METHOD FOR TREATING DRUG-RESISTANT BACTERIAL AND OTHER INFECTIONS WITH CLIOQUINOL, PHANQUINONE, AND RELATED COMPOUNDS

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a new use of clioquinol, phanquinone, combinations thereof, or other chelating agents alone or in combination in the treatment of bacterial infections and infections with other organisms that are resistant to known antibiotics or antiinfective agents.

Description of the Background Art Infections caused by multi-drug-resistant (MDR) bacteria are the result of ecological pressure exerted by the use and overuse of antibiotics, proving that the world of bacteria is an intelligent and difficult opponent. It has been disappointing to see the pharmaceutical industry stepping back from anti-infective R&D programs, because antibiotics are associated with low returns on investment. Approval of new antibacterial agents by the U.S. Food and Drug Administration (FDA) has fallen by 56% over the past 20 years. Furthermore, the regulatory approval rules represent a real hurdle that industry has to overcome, before launching a new agent onto the market. Legislative solutions, facilitating the development of new antimicrobial agents, are needed. In March 2003, the Infectious Diseases Society of America established the Antimicrobial Availability Task Force (AATF), which was responsible for ensuring future antibiotic availability through the evaluation of current trends and promotion of R&D programs.

Multiple Drug Resistant (MDR) Bacteria as Causative agents in Serious Infection

Gram-positive cocci, particularly Staphylococcus aureus, coagulase-negative staphylococci (CoNS) and Enterococcus spp., followed by Escherichia coli, Pseudomonas aeruginosa and Enterobacter spp., are the most commonly encountered pathogens in surgical site infections (SSIs) according to the National Nosocomial Infection Surveillance System reports. Although epidemiology changes over time and varies across different settings, as well as among different countries, there has been an increased prevalence of Gram- negative bacilli over time , which predominates among orthopedic and trauma patients as well as intensive care unit (ICU) surgical patients. In the latter case, P. aeruginosa is the most frequent pathogen present in SSI. Among out-

patients and non-ICU surgical patients, S. aureus predominates, whereas an increased prevalence of Acinetobacter baumannii in SSI has been observed throughout Europe.

It is easily concluded that the changing epidemiology has expanded in SSI the threat of all the highly problematic MDR pathogens, also identified as "AATF" : namely A. baumannii, extended- spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, P. aeruginosa, MRSA- and vancomycin-resistant Enterococcus faecium (VREF). Due to their multiple inherent or acquired mechanisms of resistance that enable them to escape from antimicrobial agents, all these species have been implicated in the difficult-to-treat nosocomial infections.

Bacterial enzymes Microbial proteases are predominantly extracellular enzymes that can be classified into four groups based on the essential catalytic residue at their active site. They include serine proteases (EC 3.4.21), cysteine proteases (also called thiol proteases) (EC 3.4.22), aspartate proteases (EC 3.4.23), and the metalloproteases (EC 3.4.24). Extracellular zinc-containing metalloproteases are widely distributed in the bacterial world (Hase CC & Finkelstein RA, Microbiol Rev. 1993 57:823-37). The most extensively studied are those which are associated either with pathogenic bacteria or industrially significant bacteria. They are found practically anywhere they are sought in both gram- negative and gram-positive microorganisms, be they aerobic or anaerobic. This ubiquity in itself implies that these enzymes serve important functions.

Because of the importance of Zn to enzymatic activity, it is not surprising that there is a pervasive amino acid sequence homology among members of this family of enzymes regardless of their source. The evidence suggests that both convergent and divergent evolutionary forces are at work. Within the large family of bacterial Zn-containing metalloendopeptidases, smaller family units are observed, such as thermolysin-like, elastase-like, and Serratia protease-like metalloproteases from various bacterial species. More recently a new function for Zn-containing metalloproteases was discovered: the neurotoxins of Clostridium tetani and Clostridium botulinum type B are Zn metalloproteases with specificity for synaptobrevin, an integral membrane protein of small synaptic vesicles which is involved in neurotransmission.

Most metalloproteases are Zn-containing proteins. Zn is an integral component of many proteins which are involved in virtually all aspects of metabolism of the different species of all

phyla. In all Zn enzymes whose crystal structures are known, a catalytic Zn atom is coordinated to three amino acid residues and an active water molecule, whereas structural Zn atoms are coordinated to four Cys residues (Vallee, BL et al, 1990, Biochemistry 29:5647-59). A combination of His, GIu, Asp, or Cys residues creates a tridentate active Zn site, and an activated water molecule fills and completes the coordination sphere.

Streptococcus pneumoniae (Pneumococci)

Pneumococci display large Zn metalloproteinases on their surface, including the IgA protease, which cleaves human IgAl antibodies in the hinge region, the ZmpC proteinase, which cleaves human matrix metalloproteinase 9 (MMP-9), and two other proteinases, ZmpB and ZmpD, whose substrates have not yet been identified. Surface metalloproteinases are antigenic and have been linked to virulence. The genes encoding these proteinases reside in three distinct loci: two loci specific for zmpB and zmpC, and a third, the iga locus, containing iga and zmpD. The presence of the four proteinase genes is variable in the pneumococcal strains whose genomes have been sequenced. Studies of the presence of these genes in a collection of 218 pneumococcal isolates, mostly from invasive disease, (Camilli R et al. , Microbiology, 2006, 152(Pt 2):313-21) showed that zmpB and iga were present in all the isolates examined, while zmpC and zmpD were present in a variable proportions (18 and 49 %, respectively). Isolates carrying both zmpC and zmpD were found to belong mainly to two serotypes ("st's"), 8 and 1 IA. By molecular typing, st 8 and st 1 IA isolates appeared to belong to the same clonal cluster. The presence of these two additional metalloproteinases may contribute to the fitness of particular pneumococcal clones.

Metallo-β-Lactamase (MBL)

MBLs are bacterial Zn(II)-dependent hydrolases that confer broad-spectrum resistance to beta-lactam antibiotics. These enzymes can be subdivided into three subclasses (Bl, B2 and B3) that differ in their metal binding sites and their characteristic tertiary structure. MBLs provide bacteria with an efficient and effective way of mediating resistance to beta-lactam-based antibacterial agents, particularly carbapenems. Therefore, if MBLs increase in prevalence, they could compromise the efficacy of this group of antibiotics to treat life-threatening hospital infections. The clinical ascendancy of MBLs (Timothy R. et al., Clin Microbiol Rev,, 2005, 78:306-325) has been dramatic; some reports indicate that nearly 30% of imipenem-resistant

Pseudomonas aeruginosa strains possess a metallo-beta -lactamase. Acquisition of a MBL gene will invariably mediate broad- spectrum β-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less predictable. Their clinical significance is further emphasized by their ability to hydrolyze all beta-lactams; there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo- beta -lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the MBLs encoded on transferable genes include Imp, Vim, Spm, and Gim and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation not only of surveillance but also studies of MBL inhibitor that will assure the longevity of important anti-infectives

MBLs have now been identified in a wide spectrum of clinically important pathogens (H. Ito et al. Abstracts of the 37 ^th ICAAC, abstr. C-93, 1997), such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, and Acinetobacter spp. These enzymes also disseminate through bacterial populations, demonstrated by the spread of the IMP-I metallo-beta lactamase in Japan. The spread of this enzyme is thought to be facilitated by its being encoded on an integron.. More recently, clinical isolates producing metallo-β-lactamases have been identified in Europe. One approach to combating this potential clinical problem is the use of a specific MBL inhibitor in combination with a β-lactam antibiotic. In this regard a range of different non-beta- lactam based molecules have been identified as specific inhibitors of these enzymes, e.g., a series of mercaptoacetic acid thiol ester compounds. None of these compounds exhibited significant synergy with β-lactam antibiotics, nor did they exhibit potent broad- spectrum inhibition of MBLs. However, a recently discovered series of mercaptocarboxylates have been shown to exhibit both broad- spectrum MBL inhibitory activity and antibacterial synergy with meropenem (Payne, DJ et al. Antimicrob Agents Chemother, 2002, 4(5:1880-6)

Carbapenems are the drugs of choice for the treatment of infections caused by MDR gram- negative bacilli. Carbapenemases involved in acquired resistance are of Ambler molecular classes A, B, and D. The class B enzymes, MBLs, are the most clinically promising carbapenemases,

because they are capable of hydrolyzing all beta-lactams except aztreonam. Gram-negative bacilli producing acquired MBLs have been reported in many countries and are sometimes susceptible to carbapenem in vitro under standard conditions, making them difficult to recognize.

To date there are no clinically useful pan-MBL inhibitors available, mainly due to the unawareness of key catalytic features common to all MBL types. In a recent study (Gonzalez JM et al , JMoI Biol, 2007, 575:1141-56), two double mutants of BcII, a di-Zn(II) Bl-MBL from Bacillus cereus, namely BcII-R 121H/C22 ID (BcII-HD) and BcII-R121H/C221S (BcII-HS), were designed, expressed and characterized. These mutants display modified environments at the so- called Zn2 site or DCH site, reproducing the metal coordination environments of structurally related metallohydrolases. The position of Zn2 was found to be essential for a productive substrate binding and hydrolysis.

The most clinically significant MBL producers are primarily those in which the gene encoding the enzyme is transferable, and include P. aeruginosa and Acinetobacter spp., and to a lesser extent, enterobacteria species. Thus, these broad- spectrum β-lactamases are mostly identified from bacterial species that already have a high degree of natural resistance to many antibiotic classes. Concerning beta-lactam resistance, these species express a cephalosporinase, have efficient efflux pumps, and have low intrinsic outer membrane permeability to many hydrophilic molecules. Thus, MCR may be easily observed in those species as a result of combined mechanisms of resistance. The unique problem with MBLs is their unrivalled broad- spectrum resistance profile, plus the fact that the MBL genes may be located on plasmids with genes encoding other antibiotic resistance determinants, e.g., aminoglycoside resistance genes. These MBL-positive strains are usually resistant to β-lactams, aminoglycosides, and fluoroquinolones, while usually remaining susceptible to polymyxins.

No extended survey with a series of human infections with MBL-positive isolates has been performed to determine the optimal treatment. Thus, suitable therapy for treating those infections remains unknown.

Clearly, in the absence of novel agents in the near future, the spread of MBL producers may lead to therapeutic dead ends. This would be more likely if these MBL genes spread in gram- negative isolates to outpatients. Taking into account the current distribution of these enzymes in different geographical areas (South America, southern Europe, and Southeast Asia), early detection

of MBL producers in patients from these areas should are needed along with detecting colonization with MBL producers when patients are admitted to clinical wards, in particular intensive care units and oncology units. The occurrence of an MBL-positive isolate in a localized hospital environment poses not only a therapeutic problem but also a serious concern for infection control management. It is impossible to predict what impact MBL genes will have on future antimicrobial regimens. There is little doubt that in some countries the numbers of MBL possessing P. aeruginosa and Acinetobacter spp. infections are such that the mainstay antibiotic regimens used to eradicate these bacteria can no longer be relied upon. For example, the first characterized P. aeruginosa isolate possessing blaSPM-1 was fully resistant to all antibiotics except colistin, which was deemed inappropriate due to the clinical presentation of the patient, who subsequently died of the infection. This case is now mirrored elsewhere such that it now precludes the consideration of any β-lactam or aminoglycoside treatment.

It would appear that MBL genes are first propagated in pseudomonads (usually P. aeruginosa) before appearing in Enterobacteriaceae , including S. marcescens, K. pneumoniae, C. freundii, E. coli, and Enterobacter spp. Based on circumstantial evidence it is likely that pseudomonads have transferred their plasmids to Enterobacteriaceae , probably within a clinical environment. The extent of this phenomenon is difficult to ascertain, as the majority of MBL genes in Enterobacteriaceae do confer a resistant phenotype, as judged by in vitro MICs. No animal efficacy studies have been undertaken to exam β-lactam eradication in MBL-positive Enterobacteriaceae infections, the data from which would provide a useful benchmark of their clinical importance.

The clinical significance of bacteria possessing an MBL is ultimately judged by their ability to confer in vivo β-lactam resistance and, ultimately, whether the patient fails β-lactam therapy. Many isolates possessing transferable MBL genes, particularly Acinetobacter spp. and Enterobacteriaceae, are sensitive to the carbapenems, and doubts have been raised about their in vivo resistance. Accordingly, further in vivo studies are needed to evaluate the significance of MBL genes in a range of different bacterial hosts. However, there is little doubt that these enzymes contribute significantly to β-lactam resistance even if they are not singularly responsible for it.

MBLs also represent a clinical threat due to their unrivalled spectrum of activity and their resistance to therapeutic serine beta-lactamase inhibitors. The fact that MBL and aminoglycoside

resistance genes are genetically linked merely compounds this problem. The problem of an appropriate treatment regimen is also amplified by the lack of new antimicrobials that will possess broad- spectrum potency against clinically significant P. aeruginosa and Acinetobacter spp.

