.g. Mucor circinelloides); 25 conazole, terbinafine, terconazole, tioconazole and/or Vori Nectria (e.g. Nectria haematococca); conazole, and pharmaceutically acceptable derives thereof. Paecilomyces (e.g. Paecilomyces variotii); Preferably, the antifungal agent is selected from one or more Paracoccidioides (e.g. Paracoccidioides brasiliensis); of the following compounds: acrisorcin, phenylmercuric Penicillium (e.g. Penicillium marneffei); acetate and/or tioconazole. Pichia (e.g. Pichia anomala and Pichia guilliermondii); 30 An antimalarial agent is a compound used to prevent Pneumocystis (e.g. Pneumocystis jiro veci (Pneumocystis and/or treat malaria. Examples of Suitable antimalarial carinii)); agents include one or more compounds selected from the Pseudallescheria (e.g. Pseudallescheria boydii); group consisting of amodiaquine, atovaquone, chloroquine, Rhizopus (e.g. Rhizopus Oryzae); cinchoine, cinchonidine, doxycycline, halofantrine, prima Rhodotorula (e.g. Rhodotorula rubra); 35 quine, proguanil, pyrimethamine, mefloquine, quinine, qui Scedosporium (e.g. Scedosporium apiospermum); nidine, Sulfadoxine and/or sulfamethoxypyridazine, and Schizophyllum (e.g. Schizophyllum commune); pharmaceutically acceptable derivatives thereof. Preferably, Sporothrix (e.g. Sporothrix schenckii); the antimalarial agent is mefloquine. Trichophyton (e.g. Trichophyton mentagrophytes, An antineoplastic agent is a compound used to treat Trichophyton rubrum, Trichophyton verrucosum and 40 cancer. Examples of Suitable antineoplastic agents include Trichophyton violaceum); and one or more compounds selected from the group consisting TrichospOron (e.g. TrichospOron asahi, Trichosporon of actinomycin, afimoxifene, arZoxifene, baZedoxifene, cutaneum, TrichospOron inkin and TrichospOron mucoides). bleomycin, carboplatin, cisplatin, cyclofenil, daunorubicin, Preferably, the fungi to be treated are selected from the doxorubicin, epirubicin, idarubicin, lasofoxifene, mitomy group consisting of Aspergillus filmigatus, Candida albi 45 cin C, mitoxanthrone (such as mitoxanthrone hydrochlo cans, Cryptococcus neoformans, Histoplasma capsulatum ride), ormeloxifene, plicamycin, raloxifene, sanguinarine and Pneumocystis iroveci. Sulphate, tamoxifen, toremifene (such as toremifene citrate), The biologically active compounds for use in the present and/or valrubicin, and pharmaceutically acceptable deriva invention are commercially available and/or may be pre tives thereof. Preferably, the antineoplastic agent is selected pared using conventional methods known in the art. 50 from one or more of the following compounds: bleomycin, Examples of Suitable C-adrenergic antagonists (C-block carboplatin, cisplatin, doxorubicin, mitomycin C, mitoxan ers) include non-selective C.-adrenergic antagonists, throne hydrochloride, sanguinarine Sulphate and/or tore C.-adrenergic antagonists and/or C2-adrenergic antagonists. mifene citrate. Preferred examples of C.-adrenergic antagonists include one An antipsychotic agent is a compound used to manage or more compounds selected from the group consisting of 55 psychosis, particularly associated with Schizophrenia and phenoxybenzamine, phentolamine, tolazoline, alfuZosin, bipolar disorder. Examples of Suitable antipsychotic agents prazosin, doxazosin, tamsulosin, teraZosin, atipamezole, include one or more compounds selected from the group idazoxan and/or yohimbine, and pharmaceutically accept consisting of chlorpromazine, chlorprothixene (such as able derivatives thereof. Most preferably, the C.-adrenergic chlorprothixene hydrochloride), clopenthixol, flupenthixol, antagonist is phenoxybenzamine or a pharmaceutically 60 fluphenazine, levomepromazine, mesoridazine, periciazine, acceptable derivative thereof. Such as phenoxybenzamine perphenazine, pimozide, prochlorperazine, promethazine, hydrochloride. promazine, thioridazine (such as thioridazine hydrochlo An anthelmintic agent is a compound used to treat infec ride), thiothixene, trifluoperazine (such as trifluoperazine tions with parasitic worms, including flat worms, such as hydrochloride), triflupromazine (such as triflupromazine flukes and tapeworms, and round worms, such as nema 65 hydrochloride), Zuclopenthixol, and pharmaceutically todes. Examples of Suitable anthelmintic agents include one acceptable derivatives thereof. Preferably, the antipsychotic or more compounds selected from the group consisting of agent agent is selected from one or more of the following US 9,549,965 B2 11 12 compounds: chlorprothixene hydrochloride, thioridazine guinarine Sulfate, carboplatin, cisplatin, hydroquinone, thio hydrochloride, trifluoperazine hydrochloride and/or triflu ridazine hydrochloride, trifluoperazine hydrochloride, trif promazine hydrochloride. lupromazine hydrochloride, chlorprothixene hydrochloride, An antioxidant is a compound that may protect cells perhexiline maleate, Suloctidil and vitamin B12 for the against the effects of free radicals. Examples of suitable treatment of a microbial infection; preferably a bacterial antioxidants include one or more compounds selected from infection; more preferably an infection caused by Escheri the group consisting of carotenoids such as beta-carotene, chia coli: in particular, for killing log phase and/or stationary co-enzyme Q10, hydroquinone, lutein, lycopene, selenium, phase E. coli. Vitamin A, Vitamin C and/or vitamin E, and pharmaceuti In a further embodiment of the invention there is provided cally acceptable derivatives thereof. 