Mycoplasma and Matrix Metalloproteinases (MMPs) Murine Mycoplasma pulmonis infection induces chronic lung and airway inflammation accompanied by profound and persistent microvascular remodeling in tracheobronchial mucosa (Baluk P et al, Am J Physiol Lung Cell MoI Physiol, 2004, 287:L307-17).). Because MMP-2 and MMP-9 are important for angiogenesis associated with placental and long bone development and skin cancer, were hypothesized to contribute to microvascular remodeling in airways infected with M. pulmonis. The authors compared microvascular changes in airways after M. pulmonis infection of wild-type FVB/N mice with those of MMP-9 ^{( / }} and MMP-2 ^("/") /MMP-9 ^("/") double-null mice and mice treated with the broad- spectrum MMP inhibitor AG3340 (Prinomastat). The authors concluded that despite major increases in expression, MMP-2 and MMP-9 were not essential for microvascular remodeling in M. pulmonis '-induced chronic airway inflammation. Metal Ions and Metallo-peptidase

Zn-specific metallo-regulation has been documented for bacterial yeast and mammals,. Much of this work has centered on the induction of metallothionein by Zn. For example, a Zn- sensitive inhibitor protein prevents the interaction of MTF- 1 with metal response elements preceding mammalian metallothionein genes. In Synechococcus, metallothionein expression is induced when the SmtB repressor dissociates from its operator in response to Zn. A similar repressor, ZiaA, regulates the Zn-inducible expression of a Zn efflux pump in Synechococcus strain PCC 6803. The regulation of Zn transport in Saccharomyces cerevisiae has also been well characterized. In that organism, Zap Ip activates the transcription of both low- and high-affinity Zn transporters under conditions of Zn limitation. Zinc is an essential metal ion, although excess of even this essential ion exerts toxic effects on the cell (Choudhury, R and Srivastava S, 2001, Current Science, 81:768-75). On the other hand, mechanisms to evade metal toxicity are also widespread in the microbial world. Copper in biological systems presents a formidable problem: it is essential for life, yet highly reactive and a potential source of cell damage (Magnani, D and Solioz, M., How Bacteria Handle Copper,

1

Microbiology Monographs, Springer- Verlag, 2007). Tight control of Cu is thus a cellular necessity. To meet this challenge, cells have evolved pumps for transmembrane transport, chaperones for intracellular routing, oxidases and reductases to change the oxidation state of Cu, and regulators to control gene expression in response to Cu. These systems are complemented by specific mechanisms for the insertion of Cu into enzymes. Cu homeostasis has evolved early in evolution and some components have been conserved from bacteria to humans.

The effects of Cu on trace metal and antibiotic resistance of Pseudomonas aeruginosa have been investigated (Caille O, /. Bacteriol. 2007, 189:4561-8.). Cu treatments induced resistance not only to this metal but also, surprisingly, to Zn. Proteases play a crucial role in remodeling the bacterial proteome in response to changes in cellular environment (Pruteanu M et al, J. Bacteriol., 2007, 789:3017-25). E. coli ZntR, a Zn- responsive transcriptional regulator, was identified by proteomic experiments as a likely CIpXP substrate, suggesting that protein turnover may play a role in regulation of Zn homeostasis. When intracellular Zn levels were high, ZntR activated expression of ZntA, an ATPase essential for Zn export. ZntR was degraded in vivo in a manner dependent on both the CIpXP and Lon proteases. However, ZntR degradation decreased in the presence of high Zn concentrations, the level of ZntR rises, and transcription of the zntA exporter was increased. Mutagenesis experiments revealed that Zn binding did not appear to be solely responsible for the Zn-induced protection from proteolysis. Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far

(Banerji S , Infect Immun., 2005 73:2899-2909). The gene, plaC was cloned and characterized; it encoded a protein with high homology to PIaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid: cholesterol acyltransferase activity. The reduced enzyme activities were complemented by reintroduction of an intact copy oϊplaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid: cholesterol acyltransferase activities to recombinant E. coli. PlaC was exported by the L. pneumophila type II secretion system and was activated by a factor present in the culture supernatant dependent on the Zn metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L.

pneumophila was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities which is distinct from the previously characterized phospholipase A and lysophospholipase A. Helicobacter pylori

Of the 19 species of the genus Helicobacter identified to date, Helicobacter pylori is the most important. H. pylori is a gram-negative multiflagellated unipolar spiral bacterium found almost exclusively on gastric biopsy in approximately 30 to 35% of the population less than 40 years old, with increasing incidence thereafter. The organism is responsible for chronic gastritis in humans and is associated with peptic ulcer disease. A causal role has also been proposed for gastric carcinoma in man and primary B-cell mucosa associated lymphoid tissue lymphoma of the stomach.

H. pylori express a number of surface-exposed proteins which are thought to be involved in colonization, adhesion, and virulence, including vacuolating cytotoxin and flagellin. H. pylori gastritis is characterized by protracted inflammatory cell response, abnormal gastric physiology, and gastric mucosal cell injury. A number of these surface-associated proteins are likely responsible either directly or indirectly for some of the pathological lesions observed. Microbial extracellular proteases have been demonstrated or proposed to play important roles as virulence factors in the pathogenesis of a variety of diseases. Identification, characterization, and purification of microbial proteases are prerequisites for understanding their role in the pathogenesis of infectious diseases. The researchers have identified and partially characterized a Zn-dependent, calcium-stabilized metallo-protease activity from H. pylori which is both expressed on the surface and released in a soluble form.

H. pylori require Ni as a cofactor of the enzymes urease and hydrogenase (Abraham, LO et ah, J Inorg Biochem. 2006 700:1005-14). One of the proteins that controls Ni homeostasis in this organism is NikR (ηpNikR), a homologue of Ni-dependent transcription factors from other organisms, which regulates the expression of multiple proteins such as the urease structural subunits and itself. ηpNikR binds stoichiometric Ni or Cu, and binds Ni with pM affinity in what is likely a conserved square-planar site. In vitro DNA-binding assays revealed that ηpNikR bound directly to the promoter region of the ureA operon in response to Ni, and the location of the binding site was defined. Ni induced DNA binding to the nikR promoter sequence but the complex is much weaker.

Thus, it is believed that HpNikR directly controls the expression of multiple genes by binding to separate DNA sequences employing several possible mechanisms for differential regulation. The binding constants between Ni ₂₊ and H. pylori NikR have been determined using isothermal titration microcalorimetry in order to rationalize the role of this protein as a Ni-dependent biological sensor (Zambelli B et al, Chem Commun (Camb). 2007 27:3649-51).

Microbial biofilms

Biofilms have been found to be involved in a wide variety of microbial infections, possibly as many as 80% of all infections. Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves.

It has recently been shown that biofilms are present in tissue samples taken from 80% of patients undergoing surgery for chronic sinusitis. Those patients with biofilms were shown to have been denuded of cilia and goblet cells, unlike the controls without biofilms who had normal cilia and goblet cell morphology. Biofilms were also found on samples from 2/10 healthy controls. The species of bacteria from interoperative cultures did not correspond to the bacteria species in the biofilm on the respective patient's tissue. In other words, the cultures were negative though the bacteria were present.

Hospital- acquired bacterial infections are an increasingly important cause of morbidity and mortality worldwide. Staphylococcal species are responsible for the majority of hospital-acquired infections, which are often complicated by the ability of staphylococci to grow as biofilms. Biofilm formation by S. epidermidis and S. aureus requires cell-surface proteins (Aap and SasG) containing sequence repeats known as G5 domains. G5 domains from Aap are Zn-dependent adhesion modules analogous to mammalian cadherin domains (Conrady DG et al., 2008, Proc Natl Acad Sci USA. 105: 19456-61).. The G5 domain dimerizes in the presence of Zn ions. Tandem G5 domains associate in a modular fashion, suggesting a "Zn zipper" mechanism for G5 domain-based intercellular adhesion in staphylococcal biofilms. Zn ion chelation specifically prevented biofilm formation by S. epidermidis and methicillin-resistant S. aureus (MRSA). Furthermore, individual soluble G5 domains inhibited biofilm formation in a dose-dependent manner. Thus, the complex 3D

architecture of staphylococcal biofilms results from the self-association of a single type of protein domain. Surface proteins with tandem G5 domains are also found in other bacterial species, suggesting that this mechanism for intercellular adhesion in biofilms may be conserved among staphylococci and other Gram-positive bacteria. It was concluded that Zn ion chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm- related infections. Fungal Infections

Candida species are the predominant pathogens associated with nosocomial fungal infections, particularly in intensive care units. This is further complicated by the emergence of non- Candida albicans species, such as Candida glabrata (Bille J et al, Curr Opin Infect Dis. 2005; 18:314-9; Fridkin SK et al, Clin Microbiol Rev. 1996, ;9:499-511) This trend is alarming, given that C. glabrata has variable susceptibility to the commonly used antifungal agent fluconazole, a drug of choice for the treatment of Candida infections since its introduction in the early 1990s. Fluconazole resistance among C. glabrata isolates was greatest in the United States and varied by census region. The potential for triazole cross-resistance may limit the clinical utility of second- generation triazoles, such as voriconazole, against this pathogen. The incidence of invasive aspergillosis had at least tripled among both allograft and autograft recipients. Every antifungal agent used to treat invasive aspergillosis has issues that limit its use. Amphotericin B deoxycholate, the conventional formulation of amphotericin B that has been the mainstay of treatment since the late 1950s, is notorious for infusion-related adverse effects and nephrotoxicity. While lipid-based amphotericin B formulations have helped to reduce these adverse effects, their acquisition cost may present a problem for some institutions. The second-generation triazole antifungal, voriconazole, is highly effective against invasive aspergillosis, but the potential for drug-drug interactions is of concern. Therefore, the introduction of a novel antifungal class, the echinocandins, provides clinicians with another treatment option for these pathogens.

Resistant Fungi as Complicating Causative Agents in Immunocompromised and Serious Infection The changing demographic trends as well as the revolution in therapeutic approaches have increased dramatically the incidence of serious fungal infections, see frequently in hospital intensive care and infectious disease units. Also adding to these are the growing numbers of

immunocompromised patients including HIV-infected patients, cancer patients on chemotherapy- induced neutropenia, and transplant recipients receiving immunosuppressive therapies. Of growing concern is the shift to increasing incidence of invasive mold infections, and the development of antifungal resistance. Recovery of Aspergillus species from the respiratory tract secretions of ICU patients is associated with invasive aspergillosis in almost half of the patients, and should be considered a marker for this mold infection. Monoclonal antibodies used in solid organ and stem cell transplantation, is a cause of profound immunosuppression thought to increase the risk of invasive yeast and mold infections. Since the 1990s, fluconazole has been widely used for prophylaxis and treatment of invasive fungal infections in immunocompromised patients and has resulted in a steady decrease in the incidence of Candida sepsis worldwide. However, this decrease has been "balanced" by a corresponding rise in infection with Candida glabrata and other non-albicans Candida species. Although Aspergillus species, particularly Aspergillus fumigatus, account for the largest proportion of invasive mold infections, the last decade has witnessed the emergence of new opportunistic pathogens, including non-fumigatus Aspergillus species, Fusarium species, Paecilomyces species, Scedosporium species, the dematiaceous fungi (Alternaria, Bipolaris, Curvularia, Cladosporium, and Exserohilum species), and the agents of zygomycosis (mucormycosis). Because the last pathogens listed are resistant to voriconazole in vitro, it has been suggested that the increasing prophylactic use of this agent may account for the higher prevalence of zygomycosis. Fungi and Metal Ions

Fungi are dependent on peptidases. One studied aminopeptidase (El Moudni B et ah, J Med Microbiol. 1995 43:282-8) was strongly inhibited by specific metallo-enzyme inhibitors EDTA and o-phenanthroline . Furthermore, there is evidence that a similar or identical enzyme occurs in other C. albicans clinical isolates and other Candida spp. A metallopeptidase of C. albicans could be involved in the process of dissemination of the yeast. (Imbert C et al, J Antimicrob Chemother. 2002 49:1007-10). MMPs are also responsible for collagen breakdown in inflammatory and malignant processes.

Clioquinol has been reported to inhibiting MMPs (Xilinas, WO01/82912, WO01/8291).

Fungi are dependent on metal ions (Eshwika A et al., Biometals. 2004; 17:415-22) for their growth. Antifungal Agents

The widespread use of antifungal agents in the serious infections and in the immunocompromised population has been associated with the emergence of clinically significant drug resistance among patients exposed to such agents for prolonged periods of time. Of potential concern is azole resistance in Candida species ^[ and Aspergillus species with the possibility of cross- resistance among the antifungal azoles. which has been documented.

The echinocandins (caspofungin, micafungin, and the investigational anidulafungin) inhibit the formation of the fungal cell wall and possess excellent in vitro fungicidal activity against

Candida species, including those resistant to azoles. However, emergence of resistant C. glabrata during caspofungin therapy was recently reported with a 64-fold increase in the MIC of caspofungin, raising concerns about the development of resistance to this newly introduced class of antifungal agents. Several new antifungal agents, including novel compounds in familiar classes and entirely new classes targeting previously untapped mechanisms, are in various stages of the drug development process (Chavez M et al. J Antimicrob Chemother 2000;44:697-700; Diekema DJ et al., Antimicrob Agents Chemother 1999;4J:2236-9; Espinel-Ingroff A, / Clin Microbiol, 1998; 36:2950-6). Many new triazole antifungal agents are being studied, including voriconazole, posaconazole, and ravuconazole. The echinocandin antifungals, which represent a new class of antifungal agents, possess activity against a variety of fungal pathogens. The sodarin derivatives and nikkomycins are two additional classes of antifungals in development.

Antifungal Effects of Clioquinol and Phanquinone

Candida yeasts frequently cause life-threatening systemic infections in immunocompro- mised hosts (Costa EM et al. , FEMS Immunol Med Microbiol. 2003 38: 173-80). Extracellular proteinases were characterized in 44 oral clinical isolates of C. albicans from HIV-positive (29/50) and healthy (15/50) children. These oral clinical isolates of C. albicans have complex extracellular proteolytic activity profiles, which illustrates the heterogeneity of this species. Four distinct proteolytic patterns composed of distinct serine (30-58 kDa) and metalloproteinase (64-95 kDa)

activities were discerned based on the inhibition profile with phenylmethylsulfonyl fluoride and 1 , 10-phenanthroline, respectively.