10 A vasodilator is a compound that relaxes the Smooth the use mefloquine for the treatment of a bacterial infection muscle in blood vessels, which causes the vessels to dilate. caused by Pseudomonas aeruginosa; in particular, for kill Examples of suitable vasodilators include one or more ing stationary phase Pseudomonas aeruginosa. compounds selected from the group consisting of amlo The above-mentioned classes of biologically active com dipine, arandipine, azapetine, azelnidipine, barnidipine, ben 15 pounds have Surprisingly been found to possess bactericidal cyclane, benidipine, bepridil, buflomedil, butalamine, activity and may therefore be used to treat a wide variety of cetiedil, cilnidipine, cinepazide, clentiazem, clevidipine, conditions. Particular conditions which may be treated using cyclandelate, diltiazem, efonidipine, fasudil, felodipine, Such classes of compounds according to the present inven fendiline, gallopamil, ifenprodil. isradipine, lacidipine, lido tion include tuberculosis (e.g. pulmonary tuberculosis, non flazine, lercanidipine, manidipine, moxisylyte, naftidrofuryl, pulmonary tuberculosis (such as tuberculosis lymph glands, nicardipine, nifedipine, nilvadipine, nimodipine, nisol genito-urinary tuberculosis, tuberculosis of bone and joints, dipine, nitrendipine, perhexiline (such as perhexiline tuberculosis meningitis) and miliary tuberculosis), anthrax, maleate), phenoxybenzamine, pranidipine, Verapamil, Vin abscesses, acne Vulgaris, actinomycosis, asthma, bacilliary burnine, Vincamine and/or visnadine, and pharmaceutically dysentry, bacterial conjunctivitis, bacterial keratitis, bacte acceptable derivatives thereof. Preferably, the vasodilator is 25 rial vaginosis, botulism, Buruli ulcer, bone and joint infec selected from one or more of the following compounds: tions, bronchitis (acute or chronic), brucellosis, burn perhexiline maleate, Suloctidil and/or nisoldipine. wounds, cat scratch fever, cellulitis, chancroid, cholangitis, A vitamin is an organic compound required as a nutrient cholecystitis, cutaneous diphtheria, cystic fibrosis, cystitis, by an organism. Examples of Suitable vitamins include one diffuse panbronchiolitis, diphtheria, dental caries, diseases or more compounds selected from the group consisting of 30 of the upper respiratory tract, eczema, empymea, endocardi vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, tis, endometritis, enteric fever, enteritis, epididymitis, epi vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin glottitis, erysipelis, erysipclas, erysipeloid, erythrasma, eye C. vitamin D. Vitamin E and/or vitamin K, and pharmaceu infections, furuncles, gardnerella Vaginitis, gastrointestinal tically acceptable derivatives thereof. Preferrably, the vita infections (gastroenteritis), genital infections, gingivitis, min is vitamin B12. 35 gonorrhoea, granuloma inguinale, Haverhill fever, infected In one embodiment of the invention, there is provided the burns, infections following dental operations, infections in use of a compound selected from the group consisting of the oral region, infections associated with prostheses, phenoxybenzamine hydrochloride, pyrvinium pamoate, intraabdominal abscesses, Legionnaire's disease, leprosy, pararosaniline pamoate, bithionate sodium, phenylmercuric leptospirosis, listeriosis, liver abscesses, Lyme disease, lym acetate, tioconazole, mefloquine, mitomycin C, toremifene 40 phogranuloma Venerium, mastitis, mastoiditis, meningitis citrate, sanguinarine Sulphate, carboplatin, cisplatin, hydro and infections of the nervous system, mycetoma, nocardiosis quinone, thioridazine hydrochloride, trifluoperazine hydro (e.g. Madura foot), non-specific urethritis, opthalmia (e.g. chloride, triflupromazine hydrochloride, chlorprothixene opthalmia neonatorum), osteomyelitis, otitis (e.g. otitis hydrochloride, perhexiline maleate, Suloctidil, nisoldipine, externa and otitis media), orchitis, pancreatitis, paronychia, quinacrine hydrochloride, acrisorcin, mitoxanthrone hydro 45 pelveoperitonitis, peritonitis, peritonitis with appendicitis, chloride, bleomycin, doxorubicin and vitamin B12 for the pharyngitis, phlegmons, pinta, plague, pleural effusion, treatment of a microbial infection; preferably for killing pneumonia, postoperative wound infections, postoperative multiplying, non-multiplying and/or clinically latent micro gas gangrene, prostatitis, pseudo-membranous colitis, psit organisms associated with a microbial infection. tacosis, pulmonary emphysema, pyelonephritis, pyoderma In an alternative embodiment of the invention, there is 50 (e.g. impetigo), Q fever, rat-bite fever, reticulosis, ricin provided the use of a compound selected from the group poisoning, Ritter's disease, salmonellosis, salpingitis, septic consisting of pyrvinium pamoate, pararosaniline pamoate, arthritis, septic infections, septicameia, sinusitis, skin infec bithionate Sodium, phenylmercuric acetate, tioconazole, tions (e.g. skin granulomas, impetigo, folliculitis and furun mefloquine, mitomycin C, toremifene citrate, sanguinarine culosis), syphilis, Systemic infections, tonsillitis, toxic shock Sulphate, carboplatin, cisplatin, hydroquinone, thioridazine 55 syndrome, trachoma, tularaemia, typhoid, typhus (e.g. epi hydrochloride, trifluoperazine hydrochloride, triflupromaz demic typhus, murine typhus, Scrub typhus and spotted ine hydrochloride, chlorprothixene hydrochloride, perhexi fever), urethritis, wound infections, yaws, aspergillosis, can line maleate, Suloctidil and nisoldipine for the treatment of didiasis (e.g. oropharyngeal candidiasis, vaginal candidiasis a microbial infection; preferably a bacterial infection; more or balanitis), cryptococcosis, favus, histoplasmosis, inter preferably an infection caused by S. Aureus, including 60 trigo, mucormycosis, tinea (e.g. tinea corporis, tinea capitis, MSSA and/or MRSA; in particular for killing stationary tinea cruris, tinea pedis and tinea unguium), onychomycosis, phase S. Aureus. pityriasis versicolor, ringworm and sporotrichosis; or infec In another embodiment of the invention, there is provided tions with MSSA, MRSA, Staph. epidermidis, Strept. aga the use of a compound selected from the group consisting of lactiae, Strept. pyogenes, Escherichia coli, Klebs. pneumo quinacrine hydrochloride, pararosaniline pamoate, phenyl 65 niae, Klebs. Oxytoca, Pr: mirabilis, Pr: rettgeri, Pr: vulgaris, mercuric acetate, acrisorcin, mefloquine, mitomycin C, Haemophilis influenzae, Enterococcus faecalis and Entero mitoxanthrone hydrochloride, bleomycin, doxorubicin, san COccus faecium. US 9,549,965 B2 13 14 It will be appreciated that references herein to “treatment” (3) Aminoglycosides. Such as amikacin, gentamicin, netilmi extend to prophylaxis as well as the treatment of established cin, neomycin, Streptomycin, tobramycin, amastatin, diseases or symptoms. butirosin, butirosin A, daunorubicin, dibekacin, dihy The above-mentioned classes of known biologically drostreptomycin, G 418, hygromycin B, kanamycin B, active compounds may be used alone or in combination for 5 kanamycin, kirromycin, paromomycin, ribostamycin, the treatment of microbial infections. They may also be used Sisomicin, spectinomycin, streptozocin and thiostrepton. in combination with known antimicrobial compounds. (4) (i) Macrollides, such as azithromycin, clarithromycin, In one embodiment of the invention there is provided a erythromycin, roXithromycin, spiramycin, amphotericins combination comprising at least one compound selected B (e.g. amphotericin B), bafilomycins (e.g. bafilomycin from the group consisting of an O-adrenergic antagonist, an 10 A1), brefeldins (e.g. brefeldin A), concanamycins (e.g. anthelmintic agent, an antifungal agent, an antimalarial concanamycin A), filipin complex, josamycin, mepartri agent, an antineoplastic agent, an antipsychotic agent, an cin, midecamycin, nonactin, nystatin, oleandomycin, oli antioxidant, a vasodilator, a vitamin, or a pharmaceutically gomycins (e.g. oligomycin A, oligomycin B and oligo acceptable derivative thereof, and an antimicrobial com mycin C), pimaricin, rifampicin, rifamycin, rosamicin, pound. Preferably said combination is used for killing mul 15 tylosin, Virginiamycin and fosfomycin. tiplying, non-multiplying and/or clinically latent microor (ii) Ketolides Such as tellithromycin and cethromycin ganisms associated with a microbial infection. (ABT-773). Suitable antimicrobial compounds for use in combination (iii) Lincosamines, such as lincomycin. in accordance with the present invention include one or (5) Clindamycin and clindamycin 2-phosphate. more compounds selected from the following: (6) Phenicols, such as chloramphenicol and thiamphenicol. (1) B-Lactams, including: (7) Steroids, such as fusidic acid (optionally in metal salt (i) penicillins, such as form, e.g. in Salt form with an alkali metal such as (I) benzylpenicillin, procaine benzylpenicillin, phe Sodium). noxy-methylpenicillin, methicillin, propicillin, epi (8) Glycopeptides such as Vancomycin, teicoplanin, bleo cillin, cyclacillin, hetacillin, 6-aminopenicillanic 25 mycin, phleomycin, ristomycin, telavancin, dalbavancin acid, penicillic acid, penicillanic acid Sulphone (Sul and oritavancin. bactam), penicillin G, penicillin V, phenethicillin, (9) Oxazolidinones, such as linezolid and AZD2563. phenoxymethylpenicillinic acid, azlocillin, carbeni (10) Streptogramins, such as quinupristin and dalfopristin, cillin, cloxacillin, D-(-)-penicillamine, dicloxacillin, or a combination thereof. nafcillin and oxacillin, 30 (11) (i) Peptides, such as polymyxins (e.g. polymyxin E (II) penicillinase-resistant penicillins (e.g. flucloxacil (colistin) and polymyxin B), lysostaphin, duramycin, lin), actinomycins (e.g. actinomycin C and actinomycin D). (III) broad-spectrum penicillins (e.g. amplicillin, actinonin, 7-aminoactinomycin D, antimycin A, antipain, amoxicillin, metampicillin and bacampicillin), bacitracin, cyclosporin A, echinomycin, gramicidins (e.g. (IV) antipseudomonal penicillins (e.g. carboxypenicil 35 gramicidin A and gramicidin C), myxothiazol, nisin, lins such as ticarcillin or ureidopenicillins such as paracelsin, valinomycin and viomycin. piperacillin), (ii) Lipopeptides, such as daptomycin. (V) mecillinams (e.g. pivmecillinam), or (iii) Lipoglycopeptides, such as ramoplanin. (VI) combinations of any two or more of the agents (12) Sulfonamides, such as Sulfamethoxazole, Sulfadiazine, mentioned at (I) to (V) above, or combinations of 40 Sulfaquinoxaline, Sulfathiazole (which latter two agents any of the agents mentioned at (I) to (V) above with are optionally in metal salt form, e.g. in salt form with an a B-lactamase inhibitor Such as taZobactam or, par alkali metal Such as Sodium), Succinylsulfathiazole, Sul ticularly, clavulanic acid (which acid is optionally in fadimethoxine, Sulfaguanidine, Sulfamethazine, Sulfa metal salt form, e.g. in Salt form with an alkali metal monomethoxine, Sulfanilamide and Sulfasalazine. Such as sodium or, particularly, potassium); 45 (13) Trimethoprim, optionally in combination with a sulfo (ii) cephalosporins, such as cefaclor, cefadroxil, cefalexin namide, Such as Sulfamethoxazole (e.g. the combination (cephalexin), cefcapene, cefcapene pivoxil, cefdinir, co-trimoxazole). cefditoren, cefditoren pivoxil, cefixime, cefotaxime, (14) Antituberculous drugs, such as isoniazid, rifampicin, cefpirome, cefpodoxime, cefpodoxime proxetil, cef rifabutin, pyrazinamide, ethambutol, Streptomycin, ami prozil, cefradine, ceftazidime, cefteram, cefteram piv 50 kacin, capreomycin, kanamycin, quinolones (e.g. those at oxil, ceftriaxone, cefuroxime, cefuroxime axetil, (q) below), para-aminosalicylic acid, cycloserine and cephaloridine, cephacetrile, cephamandole, cephalo ethionamide. glycine, ceftobiprole, PPI-0903 (TAK-599), 7-amin (15) Antileprotic drugs, such as dapsone, rifampicin and ocephalosporanic acid, 7-aminodes-acetoxycepha clofazimine. losporanic acid, cefamandole, cefazolin, cefimetazole, 55 (16) (i) Nitroimidazoles, such as metronidazole and tinida cefoperaZone, cefsulodin, cephalosporin C Zinc salt, Zole. cephalothin, cephapirin; and (ii) Nitrofurans, such as nitrofurantoin. (iii) other B-lactams, such as monobactams (e.g. aztreo (17) Quinolones, such as nalidixic acid, norfloxacin, cipro nam), carbapenems (e.g. imipenem (optionally in com floxacin, ofloxacin, levofloxacin, moxifloxacin, gatifloxa bination with a renal enzyme inhibitor Such as cilasta 60 cin, gemifloxacin, garenoxacin, DX-619, WCK 771 (the tin), meropenem, ertapenem, doripenem (S-4661) and arginine Salt of S-(-)-nadifloxacin), 8-quinolinol, cinoxa R04908463 (CS-023)), penems (e.g. faropenem) and cin, enrofloxacin, flumequine, lomefloxacin, oxolinic acid 1-Oxa-B-lactams (e.g. moxalactam). and pipemidic acid. (2) Tetracyclines, such as tetracycline, demeclocycline, (18) Amino acid derivatives, such as azaserine, bestatin, doxycycline, lymecycline, minocycline, oxytetracycline, 65 D-cycloserine, 1,10-phenanthroline, 6-diazo-5-oxo-L- chlortetracycline, meclocycline and methacycline, as well norleucine and L-alanyl-L-1-aminoethyl-phosphonic as glycylcyclines (e.g. tigecycline). acid. US 9,549,965 B2 15 16 (19) Aureolic acids, such as chromomycin A3, mithramycin These applications describe suitable methods for the prepa A and mitomycin C C. ration of such compounds and doses for their administration. (20) Benzochinoides, such as herbimycin A. Preferred examples of antimicrobial agents for use in (21) Coumarin-glycosides. Such as novobiocin. combination in accordance with the present invention (22) Diphenyl ether derivatives, such as irgasan. include a compound selected from the group consisting of (23) Epipolythiodixopiperazines, such as gliotoxin from 6,8-dimethoxy-4-methyl-1-(3-phenoxyphenyl)-2,3-dihydro Gliocladium fimbriatum. 