Clioquinol is known to be a potent antifungal drug. Since 1952, 186 fungal isolates were obtained from 188 cases of clinically diagnosed otomycosis ((Maher A et al., J Laryngol Otol. 1982, 96:205-13). These comprise 59 species of 26 genera of molds, and 2 genera of yeasts. This large variety of mould isolates provided ample material for in vitro evaluation of the anti-fungal activity of six antimycotic substances. The antifungal activity of clotrimazole and tolnaftate in vitro was evident. For over 94% of the 59 fungus species tested, the MIC was less than 0.1 μg/ml, and for 6 % it was between 0.4 and 1 μg/ml. As to the other 4 antimycotic substances (iodochlorhydroxyquin, fluonilid, natamycine and polymyxin B sulfate) MICs ranged from

>100μg/ml to 1 μg/ml for the majority of tested fungi. The clinical observations were mostly in accordance with these findings.

Clioquinol is used in the form of a 3% cream or ointment applied topically 2-4 times daily to treat, e.g., athlete's foot and, ringworm and jock itch. Phenanthrolines have known anti-Candida effects due to chelation of Zn. It has been known since 1985 that several chelators prevent the synchronous release of 24- to 48-hour stationary phase singlet cells of the dimorphic yeast C. albicans into either the mycelial or the budding phenotypes (Bedell GW, et al., Mycopathologia. 1985 92:161-7). The only chelator that was found to inhibit mycelium formation completely and to restrict bud formation to about 10% was 1,10- phenanthroline at minimal concentrations of 50 μM and 230 μM, respectively. The inhibition of both phenotypes could be reversed completely by the addition of 200 μM ZnSO4.

Clearly there is a need in the art for new methods for treating bacterial and fungal infections, particularly those that have developed resistance to various antibiotics or antifungal drugs. The present inventor has conceived of method that exploit one or more chelating compounds, and their combination with known antibiotics or antifungal drugs as a solution to this problem.

SUMMARY OF THE INVENTION

The present invention relates to a new use of clioquinol, phanquinone, combinations thereof, or other chelating agents with related structures and functions, alone or in combination in the

treatment of bacterial and fungal infections and infections with other organisms that are resistant to known antibiotics or antiinfective agents.

The invention pertains to the use of clioquinol and phanquinone in combination or separately, for the manufacture of a pharmaceutical composition for the antimicrobial therapeutic or empiric treatment of an infection caused by bacteria or fungi which the chelation has a bactericidal or bacteriostatic or fungicidal or fungistatic effect. Further the invention pertains to the use of clioquinol, phanquinone or other metal chelation is any two fused 6- membered rings with at least a nitrogen at position 1 and a hydroxyl at position 8 or other chelators to be used preferably in combination with other antimicrobials that act synergistically or additively in enhancing the treatment effects.

The present invention also relates to methods for treatment of fungal infections (by pathogenic yeasts or molds). including those resistant to other antifungals. The chelating compounds and their combinations have fungicidal or fungistatic effects and interact (synergistically or additively) with conventional antifungal drugs. More specifically, the present invention is directed to a method of treating a bacterial or fungal infection, comprising administering to a subject in need thereof a pharmaceutical composition that comprises an effective amount of one or more chelating compounds that chelates metal ions essential for function of (i) a bacterial or fungal metal-dependent enzyme, (ii) another bacterial or fungal metal-dependent protein, or (iii) another bacterial or fungal metal-dependent biologic activity required for metabolism, multiplication or survival of the bacteria or fungi, which compound thereby exerts bacteriostatic, bactericidal, fungistatic or fungicidal activity, thereby treating the infection. The chelating compound preferably chelates, and the bacterial or fungal enzyme, protein or other biological activity depends upon, the availability of, a metal ion selected from zinc, copper, nickel, iron, cobalt and cadmium, most preferably zinc or copper. One or more chelating compounds may be administered in combination with an antibacterial

(antibiotics) and/or an antifungal drug.

A long list of preferred chelating compounds are listed in the Detailed Description section below. More preferred among these are clioquinol; phanquinone; quinaldol; 5,7-dichloro-8- hydroxyquinoline; 5,7-diiodo-8-hydroxyquinoline; 5,7-dichloro-2- methyl- 8 -hydroxyquinoline; and

5,7-dichloro-8-hydroxy-quinaldine. Most preferred are clioquinol, phanquinone or a combination of the two.

The clioquinol is preferably highly purified and as raw material contains less than 5 ppm di- iodo-8-OH-quinoline. Alternatively, the clioquinol as a raw material containing less than 0.1 weight per cent di-iodo-8-OH-quinoline. Phanquinone is preferably administered in a daily dosage of ranging between about 5 mg and about 3 g. When the combination of clioquinol and phanquinone, is administered, the preferred daily dosage is about 500 mg to about 2 g clioquinol and 50 mg to about 1 g phanquinone.

In another embodiment, the chelating compound is the 2- 3-, or 4-substituted phenol in which a hydroxy, fluoro, carboxy, or acetamido group is substituted.

The present methods are designed to treat bacteria are multi drug-resistant, e.g., methicillin- resistant gram positive Staphylococci (MRSA) or bacteria that produce extended spectrum β- lactamases and metallo-β-lactamases, rendering them β-lactam antibiotic -resistant. In one embodiment, the β-lactam-resistant bacteria causing the infection being treated are resistant to cephalosporins, penicillins, carbapenems, carbacephems, or monobactams. The bacteria causing the infection being treated are generally metalloprotease-dependent. .

In other embodiments, the bacteria causing the infection being treated are resistant to aminoglycoside antibiotics, dihydrofolate reductase inhibitors, sulfonamides, quinolone, macrolides, rifampicin, glycopeptide antibiotics, , polypeptide antibiotics, tetracyclines, chloramphenicol, fosfomycin, clindamycin, linkomycin, fusidic acid, furazolidone, linezolid, metronidazole, isoniazid or pyrazinamide.

Preferred treatment targets also include gram positive, methicillin- sensitive Staphylococci, gram positive penicillin-resistant or intermediate resistant strains of Streptococcus pneumoniae, Streptococcus pyogenes, Enterococci, extended spectrum β-lactamase-producing or metallo-β- lactamase-producing strains of Enterobacteriacea, Acinetobacter spp., Hemophilus spp., Branhamella spp., Legionella spp. and Mycobacteria spp.

In one embodiment, the target bacteria are Acinetobacter baumanii, Enterococcus faecium, Enterococcus faecalis, Bacteroides fragilis, Mycoplasma pneumoniae, Haemophilus influenzae, Haemophilus influenzae Branhamella catarrhalis, Mycobacterium tuberculosis, Mycobacterium leprae, Legionella pneumophila, Clostridium difficile, or Mycoplasma pneumoniae.

In another embodiment, the target bacteria are Listeria, Erysipelothrix, Meningococci, Gonococci, chancroid, Brucella, Francisella (causing Tularemia), Yersinia, Pseudomonas, Aeromonas, Vibrio, Campylobacter, Treponema, Leptospira, Clostridia, Borrelia, including bacteria causing anthrax, tetanus, or gangrene In the above method, the bacteria or fungi may be ones that form a biofilm, wherein and biofilm formation is inhibited by chelating metal ion-dependent (primarily Zn-dependent) adhesion.

As indicated, in a preferred embodiment, in addition to the one or more chelating compounds, the subject is administered an amount of one or more antibiotics (for bacterial infection) of antifungals (for fungus infection) that is effective in combination with the one or more chelating compounds to treat the infection.

In such combinations, the antibiotic can be a β-lactam, preferably meropenem, ceftazidime, cefotaxime, ceftobiprole, ceftatroline, rocephin and cefepime, a polymyxin, an aminoglycoside, a quinolone, a glycopeptide, a glycylcycline, a lipopeptide, a macrolide, chloramphenicol, a dihydrofolate reductase inhibitor, a sulfonamide, rifampicin, metronidazole, clindamycin, linkomycin, fusidic acid, furazolidone, isoniazid, pyrazinamide or a combination of one or more of these antibiotics. Most preferred are meropenem, cefepime or a polymyxin.

When the infection is a fungal infection, the fungus may be a Candida species, an Aspergillus species, a Mucorales species, a Cocciodioides species, a Pneumocystis species, Paracoccidoides brasilensis, Sporothrix schenckii, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis or a dermatophyte. The invention is particularly useful against a drug-resistant fungus, e.g., one that is resistant to amphotericin B deoxycholate, a lipid associated formulation of amphotericin B, flucytosine, ketoconazole, itraconazole, fluconazole, voriconazole, caspofungin, sordarins, nikkomycins, anidulafungin or micafungin. As indicated, in addition the one or more chelating compounds, a subject being treated for a fungal infection preferably also receives an amount of one or more other antifungal drugs that is effective in combination with the one or more chelating compounds in treating the infection. Preferred antifungal drugs include amphotericin B deoxycholate, a lipid associated formulation of amphotericin B, flucytosine, ketoconazole, itraconazole, fluconazole, voriconazole, caspofungin, a

sordarin, a nikkomycin, anidulafungin, micafungin, allylamine, a triazole antifungal agent, griseofulvin, an imidazole, terbinafine, clotrimazole and any combination thereof.

The present method is used to treat infected subjects who are immunocompromised, for example, as a result of a hospital infection, a concomitant viral infection, e.g., HIV-I or -2, hepatitis B or C; a solid tumor or leukemia, burns, trauma or other injury; or incompetence or insufficient reactivity of immune cells, including several types of white blood cells, and/or blood

The subject may be is a pediatric patient, such as an infant with a weak immune system, an elderly person, a patient with a chronic disease; a long-term hospitalized patient; a septic patient; or a patient with a liver, kidney, cardiovascular or respiratory disease, all of which have a negative impact on the patient's infection resistance and ability to respond immunologically.

Non-limiting examples of infections to be treated in accordance with this invention are infections of the upper or lower respiratory tract, the skin, soft tissue, the urinary tract, the genital tract, the gastrointestinal tract, as well as intra-abdominal, intra- thoracic, pericardial, endocardial infections, gynecologic infections, infections involving the cerebrospinal spaces and fluids, orthopedic infections or infections of the eye, ear, nose, larynx or mouth, as well as sepsis that is manifest in the blood-stream or lymphatic system or the cerebrospinal fluid

Other target infections are those at a surgical site, a catheter site, or an injection site,

In the present method, the chelating compound may be administered for a treatment period of up to about six months. In preferred embodiments, the chelating compound may be administered :

(a) once to six times daily,

(b) by continuous infusion,

(c) by intermittent infusion, or

(d) orally for a period ranging from about 7 to about 21 days. The chelating compound may be administered locally subcutaneously, transdermally or intra-aurally for a period ranging from 1 day to about twelve moths.

For use in the present method, the pharmaceutical composition comprising the one or more chelating compounds, or combination of the chelating compound(s) with one or more antibiotics or antifungal agents, formulated for oral, parenteral, cutaneous or topical, subcutaneous, intramuscular, intravenous, local, intrarectal, ophthalmic, intraaural, or transdermal administration.

When formulated for parenteral administration, the active ingredient may be solubilized or diluted in a pharmaceutically acceptable dispersant, co-solvent, surfactants or complexing agents; or is diluted in carboxymethylcellulose, hydroxyethylcellulose, polyoxyethylated castor oil, glycerol- polyethylene glycol ricinoleate, a polyethylene glycol, ethoxylated glycerol or dehydrated alcohol. A parenteral pharmaceutical composition may also be a suspension or an emulsion for injection or infusion.

The chelating compound may be given concomitantly, concurrently, in adjunction to, or in combination with another agent or drug to treat a coexisting condition; examples are an antiviral drug, a anticancer chemotherapeutic or biotherapeutic (e.g., immunotherapeutic) agent, an anti- inflammatory drug, or a treatment agent or regiment for sepsis.

In one embodiment, the chelating compound(s) is/are given in combination with an effective amount of Vitamin B 12 or folic acid to protect against undesired effects of the chelating compound.

The present invention is also directed to a kit for use in the present method. The kit comprises in close confinement a pharmaceutical composition comprising the one or more chelating compounds, preferably clioquinol, phanquinone or a combination thereof, and optionally, instructions for use.

The kit may further comprise one or more antibacterial or antibiotic drugs or one or more antifungal drugs, or both classes of compounds. .

The kit may further comprise one or more additional drugs to treat an underlying condition or to promote the treatment of the infection. Such additional drugs include an antiviral drug, an anticancer chemotherapeutic or biotherapeutic agent, an anti-inflammatory drug, or a drug directed to treatment of sepsis.

The kit may further comprise an effective amount of Vitamin B 12 and/or folic acid.

The present invention is directed to the use of one or more chelating compounds that chelate metal ions for the manufacture of a composition or medicament for treating a bacterial or fungal infection in a subject, which metal ions are essential for function of (i) a bacterial or fungal metal- dependent enzyme, (ii) another bacterial or fungal metal-dependent protein, or (iii) another bacterial or fungal metal-dependent biologic activity required for metabolism, multiplication or survival of the bacteria or fungi.

Other uses of the present invention are parallel to the methods described above, and encompass the various features of the method of treatment embodiments.

DESCRIPTION OF THE PREFERRED EMBODIMENTS This invention is directed to methods of using chelators that chelate essential metal ions that necessary for the survival of pathogenic bacteria or fungi. This is the first disclosure of these compounds as antibacterial, anti-fungal or anti-infective compositions for the indicated microorganisms, in particular MDR bacteria and drug-resistant fungi.