1H-pyrrolo3.2-c-quinoline; (24) Fatty acid derivatives, such as cerulenin. 6,8-dimethoxy-4-methyl-1-(2-phenoxyethyl)-2,3-dihydro (25) Glucosamines, such as 1-deoxymannojirimycin, 1-de 1H-pyrrolo3.2-c-quinoline; 10 1-cyclopropyl-6,8-dimethoxy-4-methyl-2,3-dihydro-1H Oxynojirimycin and N-methyl-1-deoxynoirimycin. pyrrolo3.2-c-quinoline; (26) Indole derivatives, such as staurosporine. 8-methoxy-4-methyl-1-(4-phenoxyphenyl)-2,3-dihydro (27) Diaminopyrimidines, such as iclaprim (AR-100). 1H-pyrrolo3.2-c-quinoline; (28) Macrolactams. Such as ascomycin. {2-4-(8-methoxy-4-methyl-2,3-dihydro-1H-pyrrolo[3.2-c. (29) Taxoids, such as paclitaxel. 15 quinolin-1-yl)-phenyoxyethyl dimethylamine; (30) Statins, such as mevastatin. 8-methoxy-4-methyl-1-4-(pyridin-3-yloxy)phenyl-2,3-di (31) Polyphenolic acids, Such as (+)-uSnic acid. hydro-1H-pyrrolo3.2-cquinoline: (32) Polyethers, such as lasalocid A, lonomycin A, 4-methyl-8-phenoxy-1-phenyl-2,3-dihydro-1H-pyrrolo3.2- monensin, nigericin and Salinomycin. cquinoline; (33) Picolinic acid derivatives, such as fusaric acid. 1-benzyl-4-methyl-8-phenoxy-2,3-dihydro-1H-pyrrolo3.2- (34) Peptidyl nucleosides, such as blasticidine S, nikkomy cquinoline cin, nourseothricin and puromycin. 1-(indan-2-yl)-4-methyl-8-phenoxy-2,3-dihydro-1H-pyr (35) Nucleosides, such as adenine 9-B-D-arabinofuranoside, rolo3.2-cquinoline 5-azacytidine, cordycepin, formycin A, tubercidin and 4-methyl-6-phenoxy-1-phenyl-2,3-dihydro-1H-pyrrolo3.2- tunicamycin. 25 cquinoline; (36) Pleuromutilins, such as GSK-565154, GSK-275833 1-benzyl-4-methyl-6-phenoxy-2,3-dihydro-1H-pyrrolo3.2- and tiamulin. cquinoline; (37) Peptide deformylase inhibitors, such as LBM415 (NVP 1-(indan-2-yl)-4-methyl-6-phenoxy-2,3-dihydro-1H-pyr PDF-713) and BB 83698. rolo3.2-cquinoline; (38) Antibacterial agents for the skin, such as fucidin, 30 4-methyl-1-(2-phenylethyl)-8-phenoxy-2,3-dihydro-1H benZamycin, clindamycin, erythromycin, tetracycline, sil pyrrolo3.2-c-quinoline; ver sulfadiazine, chlortetracycline, metronidazole, mupi 8-methoxy-4-methyl-1-(2-phenylethyl)-2,3-dihydro-1H rocin, framycitin, gramicidin, neomycin Sulfate, poly pyrrolo3.2-c-quinolin-6-ol; myxins (e.g. polymixin B) and gentamycin. 1-(1-benzyl-piperidin-4-yl)-4-methyl-8-phenoxy-2,3-di (39) Miscellaneous agents, such as methenamine (hexam 35 hydro-1H-pyrrolo3.2-cquinoline: ine), doxorubicin, piericidin A, Stigmatellin, actidione, 1-(indan-1-yl)-4-methyl-8-phenoxy-2,3-dihydro-1H-pyr anisomycin, apramycin, coumermycin A1, L(+)-lactic rolo3.2-cquinoline; acid, cytochalasins (e.g. cytochalasin B and cytochalasin 1-(benzodioxan-2-ylmethyl)-4-methyl-8-phenoxy-2,3-di D), emetine and ionomycin. hydro-1H-pyrrolo3.2-cquinoline: (40) Antiseptic agents, such as chlorhexidine, phenol deriva 40 4-methyl-8-phenoxy-1-(1,2,3,4-tetrahydronaphthalen-1-yl)- tives (e.g. thymol and triclosan), quarternary ammonium 2,3-dihydro-1H-pyrrolo3.2-cquinoline: compounds (e.g. benzalkonium chloride, cetylpyridinium 1-cyclohexyl-4-methyl-8-phenoxy-2,3-dihydro-1H-pyrrolo chloride, benzethonium chloride, cetrimonium bromide, 3.2-cquinoline; cetrimonium chloride and cetrimonium Stearate), octeni 8-ethoxy-4-methyl-1-(4-phenoxyphenyl)-2,3-dihydro-1H dine dihydrochloride, and terpenes (e.g. terpinen-4-ol). 45 pyrrolo3.2-c-quinoline; A preferred combination of the present invention com 1-(4-methoxyphenyl)-4-methyl-8-phenoxy-2,3-dihydro prises polymyxin E (colistin) and phenoxybenzamine or a 1H-pyrrolo3.2-c-quinoline; pharmaceutically acceptable derivative thereof. Such as phe 4-methyl-1-(4-phenoxyphenyl)-2,3-dihydro-1H-pyrrolo3. noxybenzamine hydrochloride. 2-cquinoline; Preferred antimicrobial compounds for use in combina 50 4-methyl-1-(2-methylphenyl)methyl-8-phenoxy-2,3-di tion in accordance with the present invention are those hydro-1H-pyrrolo3.2-cquinoline: capable of killing clinically latent microorganisms. Methods 4-methyl-8-phenoxy-1-(4-iso-propylphenyl)-2,3-dihydro for determining activity against clinically latent bacteria 1H-pyrrolo3.2-c-quinoline; include a determination, under conditions known to those 4-methyl-8-phenoxy-1-(1-phenylethyl)-2,3-dihydro-1H skilled in the art (such as those described in Nature Reviews, 55 pyrrolo3.2-c-quinoline; Drug Discovery 1, 895-910 (2002), the disclosures of which 8-methoxy-4-methyl-1-(2-phenylethyl)-2,3-dihydro-1H are hereby incorporated by reference), of Minimum Station pyrrolo3.2-c-quinoline; ary-cidal Concentration (“MSC) or Minimum Dormicidal 6,8-dimethoxy-1-(4-hydroxyphenyl)-4-methyl-2,3-dihydro Concentration (“MDC) for a test compound. A suitable 1H-pyrrolo3.2-cquinoline; compound screening method against clinically latent micro 60 6,8-dimethoxy-1-(3-hydroxyphenyl)-4-methyl-2,3-dihydro organisms is described in WO2000028074, the contents of 1H-pyrrolo3.2-cquinoline; which are incorporated herein by reference as if the publi 6,8-dimethoxy-1-(3-hydroxy-5-methylphenyl)-4-methyl-2, cation was specifically and fully set forth herein. 3-dihydro-1H-pyrrolo3.2-cquinoline; Examples of compounds capable of killing clinically 8-methoxy-1-(4-methoxyphenyl)-4-methyl-2,3-dihydro latent microorganisms include those compounds disclosed 65 1H-pyrrolo3.2-c-quinoline; in International Patent Application, Publication Numbers 8-trifluoromethoxy-1-(4-phenoxyphenyl)-4-methyl-2,3-di WO2007054693, WO2008117079 and WO2008142384. hydro-1H-pyrrolo3.2-cquinoline:

US 9,549,965 B2 21 22 agents: C-adrenergic antagonist, an anthelmintic agent, an as from 0.5 to 95%) by weight of the total mixture; conve antifungal agent, an antimalarial agent, an antineoplastic niently from 30 to 95% for tablets and capsules and 0.01 to agent, an antipsychotic agent, an antioxidant, a vasodilator 50% (such as from 3 to 50%) for liquid preparations. and/or a vitamin, or a pharmaceutically acceptable deriva Formulations suitable for oral administration may be tive thereof, and optionally an antimicrobial agent, as a presented as discrete units such as capsules, cachets or combined preparation for simultaneous, separate or sequen tablets (e.g. chewable tablets in particular for paediatric tial use in the treatment of a microbial infection. administration), each containing a predetermined amount of As used herein the term “pharmaceutically acceptable active ingredient; as powder or granules; as a solution or derivative” means: Suspension in an aqueous liquid or non-aqueous liquid; or as (a) pharmaceutically acceptable salts with either acids or 10 an oil-in-water liquid emulsion or water-in-oil liquid emul bases (e.g. acid addition salts); and/or Sion. The active ingredients may also be presented a bolus, (b) solvates (including hydrates). electuary or paste. Acid addition salts that may be mentioned include car A tablet may be made by compression or moulding, boxylate salts (e.g. formate, acetate, trifluoroacetate, propi optionally with one or more excipients. Compressed tablets onate, isobutyrate, heptanoate, decanoate, caprate, caprylate, 15 may be prepared by compressing in a Suitable machine the Stearate, acrylate, caproate, propiolate, ascorbate, citrate, active ingredient in a free-flowing form Such as a powder or glucuronate, glutamate, glycolate, C-hydroxybutyrate, lac granules, optionally mixed with other conventional excipi tate, tartrate, phenylacetate, mandelate, phenylpropionate, ents Such as binding agents (e.g. syrup, acacia, gelatin, phenylbutyrate, benzoate, chlorobenzoate, methylbenzoate, Sorbitol, tragacanth, mucilage of starch, polyvinylpyrroli hydroxybenzoate, methoxybenzoate, dinitrobenzoate, o-ac done and/or hydroxymethyl cellulose), fillers (e.g. lactose, etoxybenzoate, salicylate, nicotinate, isonicotinate, cinna Sugar, microcrystalline cellulose, maize-starch, calcium mate, oxalate, malonate. Succinate, Suberate, sebacate, phosphate and/or Sorbitol), lubricants (e.g. magnesium Stear fumarate, malate, maleate, hydroxymaleate, hippurate, ate, Stearic acid, talc, polyethylene glycol and/or silica), phthalate or terephthalate salts), halide salts (e.g. chloride, disintegrants (e.g. potato starch, croScarmellose sodium and/ bromide or iodide salts), Sulfonate salts (e.g. benzenesul 25 or sodium starch glycolate) and wetting agents (e.g. sodium fonate, methyl-, bromo- or chloro-benzenesulfonate, Xyle lauryl sulphate). Moulded tablets may be made by moulding nesulfonate, methanesulfonate, ethanesulfonate, propane in a suitable machine a mixture of the powdered active Sulfonate, hydroxyethanesulfonate, 1- or 2-naphthalene ingredient with an inert liquid diluent. The tablets may be sulfonate or 1.5-naphthalenedisulfonate salts) or sulfate, optionally coated or scored and may be formulated so as to pyrosulfate, bisulfate, Sulfite, bisulfite, phosphate, monohy 30 provide controlled release (e.g. delayed, Sustained, or pulsed drogenphosphate, dihydrogenphosphate, metaphosphate, release, or a combination of immediate release and con pyrophosphate or nitrate salts, and the like. trolled release) of the active ingredients. Compounds for use according to the invention may be Alternatively, the active ingredients may be incorporated administered as the raw material but the active ingredients into oral liquid preparations such as aqueous or oily Sus are preferably provided in the form of pharmaceutical com 35 pensions, Solutions, emulsions, syrups or elixirs. Formula positions. tions containing the active ingredients may also be presented The active ingredients may be used either as separate as a dry product for constitution with water or another formulations or as a single combined formulation. When suitable vehicle before use. Such liquid preparations may combined in the same formulation it will be appreciated that contain conventional additives such as Suspending agents the two compounds must be stable and compatible with each 40 (e.g. Sorbitol syrup, methyl cellulose, glucose/sugar syrup, other and the other components of the formulation. gelatin, hydroxymethyl cellulose, carboxymethyl cellulose, Formulations of the invention include those suitable for aluminium Stearate gel and/or hydrogenated edible fats), oral, parenteral (including Subcutaneous e.g. by injection or emulsifying agents (e.g. lecithin, Sorbitan mono-oleate and/ by depot tablet, intradermal, intrathecal, intramuscular e.g. or acacia), non-aqueous vehicles (e.g. edible oils, such as by depot and intravenous), rectal and topical (including 45 almond oil, fractionated coconut oil, oily esters, propylene dermal, buccal and Sublingual) or in a form Suitable for glycol and/or ethyl alcohol), and preservatives (e.g. methyl administration by inhalation or insufflation administration. or propyl p-hydroxybenzoates and/or Sorbic acid). The most Suitable route of administration may depend upon Topical compositions, which are useful for treating dis the condition and disorder of the patient. orders of the skin or of membranes accessible by digitation Preferably, the compositions of the invention are formu 50 (such as membrane of the mouth, vagina, cervix, anus and lated for oral or topical administration. rectum), include creams, ointments, lotions, sprays, gels and The formulations may conveniently be presented in unit sterile aqueous Solutions or Suspensions. As such, topical dosage form and may be prepared by any of the methods compositions include those in which the active ingredients well known in the art of pharmacy e.g. as described in are dissolved or dispersed in a dermatological vehicle “Remington. The Science and Practice of Pharmacy”. Lip 55 known in the art (e.g. aqueous or non-aqueous gels, oint pincott Williams and Wilkins, 21 Edition, (2005). Suitable ments, water-in-oil or oil-in-water emulsions). Constituents methods include the step of bringing into association to of Such vehicles may comprise water, aqueous buffer solu active ingredients with a carrier which constitutes one or tions, non-aqueous solvents (such as ethanol, isopropanol, more excipients. In general, formulations are prepared by benzyl alcohol. 2-(2-ethoxyethoxy)ethanol, propylene gly uniformly and intimately bringing into association the active 60 col, propylene glycol monolaurate, glycofurol or glycerol), ingredients with liquid carriers or finely divided solid car oils (e.g. a mineral oil such as a liquid paraffin, natural or riers or both and then, if necessary, shaping the product into synthetic triglycerides such as MiglyolTM, or silicone oils the desired formulation. It will be appreciated that when the Such as dimethicone). Depending, interalia, upon the nature two active ingredients are administered independently, each of the formulation as well as its intended use and site of may be administered by a different means. 65 application, the dermatological vehicle employed may con When formulated with excipients, the active ingredients tain one or more components selected from the following may be present in a concentration from 0.1 to 99.5% (such list: a solubilising agent or solvent (e.g. a B-cyclodextrin, US 9,549,965 B2 23 24 Such as hydroxypropyl B-cyclodextrin, or an alcohol or such as a blister pack. Where the compositions are intended polyol such as ethanol, propylene glycol or glycerol); a for administration as two separate compositions these may thickening agent (e.g. hydroxymethyl cellulose, hydroxy be presented in the form of a twin pack. propyl cellulose, carboxymethyl cellulose or carbomer); a Pharmaceutical compositions may also be prescribed to gelling agent (e.g. a polyoxyethylene-polyoxypropylene the patient in “patient packs' containing the whole course of copolymer); a preservative (e.g. benzyl alcohol, benzalko treatment in a single package, usually ablister pack. Patient nium chloride, chlorhexidine, chlorbutol, a benzoate, potas packs have an advantage over traditional prescriptions, sium sorbate or EDTA or salt thereof); and pH buffering where a pharmacist divides a patients Supply of a pharma agent(s) (e.g. a mixture of dihydrogen phosphate and hydro ceutical from a bulk Supply, in that the patient always has gen phosphate salts, or a mixture of citric acid and a 10 access to the package insert contained in the patient pack, hydrogen phosphate salt). Topical formulations may also be normally missing in traditional prescriptions. The inclusion formulated as a transdermal patch. of the package insert has been shown to improve patient Methods of producing topical pharmaceutical composi compliance with the physicians instructions. tions such as creams, ointments, lotions, sprays and sterile The administration of the combination of the invention by aqueous solutions or Suspensions are well known in the art. 15 means of a single patient pack, or patients packs of each Suitable methods of preparing topical pharmaceutical com composition, including a package insert directing the patient positions are described, e.g. in WO9510999, U.S. Pat. No. to the correct use of the invention is a desirable feature of 6,974,585, WO2006048747, as well as in documents cited in this invention. any of these references. According to a further embodiment of the present inven Topical pharmaceutical compositions according to the tion there is provided a patient pack comprising at least one present invention may be used to treat a variety of skin or compound selected from the group consisting of an membrane disorders, such as infections of the skin or C.