In a first preferred embodiment, clioquinol, phanquinone or another chelating compound (one or a combination in a single pharmaceutical composition or a mixture in separate pharmaceutical compositions) is administered to a subject in need thereof , and acts as an inhibitors of, for example, metal-dependent bacterial β-lactamases. These chelating compounds (one or more) are used alone or, preferably, combined with other antibiotics of antifungals to overcome certain types of antibiotic or antifungal drug resistance. Specifically, the chelating compounds are used to overcome resistance mediated by secreted β-lactamase enzymes that otherwise inactivate most penicillins and other essential antibiotics. A primary target of the chelating compounds is any bacterial species or strain that produces MBLs, most of which are Zn-dependent.

The preferred subjects are human, though animals, particularly mammals, preferably commercially valuable mammals or pets, are intended within the scope of this invention. The present inventors have discovered that clioquinol has a strong anti-MMP effect through its action on the Zn-dependent enzyme. Based on this effect, clioquinol and the other chelating compounds described herein have potent antibacterial and specifically bactericidal effects on MMPs of mycoplasma, the bacteria responsible for infecting in the community and seriously ill hospitalized patients. The present inventor discovered that clioquinol is an inhibitor of MMP activity. Therefore clioquinol can have a bactericidal or bacteriostatic effect on pneumococci including penicillin- resistant strains.

As disclosed herein, clioquinol and the other chelating compounds potently chelate Zn ions. The high lipophilicity of clioquinol together with its high affinity for Zn allows it to inactivate

MBLs by inactivating the metallo-dependent enzyme, and thereby exert strong bactericidal effect on pathogenic strains that are resistant to (as well as those that are sensitive) to, other antibiotics. Based on the findings that Zn chelation specifically prevents pathological formation of biofilm and in particular MRSA, the fact that clioquinol and the other chelating compounds of the present invention chelate Zn, it is concluded that these drugs are inhibitors of biofilm formation, an effect that is beneficial for treatment of the relevant infections.

The invention provides a new method for the treatment of an infection caused by bacteria where agents such as the six compounds (or families of compounds) described below exert bactericidal or bacteriostatic effects on the causative bacteria. (1) Clioquinol whose chemical formula is C ₉ H ₅ ClINO (CAS RN 30-26-7, ACX number

X 1008995-7) is a potent chelating agent that chelates primarily zinc, copper, iron, nickel, cadmium and cobalt. This molecule is also known as 5-chloro-7-iodo-8-hydroxyquinoline, 5-chloro-7-iodo-8-quinolinol, 5-chloro-8-hydroxy-7-iodoquinoline, iodochlorohydroxyquinol, iodochlorohydroxyquinoline; chloroiodoquine; 7-iodo-5-chloro- 8 -hydroxyquinoline .

(2) 5,7-dichloro-2-methyl-8-hydroxyquinoline is potent chelating agent that chelates primarily zinc, copper, iron, nickel, cadmium and cobalt. It is also known by the following names: 5,7-dichloro-2-methyl-8-quinolinol, 5,7-dichloro-8-hydroxyquinaldine, and 5,7-dichloro-8- quinaldinol. (3) Phanquinone or 4,7-phenanthroline-5,6-dione, with the chemical formula C ₁₂ H _O N ₂ O ₂ (CAS RN 84-12-8, ACX number X 1064601-2) is a potent chelating agent that chelates primarily copper, zinc, iron, nickel, cadmium and cobalt.

(4) 5,7-Dichloro-8-hydroxyquinoline also known as 5,7-Dichloro-8-quinolinol has the formula C ₉ H ₅ Cl ₂ NO (CAS RN 773-76-2, ACX number X1030534-1) is a potent chelating agent that chelates primarily copper, zinc, iron, nickel, cadmium and cobalt.

(5) 5,7-Diiodo-8-hydroxyquinoline with the formula C ₉ H ₅ I ₂ NO (CAS RN 83-73-8 and ACX number X1039251-3) is a potent chelating agent that chelates copper, zinc, iron, nickel, cadmium and cobalt. It is also known as 5,7-diiodo-8-quinolinol; diiodohydroxyquin; diiodohydroxyquinoline; and diiodo-oxyquinoline

(6) More broadly, compounds having two fused 6-member rings with at least a nitrogen at position 1 and a hydrogen at position 8, preferably 8-hydroxy-quinolines, are potent chelating compounds of copper, zinc, iron, nickel, cadmium and cobalt.

Chemical entities (1) - (5) mentioned above are known drugs that have been used in the past for treating parasitic infections. They are possess potent chelating activity an affinity to zinc and copper as well as iron, nickel, cobalt, cadmium and other metal ions. All five are well tolerated in humans and cause a minimal number of drug-related adverse events that are usually benign. The five drugs are absorbed well by the human gastro-intestinal tract and achieve high concentrations n the plasma and in body fluids and tissues. The present inventor has newly discovered that these chelating agents are useful against bacteria by acting on the metabolism of the causative bacteria secreting β-lactamases and in particular MBLs that are mostly Zn-dependent. The invention provides a new method for treating an infection that caused by, or is suspected of being caused, by bacteria secreting such MBLs.

In particular clioquinol is highly active in vitro and in vivo in experimental infections by MDR bacteria like methicillin resistant Staphylococcus aureus that are public health threats and are difficult to treat in human infectious disease. Similarly clioquinol is active against extended spectrum β-lactamase producing bacteria like Acinetobacter and Enterobacteriaceae, causes of serious nosocomial infections. The combination of phanquinone or of clioquinol further combined with the antibiotic meropenem show that these classes of drugs can be used in combination for improved anti-bacterial effects.

As noted, β-lactamases render the penicillin and cephalosporin antibiotics inactive, and the secreted enzymes produced by some bacteria and are responsible for their resistance to the β-lactam antibiotics (like penicillins, cephalosporins, cephamycins, carmapenems). These β-lactamase- sensitive antibiotics have a common element in their molecular structure: a four-atom ring known as a β-lactam. The lactamase enzyme breaks that ring open, deactivating any antibacterial property. The chelating compounds (l)-(5) above are intended to be used to act as specific inhibitors of β-lactamases secreted by bacteria, most preferably, MBLs that confer resistance to β-lactam antibiotics. The chelation-mediated inhibition restores the antimicrobial activity of β-lactam antibiotics against any β-lactamase-secreting, β-lactam-resistant bacteria.

Two different families of β lactam-degrading enzymes (Rossolini, GM et al, Antimicrobial Agents and Chemotherapy, 2001, 45:837-44), which catalyze the same reaction, i.e.., the opening of the β-lactam ring by hydrolysis of the amide bond, but are structurally and mechanistically unrelated, have evolved in bacteria: (i) active- site serine- β-lactamases and (ii) MBLs. The latter enzymes were identified some 25 years later than the former, and have remained less common among pathogenic bacteria. Nevertheless, they are potentially very dangerous as resistance effectors due to their efficient hydrolysis of carbapenem antibiotics which are stable to hydrolysis by most β-lactamases. These carbapenem antibiotics often represent "last-resort" agents for chemotherapy of MDR pathogens due to their lack of susceptibility to the serine-β-lactamase inhibitors such as clavulanic acid and penicillanic acid sulfones. The recent emergence of mobile MBL genes capable of horizontal spread among nosocomial strains of Enterobacteriaceae, P. aeruginosa, and other gram-negative nonfermenters has considerably increased attention to these enzymes which constitute one of the current major threats of microbial drug resistance.

In another embodiment, the present methods are directed to the destruction, inhibition, inactivation and neutralization of the active- site serine- β-lactamases that are metal ion-dependent.

In another embodiment, the present methods are directed to antimicrobials effects mediated via other bacterial proteins/enzyme or functions that are metal ion-dependent and in which the chelation of the metal ions provided by the present invention results in a bacteriostatic or bactericidal effect. Most known MBLs are encoded by chromosomal genes of some bacterial species that are primarily members of the environmental microbiota, such as Bacillus cereus, Stenotrophomonas maltophilia, Aeromonas spp., Myroides odoratus (formerly Flavobacterium odoratum), Legionella gormanii and Chryseobacterium spp. However the present invention provides an effective antibacterial approach for those cases in which yet unknown environmental bacterial species are the most likely sources of the mobile MBL determinants that have recently appeared among gram- negative pathogens. Therefore, environmental bacteria could be an important reservoir of similar resistance determinants.

The molecular classification of β-lactamases is based on the nucleotide and amino acid sequences in these enzymes. To date, four classes are recognized (A-D), correlating with the

functional classification. Classes A, C, and D act by a serine-based mechanism, whereas class B or metallo-β lactamases (MBLs) require Zn for their action.

It is well known that chelating compounds often bind several different metal ions often with varying affinities. According to the invention, a chelating compound having specificity (i.e., relative specificity) for Cu has greater affinity for Cu than for other metal ions. Preferably, the binding constant for Zn is higher than 5.0, preferably higher than 6.0 and most preferably higher than 7.0.

A Zn-specific or -selective chelating compound preferably has greater affinity for Zn than for other metal ions, and preferably is a binding constant higher than 5.0, preferably higher than 6.0 and most preferably higher than 7.0.

The inclusion of the chelating compounds in the present methods results in inactivation of the β-lactamases, particularly the MBLs that allows to β-lactam antibiotic to kill the causative bacteria. Further the intrinsic antimicrobial activity of the chelating compounds has additional synergistic effects on the bactericidal or bacteriostatic effect of the concomitantly administered "traditional" antibiotic whether it be a β-lactam or other class of antibiotic.

Infections that are treated by the present methods using of one or more of the above chelating agents (I)- (5) or members of the additional "group" (6) include the following: respiratory tract infections, urinary tract infections, genital and gynecological infections, skin and soft tissue infections, surgical, post-operative and visceral infections, abscesses, fistulae, faschiitis, complicated skin and soft tissue infections, catheter and injection- site infections, blood stream infections, septicemia, sepsis, endocarditis, gastro-intestinal infections, hospital acquired infections, community acquired infections, superinfections, cerebro-spinal infections, any infections in compromised (e.g., immunocompromised) hosts, infections in patients with other infectious diseases, whether viral, fungal or parasitic, which hosts may also be immunocompromised, e.g. HIV, Hepatitis B and C, fungal or parasitic infections.

Examples of infections that are treated successfully by the present methods include, without limitation: Staphylococcal infections including those caused by methicillin resistant strains, Streptococcal infections including penicillin-resistant S. pneumoniae infections, MDR gram negative nosocomial infections including those caused by extended spectrum or MBL-producing strains, upper and lower respiratory infections, surgical, skin and soft tissue infections, infections

caused by Enterococcus faecalis or E. faecium, infections caused by Clostridium difficile, Legionellosis and Mycoplasma infections

Infections that are treated by the present methods include infections in children or adults, including the elderly, subjects with renal, liver or immunological insufficiency or incompetence, for example, cancer, leukemia, diabetes or AIDS.

Thos skilled in the art will appreciate that binding constants for various chelating agents the use of which is within the scope of this invention may be found in the chemical literature or may be determined readily using generally known and well-established procedures. It is therefore within the skills of the average practitioner to select suitable chelating compounds for the present invention. A non-limiting list of infectious agents that can be treated by the present methods of MDR nosocomial or community infections include: Acinetobacter baumannii, Acinetobacter baumannii Bouvet and, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter haemolyticus Bouvet and, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter Iwoffii, Acinetobacter radioresistens, Acinetobacter sp., Acinetobacter sp. deposited as Acinetobacter tartarogenes, Aeromonas caviae, Aeromonas caviae (ex Eddy), Aeromonas enteropelo genes, Aeromonas eucrenophila, Aeromonas hydrophila, Aeromonas ichthiosmia, Aeromonas punctata, Aeromonas salmonicida subsp. achromo genes, Aeromonas sobria, Aeromonas veronii, Bacillus licheniformis, Bacillus lichenisformis, Bacillus spp, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides gracilis, Bacteroides ovatus, Bacteroides ureolyticus, Campylobacter coli, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter jejuni, Campylobacter jejuni subsp. jejuni,

Campylobacter lari, Campylobacter mucosalis (Lawson et, Campylobacter sputorum, Campylobacter sputorum biovar sputorum, Chromobacterium violaceum, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter diversus, Citrobacter farmeri, Citrobacter freundii, Citrobacter gillenii, Citrobacter koseri, Citrobacter murliniae, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter sp., Citrobacter werkmanii, Citrobacter youngae, Clostridium acetobutylicus, Clostridium beijerinckii, Clostridium bifermentans, Clostridium butyricum, Clostridium cocleatum, Clostridium difficile, Clostridium novyi, Clostridium oedematiens, Clostridium perfringens, Clostridium septicum, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium tetani, Clostridum sordellii, Corvnebacterium xerosis, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanum, Corynebacterium ammoniagenes, Corynebacterium

argentoratense, Corynebacterium aurantiacum, Corynebacterium flavescens, Corynebacterium fujiokense, Corynebacterium glutamicum, Corynebacterium helvolum, Corynebacterium herculis, Corynebacterium hydrocarbooxydans, Corynebacterium liquefaciens, Corynebacterium matruchotii, Corynebacterium melassecola, Corynebacterium murisepticum, Corynebacterium mycetoides, Corynebacterium nitrilophilus, Corynebacterium paraldehydium, Corynebacterium petrophilum,