-adrenergic antagonist, an anthelmintic agent, an antifun membranes (e.g. infections of nasal membranes, axilla, gal agent, an antimalarial agent, an antineoplastic agent, an groin, perineum, rectum, dermatitic skin, skin ulcers, and antipsychotic agent, an antioxidant, a vasodilator and/or a sites of insertion of medical equipment Such as i.v. needles, 25 Vitamin, or a pharmaceutically acceptable derivative thereof, catheters and tracheostomy or feeding tubes) with any of the and optionally an antimicrobial agent, and an information bacteria, fungi described above, (e.g. any of the Staphyllo insert containing directions on the use of the invention. cocci, Streptococci, Mycobacteria or Pseudomonas organ The amount of active ingredients required for use in isms mentioned hereinbefore. Such as S. aureus (e.g. Methi treatment will vary with the nature of the condition being cillin resistant S. aureus (MRSA))). 30 treated and the age and condition of the patient, and will Particular bacterial conditions that may be treated by ultimately be at the discretion of the attendant physician or topical pharmaceutical compositions of the present inven veterinarian. In general however, doses employed for adult tion also include the skin- and membrane-related conditions human treatment will typically be in the range of 0.02 to disclosed hereinbefore, as well as: acne Vulgaris; rosacea 5000 mg per day, preferably 1 to 1500 mg per day. The (including erythematotelangiectatic rosacea, papulopustular 35 desired dose may conveniently be presented in a single dose rosacea, phymatous rosacea and ocular rosacea), erysipelas; or as divided doses administered at appropriate intervals, erythrasma; ecthyma, ecthyma gangrenosum, impetigo; e.g. as two, three, four or more sub-does per day. paronychia; cellulitis; folliculitis (including hot tub follicu Biological Tests litis); furunculosis; carbunculosis; staphylococcal scalded Test procedures that may be employed to determine the skin syndrome; Surgical Scarlet fever, Streptococcal peri-anal 40 biological (e.g. bactericidal or antimicrobial) activity of the disease; streptococcal toxic shock syndrome; pitted kera active ingredients include those known to persons skilled in tolysis; trichomycosis axillaris; pyoderma; external canal the art for determining: ear infections; green nail syndrome; spirochetes; necrotizing (a) bactericidal activity against clinically latent bacteria; fasciitis; Mycobacterial skin infections (such as lupus Vul and garis, scrofuloderma, warty tuberculosis, tuberculides, ery 45 (b) antimicrobial activity against log phase bacteria. thema nodosum, erythema induratum, cutaneous manifesta In relation to (a) above, methods for determining activity tions of tuberculoid leprosy or lepromatous leprosy, against clinically latent bacteria include a determination, erythema nodosum leprosum, cutaneous M. kansasii, M. under conditions known to those skilled in the art (such as malmoense, M. Szulgai, M. Simiae, M. gordonae, M. hae those described in Nature Reviews, Drug Discovery 1, mophilum, M. avium, M. intracellulare, M. chelonae (in 50 895-910 (2002), the disclosures of which are hereby incor cluding M. abscessus) or M. fortuitum infections, Swimming porated by reference), of Minimum Stationary-cidal Con pool (or fish tank) granuloma, lymphadenitis and Buruli centration (“MSC) or Minimum Dormicidal Concentration ulcer (Bairnsdale ulcer, Searles’ ulcer, Kakerifu ulcer or (“MDC) for a test compound. Toro ulcer)); as well as infected eczema, burns, abrasions By way of example, WO2000028074 describes a suitable and skin wounds. 55 method of screening compounds to determine their ability to Particular fungal conditions that may be treated by topical kill clinically latent microorganisms. A typical method may pharmaceutical compositions of the present invention also include the following steps: include include the skin- and membrane-related conditions (1) growing a bacterial culture to stationary phase; disclosed hereinbefore, as well as: candidiasis; sporotricho (2) treating the stationary phase culture with one or more sis; ringworm (e.g. tinea pedis, tinea cruris, tinea capitis, 60 antimicrobial agents at a concentration and or time tinea unguium or tinea corporis); tinea versicolor, and Sufficient to kill growing bacteria, thereby selecting a infections with Trichophyton, Microsporum, Epidermophy phenotypically resistant Sub-population; ton or Pityrosporum ovale fungi. (3) incubating a sample of the phenotypically resistant Compositions for use according to the invention may be Subpopulation with one or more test compounds or presented in a pack or dispenser device which may contain 65 agents; and one or more unit dosage forms containing the active ingre (4) assessing any antimicrobial effects against the pheno dients. The pack may, e.g. comprise metal or plastic foil, typically resistant Subpopulation. US 9,549,965 B2 25 26 According to this method, the phenotypically resistant counts. The persisters were then used in measurements of Sub-population may be seen as representative of clinically bactericidal activity for test compounds. latent bacteria which remain metabolically active in vivo Different concentrations of each test compound were and which can result in relapse or onset of disease. incubated with the (persister) cell suspension in 96 well In relation to (b) above, methods for determining activity plates for various periods of time (24 and 48 hours). Bac against log phase bacteria include a determination, under tericidal activity was then determined by taking CFU counts standard conditions (i.e. conditions known to those skilled in of the resulting cultures, as described above. the art, such as those described in WO 2005014585, the disclosures of which document are hereby incorporated by EXAMPLES reference), of Minimum Inhibitory Concentration (“MIC) 10 or Minimum Bactericidal Concentration ('MBC') for a test Example 1 compound. Specific examples of Such methods are described below. Methods Bactericidal Activities of Compounds Against Bacterial Strains 15 Stationary Phase S. aureus Staphylococcus aureus (Oxford Strain); Gram positive; The results obtained are summarised in Table 1 below. Reference strain. Escherichia coli K12; Gram negative; Reference strain. Pseudomonas aeruginosa NCTC 6751; Gram negative: TABLE 1. Reference strain. Log Kill Ig/ml Growth of Bacteria Bacteria were grown in 10 ml of nutrient broth (No. 2 Compound 25 12.5 6.25 (Oxoid)) overnight at 37°C., with continuous shaking at 120 Pyrwinium panoate 6.03 6.03 6.03 rpm. The overnight cultures were diluted (1000x) in 100 ml Pararosaniline pamoate 6.15 6.15 6.15 of growth medium and then incubated without shaking for 25 Bithionate sodium 6.15 6.15 O.O1 Phenylmercuric acetate 6.03 6.03 6.03 10 days. Viability of the bacteria was estimated by colony Tioconazole 6.15 6.15 O.OO forming unit (CFU) counts at 2 hour intervals at the first 24 Mefloquine 6.03 6.03 -0.01 hours and at 12-24 hours afterwards. From serial 10-fold Mitomycin C 6.15 6.15 6.15 dilutions of the experimental cultures, 100 ul samples were Toremifene citrate 6.15 6.15 6.15 30 Sanguinarine Sulfate 6.15 6.15 6.15 added to triplicate plates of nutrient agar plates (Oxoid) and Carboplatin 6.03 6.03 O.92 blood agar plates (Oxoid). Colony forming units (CFU) Cisplatin 6.03 6.03 6.03 were counted after incubation of the plates at 37° C. for 24 Hydroquinone 6.15 6.15 6.15 hours. Thioridazine hydrochloride 6.03 6.03 6.03 Trifluoperazine hydrochloride 6.03 2.22 O.OO Log Phase Cultures Triflupromazine hydrochloride 6.15 6.15 6.15 The above-described overnight cultures were diluted 35 Chlorprothixene hydrochloride 6.15 6.15 1.83 (1000x) with iso-sensitest broth. The cultures were then Perhexiline maleate 6.15 6.15 6.15 incubated at 37° C. with shaking for 1-2 hours to reach log Sulocticil 6.03 6.03 6.03 CFU 6, which served as log phase cultures. Nisoldipine 6.15 6.15 O.OO Stationary Phase Cultures Cultures incubated for more than 24 hours are in the 40 Conclusions stationary phase. For drug screening, 5-6 day old stationary All compounds exhibited significant bactericidal activi phase cultures were used. ties against stationary phase S. aureus. There were 6 log of Measurements of Bactericidal Activity Against Log Phase bacteria incubated with the compounds. Complete kill was Cultures observed at 25, 2.5 g/ml, and in Some cases at 6.25 ug/ml. The bactericidal activity of the drugs against log phase 45 cultures were determined by minimal inhibitory concentra Example 2 tion (MIC) of the drugs, which is defined as the lowest concentration which inhibits visible growth. Different con Bactericidal Activities of Compounds Against Loci centrations of each test compound were incubated with the Phase and Stationary Phase E. coli log-phase cultures in 96 well plates for 24 hours. Bacteri 50 cidal activity was then examined by taking a spectropho The results obtained are summarised in Tables 2 and 3 tometer reading (using a plate reader) at 405 nm. below. Measurements of Bactericidal Activity Against Stationary Phase Cultures TABLE 2 Different concentrations of each test compound were 55 incubated with stationary phase cultures (5-6 day cultures) Log kill Ig/ml in 96 well plates for 24 or 48 hours. Bactericidal activity was Compound 25.00 12.SO 6.25 then determined by taking CFU counts of the resulting cultures, as described above. Quinacrine hydrochloride 6.14 2.14 1.27 Pararosaniline pamoate 6.14 6.14 1.90 Measurements of Bactericidal Activity Against Persistent 60 Phenylmercuric acetate 6.14 6.14 6.14 (Clinically Latent) Bacteria Acrisorcin 6.14 6.14 6.14 An antibiotic (gentamicin) was added to 5-6 day station Mefloquine 6.14 6.14 2.48 Mitomycin C 6.14 6.14 2.27 ary-phase cultures to the final concentration of 50 to 100 Mitoxanthrone hydrochloride 6.14 6.14 6.14 ug/ml for 24 hours. After 24 hours of antibiotic treatment, Bleomycin 6.14 6.14 2.22 the cells are washed 3 times with phosphate buffered saline 65 Doxorubicin 6.14 6.14 6.14 (PBS), and then resuspended in PBS. The surviving bacterial Sanguinarine Sulfate 6.14 2.40 -0.10 cells are used as persisters. Viability is estimated by CFU US 9,549,965 B2

TABLE 2-continued organism. There was about 1.5 log reduction of the bacte rium at 40 g/ml. Suloctidil displayed no observable activity Log kill Lig/ml against stationary phase P. aeruginosa. Compound 25.00 12.SO 6.25 5 Carboplatin 6.14 6.14 6.14 Example 4 Cisplatin 6.14 6.14 6.14 Hydroquinone 6.14 6.14 6.14 Bactericidal Activities of Compounds Against Thioridazine hydrochloride 6.14 6.14 6.14 Persistent E Ii Trifluoperazine hydrochloride 6.14 6.14 -0.12 CS1S. COll Triflupromazine hydrochloride 6.14 6.14 2.10 10 The results obtained are summarised in Table 4 below.

TABLE 3 TABLE 4

E. coi 15 Log Kill g/ml Compound MIC Lig/ml Quinacrine hydrochloride Compounds 25 12.5 6.25 Pararosaniline pamoate 10 Bithionate sodium 5 Bithionate sodium 6.2 O.O3 O.O1 Phenylmercuric acetate O.15 2O Bleomycin 6.2 6.21 1.55

SRNAeloquine 10 Carboplatin 6.2 2.55 1.55 Mitomycin C O.3 Chlorprothixene hydrochloride 6.2 O.O3 O.O2 Mitoxanthrone hydrochloride Cisplatin 6.2 6.21 6.21 Bleomycin O.15 Vitamin B12 6.2 6.21 O.O3 Doxorubicin 25 Mefloquine 6.2 O.O3 O.O2 Sanguinarine sulfate Mitomycin C 6.2 6.21 6.21 Carboplatin Cisplatin Mitoxanthrone hydrochloride 6.2 6.21 6.21 Hydroquinone Pararosaniline pamoate 6.2 O.O1 -0.01 Thioridazine hydrochloride 10 Perhexiline maleate 6.2 O.04 -0.01 Trifluoperazine hydrochloride 2O Phenylmercuric acetate 6.2 6.21 6.21 Triflupromazine hydrochloride 10 30 f Chlorprothixene hydrochloride 2O Sanguinarine Sulfate 6.2 1.47 O.04 Perhexiline maleate 10 Sulocticil 6.2 O.O2 O.O2 Suloctidil 10 Thioridazine hydrochloride 6.2 O.O3 O.04 Nisoldipine Triflupromazine hydrochloride 6.2 2.34 O.O3 Vitamin B12 35 Key: — signifies MIC higher than 50 ugml Conclusions Conclusions All compounds exhibited significant bactericidal activi- All compounds exhibited significant bactericidal activi ties against stationary phase E. coli. There were 6 log of ties against persistent E. coli selected by treatment with bacteria incubated with the compounds. Complete kill was 40 getamicin. There were 6 log of bacteria, incubated with the observed at 25, 12.5 Lig/ml, and and in Some cases 6.25 compounds. Complete kill was observed at 25 ug/ml and in ug/ml. Some cases at 12.5 and 6.25 g/ml. Certain compounds were also were active against log phase E. coli showing MIC between 0.15 to 20 g/ml. Example 5 45 Example 3 Bactericidal Activity of Suloctidil and Mefloquine In vitro Activity of of Phenoxybenzamine Against Stationary Phase P. aeruginosa Hydrochloride (HTO0800157) with Polymyxin E The results obtained are summarised in FIG. 1. 50 Against Log Phased by Chequerboard Conclusions aly S1S Mefloquine was active against stationary phase P. aerugi nosa. At 80 g/ml, mefloquine removed more than 107 of the The results obtained are summarised in Table 5 below. TABLE 5

POLYMYXINE Ig/ml

2 1 O.S. O.25 O.125 0.0625 O.O3125 OO15625 O.OO78 O.OO39 O.OO19 O

HTOO8OO157 64 O.48 0.48 O.47 O.47 0.46 O.47 O48 1.45 1.44 1.47 1.57 160 |g/ml 32 O.SO OSO O.S1 O.S1 O.S1 1.27 1.52 1.52 1.53 1.51 1.49 1.57 16 O.S1 O.S2 O.S2 O.S2 O.S2 1.48 1.55 1.57 1.55 1.55 1.51 1.56 8 O.SO O.S1 O.S1 O.S2 1.51 1.54 1.57 1.56 1.58 1.55 1.57 1.56 4 O.S1 O.S2 O.S2 O.S2 1.48 1.57 1.60 1.60 1.60 1.56 1.61 160 2 O.49 OSO 0.49 O.49 125 1.46 1.45 1.57 1.49 148 1.45 1.53 1 O.SO O.S1 O.S1 O.SO 1.60 1.54 1.53 1.52 1.56 1.52 1.54 1.52 O OSO 0.49 0.49 125 148 1.52 1.42 1.53 1.55 1.55 1.53 1.53 US 9,549,965 B2 29 30 Conclusions 2. Colistin MIC reduced from 32 mg/L to 0.5 mg/L when 1. MIC for phenoxybenzamine hydrochloride alone was combined with 64 mg/L of mefloquine. >64 Lug/ml. 2. In combination with polymyxin E at 0.06 ug/ml, MIC for phenoxybenzamine hydrochloride was 64 ug/ml. 5 Time Kill Curves 3. In combination with polymyxin E at 0.25 g/ml, the Another accepted test for ascertaining the presence or MIC for phenoxybenzamine hydrochloride was reduced to 1 g/ml. absence of synergy is to use time-kill methods where the dynamic effect of a drug combination is compared to each Example 6 10 drug alone when assessing the effect on bacterial log or In vitro Activity of Mefloquine with Polymyxin E stationary-growth over time. Again, the possible results are (Colistin) NDM-1 Klebsiella pneumoniae for synergistic, additive or antagonistic effects. The following time kill curves, FIGS. 2-7, demonstrate Chequerboard Analysis 15 The results obtained from chequerboard analysis are the synergy observed between colistin and mefloquine summarised in Table 6 below. against NDM-1 Klebsiella pneumoniae. TABLE 6 Mefloquine

2S6 128 64 32 16 8 4 2 1 O.S. O.25 O

Colistin 32 0.35 0.33 0.34 0.33 0.33 0.33 0.34 0.33 O.33 O.34 0.34 0.33 16 O.33 O.33 O.34 0.34 0.33 O.33 O.35 0.34 O.67 0.68 0.68 0.78 8 O.33 O.34 0.34 0.34 O.34 0.34 0.34 O.61 O44 O.35 0.45 0.82 4 O.34 O.34 0.33 O.34 O.34 0.35 O.69 O.62 O.74 O.74 O.81 0.34 2 O.34 0.33 O.34 0.34 0.35 0.68 0.82 0.92 O.92 O.91 O.94 1.05 1 O.33 O.34 0.34 0.35 0.72 0.97 0.98 0.96 O.95 0.97 O.9S 1.03 O5 O.34 0.33 O.34 0.67 1.OO 104 1.OS 1.OO 1.04 1.08 1.06 1.08 O O.34 0.33 O.6O O.82 110 1.15 1.09 1.09 1.10 1.08 110 1.19

Synergy in the context of antimicrobials drugs is mea Example 7 Sured 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 35 In vitro Activity of Suloctidil and Colistin Against been observed is to use the “chequerboard” technique which NDM-1 Klebsiella pneumoniae leads to the generation of a value called the fractional inhibitory concentration index (FICI). Chequerboard Analysis Orhan et al J. Clin. Microbiol. 2005, 43(1):140 describes The results obtained from chequerboard analysis are the chequerboard method and analysis in the paragraph summarised in Table 7 below.

TABLE 7

Sulocticil

256 128 64 32 16 8 4 2 1 O.S 0.25 O

Colistin 32 O.39 O.38 0.36 O.36 0.35 0.36 O.36 0.36 0.36 O.35 0.36 0.36 16 O.39 O.38 0.37 O.36 0.36 0.36 O.36 0.36 O.S3 O.36 0.46 0.53 8 O42 O.38 0.36 O.35 0.36 0.36 O.36 0.37 O.S4 O.41 O.S1 O.44 4 O42 O.36 0.37 O.36 0.36 0.36 O.36 0.49 0.68 0.66 0.78 O.87 2 O41 O.37 0.37 O.36 0.36 0.63 O.62 0.67 O.80 O.80 O.78 O.95 1 0.37 O.37 0.36 O.37 O.S.S. O.63 O.74 0.92 0.86 O.84 0.83 1.02 0.5 0.37 O.36 0.37 O.S3 O.S7 O.68 O.81 0.85 0.89 O.82 0.95 0.98 O O.61 O.44 0.38 0.60 (0.6O O.7O O.94 0.93 0.98 1.03 0.97 1.03 bridging pages 140-141, and explains that the FICI value is Conclusion: a ratio of the sum of the MIC (Minimum Inhibitory Con 1. FIC index=0.094 showing synergy between Suloctidil and centration) level of each individual component alone and in 60 colistin. the mixture. The combination is considered synergistic when the XFIC 2. Colistin MIC reduced from 16 mg/L to 1 mg/L when is <0.5, indifferent when the XFIC is >0.5 to <2, and combined with 32 mg/L of Suloctidil. antagonistic when the XFIC is >2. Time Kill Curves Conclusions 65 The following time kill curves, FIGS. 8-11, demonstrate 1. FIC index=0.031 showing synergy between mefloquine the synergy observed between colistin and Suloctidil against and colistin. NDM-1 Klebsiella pneumoniae. US 9,549,965 B2 31 Example 8 In vitro Activity of Thioridazine Hydrochloride and Colistin Against NDM-1 Klebsiella pneumoniae Chequerboard Analysis The results obtained from chequerboard analysis are summarised in Table 8. TABLE 8 Thioridazine hydrochloride