Corynebacterium propinquum, Corynebacterium sp., Corynebacterium variabile, Corynebacterium vitaeruminis, Edwardsiella hoshinae, Edwardsiella ictaluri, Edwardsiella tarda, Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter amnigenus, Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter cloacae (Jordan) Hormaeche, Enterobacter cloacae, Enterobacter dissolvens, Enterobacter gergoviae, Enterobacter intermedius, Enterobacter nimipressuralis, Enterobacter sakazakii, Enterococcus avium, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Erwinia amylovora, Erwinia chrysanthemi, Erwinia cypripedii, Erwinia mallotivora, Erwinia psidii, Erwinia rhapontici, Erwinia sp., Erwinia tracheiphila, Escherichia blattae, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia intermedia, Escherichia vulneris, Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium necrogenes, Fusobacterium nucleatum subsp. nucleatum, Fusobacterium russii, Fusobacterium ulcerans, Fusobacterium varium, Haemophilus ducreyi, Haemophilus influenzae, Hafnia alvei, Klebsiella oxytoca, Klebsiella ozaenae, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. rhino scleromatis, Klebsiella sp., Moraxella (Moraxella), Moraxella (Moraxella) osloensis (Bovre, Morganella morganii, Morganella morganii subsp. morganii, Morganella morganii subsp. sibonii, Mycobacterium avium, Mycobacterium diernhoferi, Mycobacterium fortuitum subsp. fortuitum, Mycobacterium smegmatis, Mycobacterium sp., Mycoplana bullata, Mycoplana dimorpha, Mycoplana ramosa, Mycoplasma alkalescens, Mycoplasma arginini, Mycoplasma bovoculi, Mycoplasma californicum, Mycoplasma canadense, Mycoplasma capricolum subsp. capricolum, Mycoplasma citelli, Mycoplasma collis, Mycoplasma columbinum, Mycoplasma columborale, Mycoplasma cricetuli, Mycoplasma equigenitalium, Mycoplasma equirhinis, Mycoplasma faucium, Mycoplasma feliminutum, Mycoplasma lagogenitalium, Mycoplasma mobile, Mycoplasma ovipneumoniae, Mycoplasma putrefaciens, Mycoplasma subdolum, Mycoplasma testudinis, Mycoplasma verecundum, Neisseria macacae, Neisseria mucosa, Nocardia asteroides, Nocardia canicruria, Nocardia corallina, Nocardia sp., Pasteurella multocida subsp. multocida, Pectinatus cerevisiiphilus, Proteus mirabilis, Proteus

mitajiri, Proteus morganii, Proteus myxofaciens, Proteus penneri, Proteus rettgeri, Proteus vulgaris, Protomyces inundatus, Providencia alcalifaciens, Providencia heimbachae, Providencia rettgeri, Providencia rustigianii, Providencia sp., Providencia stuartii, Pseudomonas acidophila, Pseudomonas aeruginosa, Pseudomonas aeruginosa subsp. erythrogenes, Pseudomonas agarici, Pseudomonas alcaligenes, Pseudomonas alkanolytica, Pseudomonas amyloderamosa, Pseudomonas anguilliseptica, Pseudomonas aromatica, Pseudomonas asplenii, Pseudomonas aurantiaca, Pseudomonas auricularis, Pseudomonas avellanae, Pseudomonas azelaica, Pseudomonas boreopolis, Pseudomonas cannae, Pseudomonas caricapapayae, Pseudomonas chlororaphis, Pseudomonas cichorii, Pseudomonas cissicola, Pseudomonas citronellolis, Pseudomonas coenobios, Pseudomonas corrugata, Pseudomonas cruciviae, Pseudomonas dacunhae, Pseudomonas diazotrophicus, Pseudomonas ficuserectae,

Pseudomonas flavescens, Pseudomonas fleet ens, Pseudomonas floridana, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas fulva, Pseudomonas huttiensis, Pseudomonas lanceolata, Pseudomonas lundensis, Pseudomonas luteochromogenes, Pseudomonas luteola, Pseudomonas marginalis, Pseudomonas meliae, Pseudomonas mendocina, Pseudomonas mephitica, Pseudomonas mesoacidophila, Pseudomonas monteilii, Pseudomonas mucidolens, Pseudomonas mutabilis,

Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas oryzihabitans, Pseudomonas papaveris, Pseudomonas perolens subsp. gdansk, Pseudomonas pictorum, Pseudomonas pseudoalcaligenes, Pseudomonas pseudoalcaligenes subsp. pseudoalcaligenes, Pseudomonas putida, Pseudomonas reptilivora, Pseudomonas resinovorans, Pseudomonas savastanoi, Pseudomonas septica, Pseudomonas sp., Pseudomonas spinosa, Pseudomonas stanieri, Pseudomonas straminea,

Pseudomonas stutzeri, Pseudomonas synxantha, Pseudomonas syringae, Pseudomonas syringae subsp. syringae, Pseudomonas taetrolens, Pseudomonas tolaasii, Pseudomonas turbinellae, Pseudomonas tuticorinensis, Pseudomonas veronii, Pseudomonas viridiflava, Pseudomonas viridilivida, Salmonella choleraesuis subsp. arizonae, Salmonella choleraesuis subsp. choleraesuis, Salmonella choleraesuis subsp. diarizonae, Salmonella choleraesuis subsp. houtenae, Salmonella choleraesuis subsp. salamae, Salmonella enterica, Salmonella enteritidis, Salmonella infantis, Salmonella pullorum, Salmonella sp., Salmonella typhi, Salmonella typhimurium, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia marcescens, Serratia marcescens Bizio deposited, Serratia odorifera, Serratia plymuthica, Serratia proteamaculans subsp. proteamaculans, Serratia proteamaculans subsp. quinovora, Serratia rubidaea, Serratia sp., Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shigella sp., Staphylococcus aureus, Staphylococcus aureus subsp.

aureus, Staphylococcus auricularis, Staphylococcus bovis, Staphylococcus capitis subsp. capitis, Staphylococcus caprae, Staphylococcus chromogenes (Devriese et, Staphylococcus cohnii subsp. cohnii, Staphylococcus epidermidis, Staphylococcus faecalis, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis subsp. hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus lentus (Kloos et, Staphylococcus mitis, Staphylococcus muscae,

Staphylococcus pulvereri, Staphylococcus saprophyticus, Staphylococcus sciuri subsp. carnaticus, Staphylococcus sciuri subsp. rodentium, Staphylococcus sciuri subsp. sciuri, Staphylococcus simulans, Staphylococcus sp., Staphylococcus warneri, Staphylococcus xylosus, Streptobacillus moniliformis, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus ferus, Streptococcus intermedius, Streptococcus lactis, Streptococcus mitis, Streptococcus mutans,

Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguis, Streptococcus sp., Veillonella dispar, Veillonella parvula, Vibrio adaptatus, Vibrio aerogenes, Vibrio aestuarianus, Vibrio alginolyticus, Vibrio algosus, Vibrio anguillarum, Vibrio campbellii, Vibrio cholerae, Vibrio cincinnatiensis, Vibrio cyclitrophicus, Vibrio cyclosites, Vibrio diazotrophicus, Vibrio fischeri, Vibrio fluvialis, Vibrio furnissii, Vibrio gazogenes, Vibrio harveyi, Vibrio hollisae, Vibrio ichthyoenteri, Vibrio lentus, Vibrio logei, Vibrio marinagilis, Vibrio marinofulvus, Vibrio marinovulgaris, Vibrio mediterranei, Vibrio metschnikovii, Vibrio mimicus, Vibrio mytili, Vibrio natriegens, Vibrio navarrensis, Vibrio nereis, Vibrio nigripulchritudo, Vibrio ordalii, Vibrio parahaemolyticus, Vibrio pectenicida, Vibrio penaeicida, Vibrio ponticus, Vibrio proteolyticus, Vibrio salmonicida, Vibrio shiloii, Vibrio sp., Vibrio splendidus, Vibrio tubiashii, Vibrio tyrosinaticus, Vibrio vulnificus, Yersinia aldovae, Yersinia enterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia rohdei, Yersinia ruckeri.

Preferred chelating compounds used in the present methods include, but are not limited to: clioquinol; phanquinone (4,7-phenanthroline-5,6-dione); 5,7-dichloro-8-hydroxy-quinaldine; 5,7- diiodo-8-hydroxyquinoline; an 8-hydroxy-quinoline derivatives in which the phenol ring is 2-, 3-, and 4-substituted with hydroxy, fluoro, carboxy, and/or acetamido groups; 5,7-dibromo-8-hydroxy- quinoline; 2-, 3-, and 4-substituted phenol derivatives substituted with hydroxy, fluoro, carboxy, and acetamido moieties; 2- methyl- 8 -hydroxyquinoline; 2-methyl-8-hydroxy-quinaldine; 5,7- dichloro-2-methyl-8-hydroxyquinoline; 5,7-dichloro-8-hydroxy-8-quinolinol; 5-iodo-4'-chloro-3'- quinaldine, 5 -methyl- oxine (5-methyl-8-hydroxyquinoline); 5-methyl-oxine derivatives; 2- mercaptopyridine-n-oxide; 5-formyl-8- hydroxyquinoline; 5-iodo-8-hydroxyquinoline; 5-fluoro-8-

hydroxyquinoline; 5-acetyl-8-hydroxyquinoline; 5-methoxymethyl-8-hydroxyquinoline; ethyl-5-(8- hydroxy-8-quinolinolium l'-quinolyl)acetate; methyl-5(8- hydroxyquinolyl) acetate; ethyl-5-(8- hydroxyquinolyl)acetate; 2,7,8-trihydroxyquinoline; a 4-hydroxyquinoline-3-carboxylate; a 5- phenylethyl-4-hydroxyquinoline-3-carboxylic acid; dibromo-8-benzoyl-oxyquinoldine; an arylglyoxal n-7-amino-5-substituted 8 -hydroxyquinoline hemiacetal; a 5-phenylglyoxylidenamin-8- hydroxyquinoline; α,α'-dipyridyl-8-hydroxyquinoline; 2,2',2"-tripyridine; 2,6-dihydroxyquinoline; 5-formyl-l-methoxycarbonyl-4,6,8-trihydroxyphenazine; a 5- or 7-methylthio-8-hydroxyquinoline derivative; a 4-hydroxy-5-chloro-7-((β-quinoline-2-carboxylic acid; a 3-hydroxyquinoline-2- carboxylic acid; a 5-phenylethyl-4- hydroxyquinoline-3-carboxylic acid; a 2-n-alky-l-4- hydroxy- quinoline derivate; o-acetyl-8-hydroxyquinoline; S-acetyl-8- mercaptoquinoline; 5,7 dibromo-8- benzoyl-oxyquinoldine; a 5 -phenylglyoxylidenamin- 8 -hydroxyquinoline; ethyl 6,7-di-isobutoxy-4- hydroxyquinoline-3-carboxylate; 5-chloro-8-hydroxyquinoline; a 5-chloro-8-hydroxy-8-quinol- inolium 3'-quinoline (chloroxine) carboxylic acid ester; m-phenylenediamine; laH-oxazirino[2,3-a- quinoline-la-carbonitrile or a substituted derivative thereof to a corresponding 3 -hydroxyquinoline derivative; 6,7-dimethoxy-4-hydroxyquinoline HCl; 4-nitroquinoline 1 -oxide; nitroxoline; 8- hydroxyquinoline-7-carboxylic acid; hydroxy- 8 -quinolinolium 4',7'-dibromo-3'-chlorquinaldol, iodoquinol; 8-hydroxy-5-quinolinesulfonic acid; benzoxiquine; 8-(methylmercurioxy)quinoline; cloxyquin; oxyquinoline sulfate; chlorohydroxyquinoline; 8-hydroxyquinoline8-hydroxyquinoline citrate; oxyquinoline; bromoxyquinoline; 8-hydroxy-7-iodo-5-quinolinesulfonic acid; a derivative of 1,2,3,4-tetrahydroquinoline; 2-heptyl-4- hydroxyquinoline-n- oxide; 2-n-heptyl-4- hydroxyquinoline-n-oxide; a 4-hydroxyquinoline-3-carboxylic acid that is 7-substituted with benzyloxy, phenethyloxy or phenoxyethyloxy; 6-hydroxyquinoline; 6-nitroquinoline; 8- nitroquinoline; 6-chloroxine; 4-(2-(4-(dimethylamino)phenyl)vinyl)-8-quinolinol; 8-hydroxy-2- methylquinoline; 8-methylquinoline; 8-hydroxy-quinoline; 5,7-dibromoquinoline; 2-n-nonyl-4- hydroxy[3-3h]quinoline; 2n-nonyl-48-quinolinol n-oxide; 5-chloro-7-(2-((3-(diethylamino)- propyl)amino)ethyl)-8-quinolinol; 5-bromo-8-quinolinol; 8-quinolinol hydrogen sulfate; 2-(2-(2- pyridyl)vinyl)-8-quinolinol methylchloride; 8-quinolinol salicylate; 8-quinolinol acetate; 5- acetyloxine; 5-nitroso-8-quinolinol; 4-methyl-8-quinolinol; nitroxoline; 5-(chloromethyl)-8-quinol- inol; 5-(phenylazo)-8-quinolinol; clamoxyquin HCl; 5-((p-hydroxyphenyl)azo)-8-quinolinol; 4,8- dimethyl-2-hydroxyquinoline; -n-oxide; 8-hydroxyquinoline-5-sulfonic acid; a 2,2',2"-terpyridine