2S6 128 64 32 16 8 4 2 1 O.S. O.25 O

Colistin 32 OSS O-39 O.39 O.39 O.39 O.39 O.39 O.39 O.39 O.49 0.42 0.41 16 O.S4 O.39 O.39 O.39 O.39 O.39 O.39 O.39 O.39 O.39 O.39 O.39 8 O.S2 O.39 O.39 O.38 OSO 0.48 0.48 0.48 0.48 0.52 O.S7 O.S7 4 O.S3 O.39 O.39 O.40 0.46 0.46 0.41 OSS O-47 O.S4 OSS O.S4 2 O.S2 O.39 O.39 O.46 O.S2 O.62 0.81 0.77 O.80 O.80 O.73 0.87 1 O.S3 O.4O O.39 O.6O O.77 O.73 0.74 0.86 O.80 O.77 0.82 0.94 O.S. O.S2 0.46 0.6O O. 68 0.78 O.80 O.80 1.34 0.81 0.77 O.80 O.94 O 141 0.76 O.73 0.93 0.96 O.96 O.96 O.96 O.97 0.97 0.98 1.03

Conclusion: 1. FIC index=0.16 showing synergistic activity between thioridazine hydrochloride and colistin. 25 2. Colistin MIC reduced from 16 mg/L to 0.5 mg/L when combined with 256 mg/L of thioridazine hydrochloride. Time Kill Curve The following time kill curve, FIG. 12, demonstrates the synergy between thioridazine hydrochloride and colistin 30 against NDM-1 Klebsiella pneumoniae. Example 9 In vitro Activity of Chlorprothixene Hydrochloride 35 and Colistin Against NDM-1 Klebsiella pneumoniae Chequerboard Analysis The results obtained from chequerboard analysis are summarised in Table 9. TABLE 9 Chlorprothixene hydrochloride

2S6 128 64 32 16 8 4 2 1 O.S. O.25 O

Colistin 32 O40 0.40 0.4O O.39 O.39 O.65 0.6S O.S2 0.4O O.S9 O.39 O.39 16 O41 O.39 O.4O O.41 O40 0.4O O.40 O39 O.40 0.40 0.40 O40 8 O40 0.40 0.40 0.41 O.S9 O.67 O.47 0.76 O.76 O.72 O.72 O.68 4 O.39 O.39 O.40 0.45 O.S3 O.S8 O.6O O.67 O.69 O.65 0.77 O.80 2 O4O O.39 O.40 0.46 0.77 O.81 0.87 0.83 0.87 0.81 0.90 1.07 1 O40 0.40 0.4O O.78 O.91 O.93 0.93 0.95 0.85 0.89 0.98 1.13 O5 O40 0.39 O.62 0.81 0.90 O.94 0.94 0.94 O.97 O.95 O.94 112 O O4O O.62 0.7O 1.OS 1.1O 113 1.12 1.12 113 1.15 114 1.18

55 Conclusion: 1. FIC index=0.05 showing synergistic activity between chlorprothixene hydrochloride and colistin. 2. Colistin MIC reduced from 16 mg/L to 0.5 mg/L when 60 combined with 128 mg/L of Chlorprothixene hydrochloride. Time Kill Curve The following time kill curve, FIG. 13, demonstrates the 65 synergy between chlorprothixene hydrochloride and colistin against NDM-1 Klebsiella pneumoniae. US 9,549,965 B2 33 34 Example 10 In Vitro Activity of Triflupromazine and Colistin Against NDM-1 Klebsiella pneumoniae Chequerboard Analysis 5 The results obtained from chequerboard analysis are summarised in Table 10. TABLE 10 Triflupromazine

2S6 128 64 32 16 8 4 2 1 O.S. O.25 O

Colistin 32 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 16 O.S7 O.43 O.40 O.S1 O.4O O.40 O.39 O.40 0.43 O.4O OSO O.69 8 O40 O40 0.4O O.49 O40 0.46 O.46 O.6O O.67 0.63 0.62 0.63 4 O40 0.40 0.40 O.S6 O.63 0.75 0.65 0.77 O.7O 0.68 0.86 O.89 2 O.4O O.39 O.4O O.S8 O.79 0.83 0.85 0.86 O.83 0.88 O.94 1.09 1 O40 0.40 O.48 0.78 O.86 O.86 0.87 0.89 O.86 O.90 O.99 112 O.S. O.4O O.39 O.S8 0.83 0.90 O.93 0.94 O.94 O.95 0.98 0.97 1.14 O O.41 0.76 O.73 1.08 1.11 1.17 114 1-13 1.14 1-13 1.11 115

Conclusion: 25 1. FIC index=0.02 showing synergistic activity for triflu promazine and colistin. 2. Colistin MIC reduced from 32 mg/L to 0.5 mg/L when combined with 128 mg/L of Triflupromazine. Time Kill Curve 30 The following time kill curve, FIG. 14, demonstrates synergy between triflupromazine hydrochloride and colistin against NDM-1 Klebsiella pneumoniae. Example 11 35 In vitro Activity of Trifluoperazine Hydrochloride and Colistin Against NDM-1 Klebsiella pneumoniae 40 Chequerboard Analysis The results obtained from chequerboard analysis are summarised in Table 11 below. TABLE 11 Trifluoperazine hydrochloride

2S6 128 64 32 16 8 4 2 1 O.S. O.25 O

Colistin 32 0.63 0.39 0.39 0.39 0.46 0.39 0.43 0.49 O.39 O.39 O.41 16 O.61 O.39 O.39 O.39 O.39 O.39 O.40 0.47 O.40 O40 O.48 8 O.6O O.39 O.39 O.39 O.46 O.SO 0.57 0.41 O.S2 O.S2 O.S3 4 O.59 O.4O 0.39 O.4O O.43 O.S2 O42 (0.46 O.82 0.74 0.90 2 O.S8 O.39 O.39 O.39 O.41 O.47 0.46 0.83 O.86 0.78 O.91 1 O.S7 O.40 0.40 0.48 0.76 O.73 0.74 0.81 O.74 0.81 0.93 O.S. O.S6 O.4O O.38 0.78 O.80 O.75 0.79 0.83 O.80 O.83 0.96 O 1.33 O.87 0.77 0.95 0.98 0.97 0.95 0.96 O.95 0.96 1.OO

Conclusion: The invention claimed is: 1. FIC index=0.04 showing synergistic activity for trifluop 1. A synergistic combination comprising phenoxyben erazine hydrochloride and colistin. 60 Zamine, or a pharmaceutically acceptable salt and/or Solvate thereof; and polymyxin E (colistin). 2. Colistin MIC reduced from 16 mg/L to 0.5 mg/L when 2. A synergistic combination according to claim 1, combined with 256 mg/L of trifluoperazine hydrochloride wherein the phenoxybenzamine is phenoxybenzamine Time Kill Curve hydrochloride. The following time kill curve, FIG. 15, demonstrates the 65 3. A method of treating a microbial infection which synergy between Trifluoperazine hydrochloride and colistin comprises administering to a mammal, the synergistic com against NDM-1 Klebsiella pneumoniae. bination according to claim 1. US 9,549,965 B2 35 36 4. A method of treating a microbial infection according to claim 3, wherein the infection is a bacterial infection. 5. A product comprising the synergistic combination of claim 1 for simultaneous, separate or sequential use in the treatment of a microbial infection. 5 6. A pharmaceutical composition comprising the syner gistic combination of claim 1 and a pharmaceutically accept able carrier.