complex; 8-hydroxyquinoline-5- sulfonic acid; 5-nitro-8-8-((tributylstannyl)oxy)quinoline; 2-(2-(5- nitro-2-furyl)vinyl)-8-quinolinol acetate; 5-(2-(6-ethoxy- 1-methyl- lλ(5)-quinolin-2-yl)vinyl)-8- quinolinol; 5-(2-(l,6-dimethyl-lλ(5)-quinolin-2-yl)vinyl)-8-quinolinol; 5-benzyl-8-quinolinol; oxyquinoline benzoate; 8-quinolinol dihydrogen phosphate; 7-bromo-5-chloro-8-quinolinol; 8-quin- olinol oxalate; 5-methyl-7-(2-(l-methyl-lλ(5)-quinolin-4-yl)vinyl)-8-quinol inol; 2-methyl-8-quinol- inol HCl; 5-iodo-8-quinolinol; 8-quinolinol methylcarbamate; 7-(2-(6-ethoxy-l-methyl-l-λ(5)- quinolin-2-yl)vinyl)-5-methyl-8-quinolinol; 8-hydroxyquinoline zinc salt; 8-quinolinol potassium sulfate; 2-methyl-8-quinolyl methylcarbamate; 8-hydroxyquinoline glucuronide, bromoquinaldol, 5- chloro-7-iodo-8-quinolinol hydrogen sulfate; 5,7-dichloro-8-quinolinol benzoate, 7-amino-5-fluoro- 8-quinolinol; 7-nitro-8-quinolinol; 7-amino-5-iodo-8-quinolinol; 8-quinolinol phosphate; 5-amino- 8-hydroxyquinoline; 2-mercapto-quinoline-n-oxide; 5,6-benzoquinoline; 5-methyl-8-hydroxy- quinoline-4-hydroxy-quinoline; 26-((-n-alkyl-4-hydroxyquinoline; ethylenediaminetetraacetic acid; o-phenanthroline; 1,10-phenanthroline; 2,9-dimethyl-l,10-phenanthroline; (2'-hydoxyphenyl)- pyridine; rhodotorulic acid; mycobactin p; 8-hydroxyquinoline-7-carboxylate; a 5-substituted 8- hydroxyquinoline; 5-chloro-8-hydroxyquinoline; 4-hydroxyquinoline; a 6-substituted-4- hydroxyquinoline-S-carboxylic acid; a 7-substituted-4-hydroxyquinoline-S-carboxylic acid; a δ- substituted-4-hydroxyquinoline-S-carboxylic acids; 5-7-dibromo-8-hydroxyquinoline; 2,2'- bipyridine; an 8-hydroxyquinoline ester; a halogen derivative of salicylanilide; batimastat; a hydroxamic acid;a fenamate; mebiquine; 8-chloro-2-hydroxyquinoline; 5-chloro-7-((3,4-dihydro-l- methyl-7-phenyl-2(lh)-isoquinolyl)methyl)-8-quinolinol; 5-((p-methoxybenzoyl)methylenamino)- 8-quinolinol; 5-(n-acetaminophenylazo)-8-oxyquinoline; 5-((p-phenylthiobenzoyl)methylenamino)- 8-quinolinol; 5-((p-chlorobenzoyl)methylenamino)-8-quinolinol; 5-((p-phenylbenzoyl)meth- enamino)-8-quinolinol; 5-((p-nitrobenzoyl)methylenamino)-8-quinolinol; 5-((p-methylbenzoyl)- methylenamino)-8-quinolinol; 7-nitro-8-quinolinol salicylate; 7-nitro-8-quinolinol 3-(phenylthio)_ benzoate; 7-nitro-8-quinolinol m-isopropoxybenzoate; 7-nitro-8-quinolinol 4-(phenylthio)benzoate; 7-nitro-8-quinolyl 2,3-dimethoxycinnamate; 7-nitro-8-quinolinol o-(benzyloxy)benzoate; 7-nitro-8- quinolinol 2,4-dimethoxybenzoate; 7-nitro-8-quinolinol 2-(phenylthio)benzoate; 7-nitro-8-quinolyl 2,3-dimethoxy-α-methylcinnamate; 7-nitro-8-quinolinol m-(benzyloxy)benzoate; 7-nitro-8-quinol- inol p-isopropoxybenzoate; 7-nitro-8-quinolinol 4-propoxybenzoate; 7-nitro-8-quinolinol m- butoxybenzoate; 7-nitro-8-quinolinol; 2,6-dimethoxybenzoate; 7-nitro-8-quinolinol 4-biphenylcar-

boxylate; 7-nitro-8-quinolinol p-butoxybenzoate; 7-nitro-8-quinolinol m-ethoxybenzoate; 7-nitro-8- quinolinol o-ethoxybenzoate; 7-nitro-8-quinolinol benzoate; 7-nitro-8-quinolinol o-bromobenzoate; 7-nitro-8-quinolinol p-bromobenzoate; 7-nitro-8-quinolinol m-chlorobenzoate; 7-nitro-8-quinolinol p-chlorobenzoate; 7-nitro-8-quinolinol m-bromobenzoate; 7-nitro-8-quinolinol p-ethoxybenzoate; 7-nitro-8-quinolinol 4-phenoxybenzoate; 7-nitro-8-quinolinol p-nitrobenzoate; 7-nitro-8-quinolinol o-nitrobenzoate; 7-nitro-8-quinolinol 3-phenoxybenzoate; 7-nitro-8-quinolinol 2,4-dichloroben- zoate; 7-nitro-8-quinolinol o-chlorobenzoate; (l,l'-biphenyl)-2-carboxylic acid, 7-nitro-8-quin- olinyl ester; 7-nitro-8-quinolyl p-toluate; 7-nitro-8-quinolyl m-toluate; 7-nitro-8-quinolinol 2- chloro-4-nitrobenzoate; 7-nitro-8-quinolinol 2-chloro-6-nitrobenzoate; 7-nitro-8-quinolyl p-anisate; 7-nitro-8-quinolinol 3,4,5-trimethoxybenzoate; 7-nitro-8-quinolinol 3,4-dichlorobenzoate; 7-nitro-8- quinolinol 3,4-dimethoxybenzoate; 7-nitro-8-quinolinol 3,5-dimethoxybenzoate; 7-nitro-8-quinol- inol 4-(l,l-dimethylethyl)benzoate; 7-nitro-8-quinolyl o-veratrate; 7-nitro-8-quinolyl p-anisate; 7- nitro-8-quinolyl m-anisate; 7-nitro-8-quinolinol acetylsalicylate; 7-nitro-8-quinolinol 2,5- dimethoxybenzoate; 7-nitro-8-quinolinol 2-iodobenzoate; 7-nitro-8-quinolinol 2-phenoxybenzoate; 7-nitro-8-quinolinol m-propoxybenzoate; 8-hydroxyquinoline glucoside; 8-hydroxyquinoline-5,7- disulfonic acid; 5-(p-tolylazo)-8-quinolinol; 7-chloro-5-iodo-8-quinolinol; 5,7-dimethyl-8-quinol- inol; 8-quinolinolium salicylate; naphthylazoxine; 5,7-dichloro-8-quinolinol acetate; 7-nitro-8-quin- olinol 2,3,5, 6-tetramethylbenzoate; 4-aminosalicylic acid oxine; clioquinol fluocinolone acetonide; clioquinol βmethasone 17-valerate; 2-(2-hydroxy-5-n-hexylphenyl)-8-quinolinol-4-carboxylic acid; 8-hydroxyquinoline-4-carboxylic acid; 8-quinolinol succinate; di-8-oxyquinoline-n-aminosalicylic acid; 7-(2-ethyl-l-hexenyl)-8-quinolinol; disodium 8-hydroxy-7-((6-sulfonato-2- naphthyl)azo)quinoline-5-sulfonate; 5-chloro-7-iodo-8-quinolinol 4-methylbenzenesulfonate; dimethyl- 8 -quinolyl methylcarbamate HCl; oxyquinoline methiodide methylcarbamate; 5-chloro-8- quinolium 4'-chloro-3'-hydroxy-2'-naphthoate; 8-quinolinol 5-chloro-3'-hydroxy-2'-naphthoate; 8- quinolinol 5-iodo-4'-chloro-3'-hydroxy-2'-naphthoate; 8-quinolinolium l'-hydroxy-2'-naphthoate; 5-chloro-7-((β-hydroxy-3-nitro-α-oxo)phenethylamino)-8-qui nolinol; 8-quinolinolium 3'-hydroxy- 2'-naphthoate; 8-quinolinolium 4',7'-dibromo-3'-hydroxy-2'-naphthoate; 8-quinolinol monophosphate; 5-(p-acetamidophenylazo)-8-quinolinol HCl; 8-quinolinol 5-iodo-4'-chloro-3'- hydroxy-2'-naphthoate copper (ii) salt; 8-quinolinol 5-chloro-3'-hydroxy-2'-naphthoate copper (ii) salt; 8-quinolinolium 4',7'-dibromo-3'-hydroxy-2'-naphthoate copper (ii) salt; 8-quinolinolium T-

bromo-3'-hydroxy-2'-naphthoate copper (ii) salt; 8-quinolinolium salicylate copper (ii) salt; 8-quin- olinolium l'-hydroxy-2'-naphthoate copper (ii) salt; 8-quinolinolium 3'-hydroxy-2'-naphthoate, copper (ii) salt; 7-(4-ethyl-l -methyl- l-octenyl)-8-quinolinol; n-methyl-2,2'-imino-di(8-quinolinol); 8-hydroxyquinoline magnesium salt; 8-quinolinol alkyl(Ci ₂ -i ₆ ) ester; 7-(2-ethylhexyl)-8-quinolinol; 5-chloro-8-quinolinol 7-triisobutenyl ester; 7-(tetrapropenyl)-8-quinolinol; 8-chloroquinoline zinc complex; 2-amino-8-quinolinol; 7-(4-ethyl-l-methyleneoctyl)-8-quinolinol; 7-(4-ethyl-l-methyl- octyl)-8-quinolinol; 7-(α-2-thiazolylaminobenzyl)-8-quinolinol; 7-(α-(m-trifluoromethylani- lino)benzyl)-8-quinolinol; 7-(α-(3-methyl-2-pyridylamino)benzyl)-8-quinolinol; 7-(3-quinolyl- aminobenzyl)-8-quinolinol; 7-amino-5-chloro-8-quinolinol HCl; 7-(α-2-benzothiazolyl- aminobenzyl)-8-quinolinol; 7-((4-methyl-l-piperazinyl)methyl)-5-nitro-8-quinolinol; 5-nitro-7-(l- piperidinylmethyl)-8-quinolinol; 7-(4-morpholinylmethyl)-5-nitro-8-quinolinol; 5-nitro-7-(l-pyrrol- idinylmethyl)-8-quinolinol; 7-((diethylamino)methyl)-5-nitro-8-quinolinol; 7-((bis(l-methylethyl)- amino)methyl)-5-nitro-8-quinolinol; quinoderm hydrocortisone, quinoderm; n-butyl-2,2'-imino-di(8- quinolinol); 6-((4-(diethylamino)-l-methylbutyl)amino)-2,4-dimethyl-8-qui nolinol; 3-ethyl-8-quin- olinol; and 8-quinolinol hemisulfate salt hemihydrate.

In addition several other chelating compounds are intended, such as ethylenediaminetetraacetic acid; o-phenanthroline; 1,10-phenanthroline; 2,9-dimethyl-l,10-phenanthroline; (2'- hydoxyphenyl)pyridine; rhodotorulic acid; mycobactin p; 8-hydroxyquinoline -7-carboxylate; a 5- substituted 8- hydroxyquinoline; 5-chloro-8-hydroxyquinoline; 4-hydroxyquinoline; a 6-substituted-4- hydroxyquinoline-S-carboxylic acid; a 7-substituted-4-hydroxyquinoline-S-carboxylic acid; a δ- substituted-4-hydroxyquinoline-S-carboxylic acids; 5-7-dibromo-8-hydroxyquinoline; 2,2'-bipyridine; an 8-hydroxyquinoline ester; a halogen derivative of salicylanilide; batimastat; a hydroxamic acid and a fenamate.

The preferred chelating compounds are clioquinol, 5,7-dichloro-8-hydroxy-quinaldine, phanquinone, 5,7-dichloro-8-hydroxyquinoline, 5,7-di-iodo-8-hydroxyquinoline and compounds having two fused 6-member rings with at least a nitrogen at position 1 and a hydrogen at position 8.

In one preferred embodiment the invention relates to the use of a combination of at least one copper specific chelator and at least one zinc specific chelator for the treatment of nosocomial pneumonia.

Thus, one or more Cu-specific chelator(s) may be administered to the subject in need of such treatment simultaneously with, or sequentially with, the administration of the one or more Zn- specific chelator(s). It is preferred to administer the one or more Cu specific chelator(s) and the one or more Zn specific chelator(s) with a timing so that a therapeutic concentration of each is reached at least in part of the period of the treatment interval.

The one or more copper specific chelator(s) and/or the one or more zinc specific chelator(s) is (are) according to the invention administered to a patient subject in need therefore in form of one or more pharmaceutical composition(s). The pharmaceutical compositions are prepared according to well known procedures for formulating pharmaceutical compositions as it will be well known within the area. Techniques and formulations useful herein are well-known in the pharmaceutical art and may be found, for example, in Gennaro, AR, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 21 ^st Ed, 2005 (or latest edition); Rowe, R.C. et ah, Handbook of Pharmaceutical Excipient, APhA Publications, 5 ^th ed, 2005 (or latest edition); Ansel, HC & Stoklosa, MJ, Pharmaceutical Calculations, Lippincott Williams & Wilkins Publishers, 12 ^th Ed, 2005 (or latest edition); Watson, D (ed), Pharmaceutical Analysis: A Textbook or Pharmacy Students and Pharmaceutical Chemists, Publisher: Churchill Livingstone; 2 ⁿ ed, 2005 (or latest edition); or Goodman & Gilman's The Pharmacological Basis of Therapeutics, Brunton, LL et ah, eds, 11 ^th edition, McGraw-Hill Professional, New York, 2005

The pharmaceutical compositions according to the invention is explained in further details below with reference to clioquinol and phanquinone even thought the skilled person will appreciate that the disclosure applies likewise to other chelators according to the invention.

The pharmaceutical composition manufactured using, for example, clioquinol, comprises one or more pharmaceutical acceptable carriers and, optionally, one or more further active constituent(s). The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof. In a preferred embodiment, the clioquinol and, optionally, further active constituents in the pharmaceutical composition are purified.

Preferably, formulations of a concentrated (stock) solutions guarantees long term storage of clioquinol (or other chelating agents) or a salt thereof, without changes in the parent compound or

the formation of by-products in the concentrate solution which would preferably be injected as i.v. infusion after dilution with an appropriate diluent.

Clioquinol can be dissolved in physiologically acceptable solvents e.g. water or an organic solvent. Examples of solvents are alcohol, physiological saline, an aliphatic amide, a glycol, a polyalcohol, esters of a polyalcohol, a polyglycol, a polyether, a sugar alcohol or mixtures thereof, with the use of NaOH to adjust the pH eventually. A preferred mixture is water and ethanol in a v/v ratio of 90:10 to 70:30, preferably 80:20 to which NaOH can be added until a clear solution is reached within a preferred pH range of 8 to 9.

In one embodiment, the physiologically acceptable solvent is a mixture of water and organic solvents, preferably a mixture comprising from about 100% to about 40%, more preferably from about 90% to about 50%, more preferably from about 80% to about 60%, by volume of water in a pH range of 7 to 10.

To stabilize the solution, a physiologically acceptable excipient including, for example, a wetting agent, preferably N-methylpyrrolidone, a polyvinylpyrrolidone such as, for example, Povidone 12 PF, Povidone 17 PF, Povidone 25, Povidone 30, Povidone 90 F, a cyclodextrin, such as, for example, 2-hydroxypropyl-β-cyclodextrin, macrogol hydroxystearate, and macrogol glycerol ricinoleate, can be added.

A concentrated stock solution may be prepared by dissolving clioquinol in water or isotonic sodium chloride solution or alcohol or in a mixture thereof by adjusting the pH in a range of 7 to 10 with NaOH. Mixtures of water/ethanol, water/propylene glycol, isotonic sodium chloride solution/ethanol or isotonic sodium chloride solution/propylene glycol, such as a mixture of from about 100% to about 40%, particularly from about 90% to about 50%, particularly from about 80% to about 60%, by volume of water and adding sodium hydroxide to adjust the pH are prepared.

Clioquinol can be formulated as ready-to-use injection solution, wherein a stock solution as described above is completed to the desired volume with a physiologically acceptable solvent such as injectable water; glucose solution; a full electrolyte solution with or without amino acids, lipids, vitamins, trace elements and other minerals; a Ringer-lactate solution; a Ringer- acetate solution; a

NaCl solution (isotonic, hypotonic or hypertonic) for infusion.

Finally, the solution in the ampoules can be sterilized either through aseptic filtration or heat sterilization.

It will be appreciated that the amount of clioquinol and, optionally, further active constituents required for said treatment or prevention will vary according to the route of administration, the disorder to be treated, the condition, age, the file history of the subject, and the galenic formulation of the pharmaceutical composition, etc. When treating a patient diagnosed as having a pathological condition influenced by a Zn- or

Cu- dependent protein, the amount of clioquinol or phanquinone (or other chelator) is preferably the amount that is effective to provide at least a partially modulation or inhibition of the activity of such protein/enzyme

In general, a suitable therapeutically effective amount of clioquinol in the pharmaceutical composition for parenteral use is, for example, 500 mg to 6 g, to more preferred 1 to 3 g to 500 mg. The amounts of phanquinone for oral use, is for example 5 mg and 2 g, to more preferred 500 mg to Ig. The combination of clioquinol and phanquinone for a suitable therapeutically effective amount in the pharmaceutical composition for parenteral use is, for example, 100 mg to 1 g, and more preferred 250 mg to 500 mg. of clioquinol and 50 mg to 250 mg and more preferred 50 mg to 100 mg of phanquinone. The most suitable formulation that will modulate simultaneously on the target zinc and copper dependent proteins that are deregulated or pathologically increased, is a combination of both clioquinol and phanquinone. The daily effective amount for administration in the pharmaceutical composition comprises 100 mg to 6 g clioquinol and 5 mg to 2 g phanquinone.

In general, a suitable therapeutically effective amount of clioquinol in the pharmaceutical composition for oral use is, for example, about 10 mg to about 1O g, more preferably about 100 mg to about 1 g, more preferably about 250 mg to about 500 mg.

The amounts of phanquinone for oral use, is for example about 5 mg to about 500 mg, more preferred about 50 mg to about 100 mg.

Clioquinol is preferably combined with Vitamin B 12 in a kit or in a combined formulation or in two separate formulations to be co-administered for use in certain patients, particularly when given over prolonged periods or when clioquinol is to be given in high doses. Preferably, 1 to 5 mg of vitamin B 12 is used in such combined formulations or mixtures.

The combination of clioquinol and phanquinone for a suitable therapeutically effective amount in the pharmaceutical composition for oral use is, for example, about 100 mg to about 1 g,

preferably, about 250 mg to about 500 mg of clioquinol and about 5 mg to about 500 mg, and more preferably about 50 mg to about 100 mg of phanquinone.

The most suitable formulation that will modulate simultaneously on the target Zn-dependent and/or other metal-dependent enzymes of the MBLs is a combination of both clioquinol and phanquinone.

The daily effective amount for administration in the pharmaceutical composition comprises about 100 mg to about 6 g clioquinol and about 5 mg to about 2 g phanquinone.

The administration, for example, can be at high dosages for periods of one week to three weeks and at low dosages for periods up to three months. The high dosage administration is preferably parenteral and the low dosages are preferably oral. The combination therapy with these (or other) chelating agents will effect stronger metal chelation and more potent inhibition of metal dependent target proteins and will broaden the spectrum of the chelating effect.

In general, a suitable therapeutically effective amount of clioquinol in the pharmaceutical composition is, for example, about 100 mg to about 6 g, preferably about 250 mg to about 1 g. The amounts of 5,7-dichloro-8-hydroxy-quinaldine, phanquinone, 5,7-dichloro-8- hydroxyquinoline, or 5,7-di-iodo-8-hydroxyquinoline are preferably about 5 mg to about 1 g, more preferably about 50 mg to about 100 mg.

The amounts of 5,7-di-iodo-8-hydroxyquinoline are preferably from about 50 mg to about 5 g, more preferably, about 500 mg to 1 g. If the pharmaceutical composition also comprises further active constituents they may be present as part of a single composition for administering these compounds in combination, concurrently, or in separate compositions for administering substantially simultaneously but separately, or sequentially. If the active constituents are administered sequentially, the further active ingredients may be administered prior or subsequent to the administering of the chelating agent, e.g., clioquinol.

As is well-known in the art, and disclosed in the references cited above, pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the active constituents. An aqueous or oily carrier may be used. Such pharmaceutical carriers may be sterile liquids such as water and oils including those of petroleum animal, vegetable or synthetic origin such as castor oil, peanut oil, soybean oil, mineral oil, sesame oil and the like. Formulations

for parenteral administration also include a lyophilized powder comprising clioquinol and, optionally, further active constituents that is to be reconstituted by dissolving in a pharmaceutically acceptable carrier that dissolves the active constituents, e.g. an aqueous solution of carboxymethyl cellulose and lauryl sulfate. Solutions can be dry, soluble products ready to be combined with a solvent just prior to use, suspensions ready for injections, dry, insoluble products ready to be combined with a vehicle just prior to use, emulsions, liquid concentrates ready for dilution prior to administration.

The solubility of clioquinol as well as 5,7-dichloro-8-hydroxy-quinaldine, phanquinone, 5,7- dichloro-8-hydroxyquinoline, 5,7-di-iodo-8-hydroxyquinoline can be increased by pH adjustment or the use of water miscible co- solvents or surfactants or complexing agents or the change of the dosage form to dispersed system (suspension, emulsion, liposome). Parenteral preparations for intravenous or intramuscular or subcutaneous or intraspinal, or intracisternal or intrathecal or intraarterial or intra- articular injection or infusion may be prepared by dilution to the desired concentration with an aqueous solvent or emulsifying agent, or the use of a cosolvents to increase solubility like water containing dissolved carboxymethylcellulose or polysorbate, such as polysorbate 80, ethyl oleate, Tween 20, or castor oil or the like. Prior to the dissolution, the quinoline chelators may initially be pre-dissolved in an organic solvent, preferably an aprotic solvent like DMSO, DMF, and the like. Parental formulations are preferably made isotonic by adjusting with suitable electrolytes. Clioquinol suspension for injection consists of insoluble solid particles dispersed in a liquid medium, with the solid particles accounting for 0.5- 30% of the suspension. The vehicle may be aqueous, oil, or both.

Excipients in injectable suspensions include antimicrobial preservatives, surfactants, dispersing or suspending agents, and buffers. Surfactants wet the suspended powders and provide acceptable syringe ability while suspending agents modify the viscosity of the formulation. Clioquinol (or other chelator) emulsion for injection examples include oil-in- water sustained-release depot preparations, which are given intramuscularly.

When the pharmaceutical composition is a capsule, it may contain a liquid carrier, such as fatty oil e.g., cacao butter. Suitable pharmaceutical excipients include starch, glucose, lactose sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The

compositions may be solutions suspensions emulsion tablets, pills, capsules, powders, sustained release for emulations and the like. The composition may be formulated as a suppository, with traditional binders and carriers such as triglycerides.

In yet another embodiment, the clioquinol or phanquinone (or other chelator) may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials may be used. In yet another embodiment, a controlled release system may be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose.

In one embodiment of the pharmaceutical composition, clioquinol and the, optionally, further active constituents, are comprised as separate pharmaceutical entities. For example, one entity may comprise clioquinol and another entity may comprise phanquinone. The two entities may be administered simultaneously or sequentially. For example, the entity comprising clioquinol can be administered, followed by phanquinone administered within a day, week, or month of clioquinol administration. If the two entities are administered sequentially, the entity comprising clioquinol is preferably administered for one to three weeks followed by a wash out period of one to four weeks. After the wash out period, the treatment may be repeated.

The pharmaceutical composition may be provided as a pack or kit comprising one or more entities containing one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally, associated with such entities may be a notice in the form described by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

An example is a 75 year old patient, with chronic bronchitis, smoker and bronchiectatic bronchi diagnosed with a community acquired S. pneumoniae lobar pneumonia. The patient is hospitalized and treated with 500 mg clioquinol t.i.d. combined with 50 mg phanquinone t.i.d. The patient is weak and with nutritional deficits and the treatment is supplemented with 20 μg of cyanocobolamin daily.

Another patient in the general practice is a 30 year old man diagnosed of S. pneumonia bronchopneumonia. The patient is treated with 500 mg clioquinol orally b.i.d. for ten days.

An exemplary use clioquinol and/or phanquinone as the chelating agent is in intensive care severe sepsis patients. A patient has been hospitalized for the past seven days with underlying cancer and a currently nosocomial infection, No bacteria have been isolated from repetitive bronchial lavage and blood cultures despite t elevated white blood cell counts, fever and tachycardia. An empiric antibiotherapy consisting of cefepime and tobramycin has been initiated since 72 hours. To this regimen is added a combination of clioquinol 6 g daily and phanquinone 2 g daily by intravenous infusion administered by a separate line.

Another example is a patient hospitalized in the internal medicine unit for community acquired pneumonia. A standard treatment has been administered consisting of rocephin and vancomycin. Vancomycin has been added since the patient comes from an region endemic for MRSA and is suspected of being a carrier. The patient's condition is deteriorating and the bacteriological results after 96 hours of antibiotherapy indicate isolation from bronchial specimens of an Acinetobacter multiresistant strain. Addition of clioquinol 2 g daily by intravenous infusion is initiated and the rocephin replaced with colistin. Another exemplary patient is in a surgical intensive care unit; the patient is developing peritonitis. Empiric treatment consisting of meropenem, clioquinol and metronidazole is started. The patient after 24 hours does not respond to the treatment and develops the clinical picture of severe sepsis. Clioquinol, 1 g daily, is administered every four hours by a 60 minute intravenous infusion. Concurrent treatment is administered to the patient following the standard procedures for the management of the sepsis and the intercurrent respiratory organ dysfunction. Mineral and multiple vitamin supplements will be added accordingly.

In a patient a MBL producing strain of P. aeruginosa has been isolated after long hospitalization. The strain is resistant to most antibiotics. The patient is treated with meropenem and a combination of 0.5 g phanquinone and 4 g of clioquinol daily by intravenous infusion is initiated. The patient after seven days treatment is continued on oral clioquinol and phanquinone treatment for another four weeks (2 g of clioquinol daily in four 500 mg doses and 1 g of phanquinone daily in two 500 mg doses).

Another exemplary patient presents with a fever of unknown origin. The patient has a normal blood count and no particular clinical signs and symptoms except an evening fever. Empirical treatment with meropenem has been initiated and the patient is hospitalized for three

days. All laboratory tests show nothing of relevance. Phanquinone 100 mg, three times daily, has been initiated.

EXAMPLES

Effectiveness of Clioquinol Against Multidrug Resistant Staphylococcal Aureus

Minimal inhibitory concentrations (MIC) of clioquinol was tested against S. Aureus isolates from various human sources. These MIC analyses (and those in later examples) were performed in accordance with the Clinical Laboratory Standards Institute(CLSI) Guidelines with slight modifications due to the relative insolubility of clioquinol.

The agar dilution method (and not broth dilution) was employed to avoid clioquinol precipitation. Muller Hinton agar was used for Gram negative and for Staphylococcus aureus. When Streptococcus pneumoniae was tested, 7% sheep blood was added. The compounds were dissolved in dimethylsulfoxide (DMSO) with no addition of water. Clioquinol stock was prepared by dissolving 20mg in 2ml DMSO followed by serial doubling dilutions in the agar. (Dilutions were made overnight.).. Cultures were set up to yield to give 10 ⁴ (to 10 ⁵ ) cfu/ml. As a standard, S. aureus ATCC 29213 was used, and yielded an MIC of 6.25 μg/ml.

Abbreviations used in Tables: MRSA -Methicillin Resistant S. aureus ; BAL - Bronchoalveolar lavage; DTA - Deep Tracheal aspirate; MDR Multi-drug-resistant

EXAMPLE 2

MIC of Clioquinol for Clinical Strains of MRSA, ESBL E. colu MDR A. baumannii and

Penicillin-resistant Streptococcus pneumoniae

MIC for Clioquinol was determined as described above.

This approach is based on that described by White and colleagues (R. L. White et αl. , , Antimicrob Agents Chemother, 1996, 40:1914-18

Synergy of chelating drugs with "standard" antibiotics was determined based on MIC values as follows.

Fractional inhibition concentration (FIC) values were calculated according to Eliopoulos, G. M. & Moellering, R. C. (1991). In Antibiotics in Laboratory Medicine, (Lorian, V., Ed.), pp. 432- 92. Williams and Wilkins Co., Baltimore, MD.

FIC = [A]ZMICA + [B]ZMIC _B ,

where MIC _A and MIC _B are the MICs of compounds A and B, respectively, and [A] and [B] are the concentrations at which compounds A and B, in combination, inhibit bacterial growth. Results appear in Table 3.

Table 3

* FIC: fractional inhibitor concentration index

EXAMPLE 4 Cumulative Killing Effect of Clioquinol Against MRSA Strains

Eight MRSA clinical strains were used. The starting inoculum was 5.0 x 10 ⁵ — 1.0 x 10 ⁶ cfu/ml at log-phase. Exposure was to 1/4 x MIC; 1/2 x MIC; 1 x MIC; 4 x MIC. The results express the estimation of growth at standard time intervals. The killing-effect was >3 logio decrease from the starting inoculum.

EXAMPLE 5 In vivo Effects of Clioquinol

Based on the in vitro potency of clioquinol against MRSA, reflected as a concentration- dependent killing effect observed at 24 hrs of growth with eight MRSA strains (see Example 4), in vivo murine studies were performed.

Intraperitoneal (IP) injection of 100 μl of DMSO had no effect on the survival of C57BL/6 (male) mice, whereas IP injection of 200 μl of the detergent Tween 20 resulted in the death of all animals. After the preliminary testing of solvents for clioquinol, five mice were injected IP with 50mg/kg of clioquinol that diluted in DMSO at a final volume of 100 μl. All mice survived.

To study sepsis, 4 mice were injected IP with 50mg/kg of clioquinol diluted in DMSO in a final volume of 100 μl. Two were sacrificed at 10 minutes after injection and two were sacrificed at 15 minutes. Blood was sampled from the inferior vena cava and serum levels of clioquinol were measured by an HPLC assay. Results shown in Table 5 indicate that significant serum levels are attained rapidly.

TABLE 5 Time course of clioquinol distribution in 4 P5015-nfected mice

The mice were rendered neutropenic by IP injection of 150mg/kg cyclophosphamide on days 1 and 3 of the experiment. On the 5 ^th day, animals were infected subcutaneously (SC) with 10 ⁸ CFU units of a highly lethal strain of methicillin-resistant Staphylococcus aureus (Strain P5015J. During the process of sepsis development, five control animals were injected IP with DMSO b.i.d for two days. The experimental clioquinol group was injected 50mg/kg of clioquinol b.i.d. for two days. The animals were sacrificed 48 hours later. Results shown in Table 6 indicate a clear antibacterial effect of clioquinol measured as prolonged survival.

TABLE 6 Clioquinol Prolongs Survival in Neutropenic Mice Challenged with MRSA

Statistical analysis: Log Rank 6.7, p=0.009

Lung and liver tissues from these animals were examined for numbers of viable bacteria. Results in Table 7 show that bacteria were eradicated from lungs and liver (10-fold drop) presumably related to the pharmacokinetics of clioquinol.

TABLE 7. Clioquinol Reduced Growth of MRSA in Liver and Lung

CFU: Colony forming units (CFU) of live Methicillin-resistant Staphylococcus aureus

The sepsis model in mice showed that there is a prolongation of survival after infection with MRSA. Further that there is a eradication of bacterial isolates from lungs and liver that is probably related to the pharmacokinetics of clioquinol and that the solvent or vehicle used is not toxic.

EXAMPLE 6 Preparation of a Clioquinol and Pharmaceutical Compositions

Preparation 1

In a glass container, nitrogen-gassed aqueous solvent for injection equal to approximately 70% of the final volume to be manufactured was added (according to the following amounts specified for about 1 liter). • 200 mL of organic solvent was added and dissolved in the aqueous solvent while mixing.

• With strong mixing, 200 g 5-chloro-7-iodo-quinolin-8-ol or and ingredients were slowly added. The mixture was stirred and 1OM sodium hydroxide was added until all the crystals were dissolved.

• Aqueous solvent for injection was added to bring the solution to the final of 1 liter volume. The solution was sterilized by filtration through a sterile 0.2 μm-rated filter into a sterile receiving flask. Sterile, depyrogenated, brown glass ampoules or vials were filled with the solution, purged with nitrogen, and the ampules optionally sealed.

Preparation 2

Clioquinol (2 g, 6.5 mM) was suspended in a mixture of water (5 mL) and ethanol (3 mL) under nitrogen. To this suspension 1OM sodium hydroxide (0.325 mL) was added at room temperature. The suspension was stirred at room temperature until dissolution and the volume adjusted to 10 mL with water. The solution was sterilized by filtration.

Preparation 3

Clioquinol (2 g, 6.5 mM) was suspended in a mixture of water (4 mL) and propyleneglycol (3 mL) under nitrogen. To this suspension 1OM NaOH (0.65 mL) was added at room temperature. The suspension was stirred at room temperature until dissolution and the volume adjusted to 10 mL with water. The solution was sterilized by filtration.

Preparation 4

Clioquinol (2 g, 6.5 rnM) and 2-hydroxypropyl-β-cyclodextrin (0.5 g) were suspended in a mixture of water (4 rnL) and propylene glycol (2 rnL) under nitrogen. To this suspension 1OM NaOH (0.325 rnL) was added at room temperature. The suspension was stirred at room temperature until dissolution and the volume adjusted to 10 mL with water. The solution was sterilized by filtration. Preparation 5

Clioquinol (2 g, 6.5 mM) was suspended in a mixture of water (5 mL) and ethanol (2 mL) under nitrogen. To this suspension 1OM sodium hydroxide (0.487 mL) was added at room temperature. The suspension was stirred at room temperature until dissolution and the volume adjusted to 10 mL with water. The solution was sterilized by filtration. Preparation 6

Clioquinol (2 g, 6.5 mM) was suspended in a mixture of isotonic NaCl solution (5 mL) and ethanol (2 mL) under nitrogen. To this suspension 1OM NaOH (0.325 mL) was added at room temperature. The suspension was stirred at room temperature until dissolution and the volume adjusted to 10 mL with isotonic sodium chloride solution. The solution was sterilized by filtration. Preparation 7

Clioquinol, 250 g, was mixed with 200 g Sapamine(R) (N-(4'- stearoyl amino-phenyl)- trimethylammonium methyl sulfuric acid) and 1025 g lactose monohydrate for a period of minutes. 5 g of boiling water was added in one go to a mixture of 100 g corn starch in 100 g cold water. The maize suspension, cooled to 40°C, was added to the clioquinol-containing powder mixture under continuous stirring. The mixture was granulated using a 2.5 mm sieve and desiccated for 18 hours at 40°C. The dry granules were mixed with 400 g cornstarch and 20 g Mg stearate. The final mixture was formulated into tablets having a diameter of 8.0 mm and a weight of 200 mg.

EXAMPLE 7

Inhibition of MMPs by Clioquinol

An enzyme assay was conducted with five of the enzymes belonging to the MMP group: MMP-I, MMP-2, MMP-3, MMP-7, and MMP-9 at various concentrations.

MMP-I, MMP-3, and MMP-7 were initially pre-incubated for 60 min at 37°C and MMP-2 and MMP-9 were pre-incubated for 60 min at 25°C in an aqueous vehicle of 5OmM MOPS, 1OmM CaCi2.2H ₂ O, 10 μM ZnCl ₂ , 0,05% Brij 35, pH 7.2 and 100 μM clioquinol.

A "classical" substrate for MMP activity, a fluorogenic peptide substrate known as FS-I was used ((7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dini trophenyl]-L-2,3- diamino-propionyl)-Ala-Arg-NH) ₂ ) which when hydrolyzed by an MMP, yield a fluorescent product. The FSl was added to a concentration of 25 μM. MMP-I was incubated for 2 hours at 37°C, MMP-2 was incubated for 3 hours at 25°C, MMP-3 was incubated for 90 min at 37°C. MMP- 7 was incubated for 90 min at 37°C, and MMP-9 was incubated for 2 hours at 25°C. The activity of the enzymes was measured fluorimetrically.

The enzyme assay was repeated for MMP-2 - at a 10- and 100-times higher clioquinol concentration. At a clioquinol concentration of 1 mM the inhibition was 26% and at a clioquinol concentration of 10 mM the inhibition was measured as 101% ; the inhibition was highly concentration-dependent. EXAMPLE 7

Phanquinone: Single Case Reports of Side Effects, Overdosing, Teratogenicity and Interactions

From data contained in the single (spontaneous) case reports and from medical publications on phanquinone available till the withdrawal of the drug from the market, the following conclusions may be drawn.

The most frequently reported unwanted effects were related to the gastrointestinal system, e.g., nausea, vomiting, abdominal pain (variously described) and diarrhea. The only other unwanted effects reported with relative frequency are were irritability and headache. There was no evidence, that phanquinone had any adverse effect on the nervous system, and the other reported symptoms were non-specific in nature and infrequent. There was no evidence that any system, other than the gastrointestinal was prone to be affected adversely by phanquinone with significant frequency.

The dosage of phanquinone employed was most often 200-400 mg/day for 7 to 10 days. When the daily dose was >600 mg/day or the duration of treatment was longer than two weeks, the incidence, but not the severity, of unwanted effects appeared to increase.

Phanquinone was generally reported as being well-tolerated and that the unwanted effects observed so mild in degree that discontinuation of treatment was rarely necessitated. There have been no reports of deaths related to treatment with phanquinone and no reports of fetal malformations, drug interactions or carcinogenicity.

EXAMPLE 8

Preparation of a Pharmaceutical Composition Comprising Phanquinone

Phanquinone (250 g) of was mixed with 200 g sapamine(R) (N-(4'- stearoyl amino-phenyl)- trimethylammonium methyl sulfuric acid) and 1025 g lactose mono-hydrate for a period of 5 minutes. 300 g of boiling water was added in one go to a mixture of 100 g maize starch in 100 g cold water. The maize suspension, cooled to 40°C. was added to the phanquinone containing powder mixture under continuous stirring. The mixture was granulated using a 2.5 mm sieve and desiccated for 18 hours at 40°C. The dry granules were mixed with 400 g corn starch and 20g Mg stearate. The final mixture was formulated into tablets having a diameter of 8.0 mm and a weight of 200 mg. EXAMPLE 9

Clioquinol and phanquinone are administered to a group of patients receiving intensive care for severe sepsis vs. group that is given standard therapy. The patients suffer from a MRSA nosocomial pneumonia and fail an initial round of vancomycin treatment (standard). The antibiogram shows that their MRSA are sensitive to both clioquinol and phanquinone. A combination of clioquinol 6 g daily and phanquinone 2 g daily are administered by intravenous infusion for 120 hours - in addition to the standard procedures as defined in the Guidelines for sepsis.

Another group of intensive care patients with sepsis are given clioquinol 2 g daily by intramuscular injection added to the standard procedures (per the Guidelines for sepsis). Another group of patients in intensive care have acute pancreatitis. These patients receive the same dosages as in severe sepsis (above) but for a duration of seven days followed by oral administration of clioquinol 500 mg three times daily for another four weeks. Mineral and multiple vitamin supplements are added accordingly.

Patients in all three groups clioquinol groups show improvement compared to patients who receive standard antibiotic and supplementary therapy.

EXAMPLE 10

A group of patients with colorectal cancer with liver metastasis have extensive intraabdominal postoperative infections after surgery. One subgroup of patients receive standard metronidazole and ceftriaxone antibiotherapy. A second subgroup receives a daily regimen of 2 g clioquinol plus 500 mg phanquinone intravenous infusions each lasting two hours.

A parenteral regimen involves three daily doses per os of 750 mg clioquinol concurrently with parenteral metronidazole/ceftriaxone, during the periods when no parenteral drugs are administered. Mineral and vitamin B 12 supplements multiple vitamin supplements are added accordingly.

The group of patients receiving the IV antibiotics and per os clioquinol with supplementation have better outcomes than the patients receiving conventional therapy.

EXAMPLE 11 A patient with stage III lung cancer (tumor that have invaded the chest wall and nearest lymph nodes and subject to a wedge resection and receives hyperfractionated radiation therapy) develops acute respiratory syndrome with pneumonia. A gram negative Acinetobacter is isolated that is resistant to several classes of antibiotics.

The patient is treated with a combination of cefepime and amikacin to which is added clioquinol 6 g daily alone or in combination with phanquinone 2g daily, by intravenous infusions for one week. The antibiotic treatments are given together with paclitaxel and carboplatin standard protocol. This patient receives mineral and multiple vitamin supplements. The patients condition improves in response to the treatment with the chelating drugs and he survives.

The references cited above are all incorporated by reference in their entirety herein, whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.