FIELD OF THE INVENTION:

The present invention relates to a combination of (i) a cationic polymer, and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of Gram negative bacterial infections especially due to antibiotic-resistant bacteria, e.g. a Pseudomonas aeruginosa drug resistance strain. The present invention also provides a cationic polymer, for use in a method for enhancing sensitivity to an antibiotic of a patient suffering from bacterial infection, especially from antibiotic-resistant bacterial infection.

BACKGROUND OF THE INVENTION:

Since their discovery, antibiotics have revolutionized the medical treatment of patients with bacterial infections by saving numerous lives. They represent a major therapeutic medical tool, which can be used in several clinical settings, including infections, chemotherapies, transplantation, and surgery for examples.

However, antimicrobial resistance (AMR) has been observed at dangerously high levels worldwide (Spellberg et al. (2013) Engl. J. Med. 368:299-302) and alternative therapeutic strategies are urgently needed. Among the different resistance phenomena, the AMR involving the broad-spectrum cephalosporins, one of the major class of antibiotic used worldwide, has become a major public health issue (Rossolini et al. (2008) Clin. Microbiol. Infect. 14(suppl 1):33-41). For this b-lactam family, the main resistance mechanism in enterobacteria, is characterized by the production of Extended-Spectrum b-lactamases (ESBLs) with the most widespread type of ESBL in European countries, CTX-M-15 (Bevan et al. (2017) J. Antimicrob. 72:2145-2155). But the mechanisms underlying the AMR (to inactivate antibiotics or to prevent them from reaching their target) are multiple and also affect chronic bacterial infection like pulmonary bacterial infection.

In particular, cystic fibrosis (CF) patients are confronted with recurring bronchial infections which result from a thickening of mucus, a deterioration of the mucociliary clearance and a decrease of immune defenses (Matsui et al. , 2005). The pathogens at the origin of these infections are opportunist bacteria which are different according to the age of patient. After the birth, more than 65% of CF patients are affected by Staphylococcus aureus ( S . aureus), a Gram positive bacteria. The antibiotics used to eliminate this bacteria as well as the pulmonary lesions that this one causes, support the establishment of another germ which is Pseudomonas aeruginosa ( P . aeruginosa ) (Govan & Nelson, 1993; Ratjen et al, 2001). P. aeruginosa is an aerobic Gram-negative bacteria, which is largely widespread in the environment and colonizes most of the CF patients at the adolescence. At the time of the primary infection by P. aeruginosa, the isolated strains are generally not mucoid and sensitive to antibiotics. However, P. aeruginosa possess several virulence factors adapted to the pulmonary environment of CF patients and these patients can also be colonized by strains which are resistant to antibiotics. In this context, the infection becomes chronic at nearly 80% of CF patients at the adulthood. This colonization then involves an increase in the inflammatory response as well as a loss of the respiratory functions and constitutes a bad index of morbidity and mortality (Emerson et al., 2002).

The only current means to fight against this chronic infection is the antibiotherapy, with administration by inhalation (Flume et al, 2014). During pulmonary exacerbations, the treatments are more intensive and adjusted according to the profile of resistance of the bacteria (antibiotic association, oral or intravenous administration) (Balls et al, 2011; Gibson et al., 2003). Unfortunately, this antibiotic pressure involves the emergence of other resistant strains of P. aeruginosa and then the effectiveness of antibiotics appears limited. Thus, it becomes urgent to find new therapeutic strategies to fight these resistant strains ofP. aeruginosa. In this context, the comprehension of their mechanisms of resistance to antibiotics is essential as well as the development of new treatments.

P. aeruginosa is an opportunist pathogen which is naturally sensitive only to a small number of antibiotics. Indeed, P. aeruginosa possess natural and acquired resistances to certain antibiotics. Concerning its natural resistances, four principal ones are described. The first one concerns its external membrane through which only few molecules can pass thanks to the porins which are a unique way for several antibiotics to access to their target. Thus some aminosides, carbapenems and others b-lactamins have a limited passage. The second natural resistance is the synthesis of different b-lactamases which hydrolyze b-lactamins (cephalosporinase inductible AmpC or oxacillinase called OXA-50 or PoxB). The third resistance is the production of different active efflux systems such as the MexAB-OprM efflux pump (constitutive expression) which generates a resistance to several b-lactamines, fluoroquinolones, tetracyclines, trimethoprime and chloramphenicol, as well as the MexXY/OprM efflux pump (inducible expression by antibiotics) which generates a resistance to aminoglycosides, fluoroquinolones, switterionic cephalosporins or tetracyclines (Li et al, 1995; Surfaces et al., 1999; Jeannot et al, 2005). The purpose of all these mechanisms are to inactivate antibiotics or to prevent them from reaching their target. The fourth natural mechanism of resistance to antibiotics is the capacity of P. aeruginosa to adjust to its environment, by establishing a biofilm. Initially, bacteria adhere to the cells and form successive layers which are then covered with an exopolysaccharidic matrix. The formation of a biofilm takes between 5 and 7 days (Hoiby et al., 2011). The biofilm is a very particular environment, with a gradient of nutrients and availability of oxygen. Thus, the bacteria of the biofilm located in depth will have a reduced metabolic activity but will be protected from phagocytosis and less sensitive to the antibiotics (Lyczak et al. , 2002). Moreover, this lifestyle would facilitate the horizontal gene transfer (acquisition of genes of resistance to antibiotics) (Flemming & Wingender, 2010).

Among CF patients, many P. aeruginosa strains responsible of chronic infections possess acquired resistances, which is a consequence of the pressure of selection induced by the large use of antibiotics. Three principal mechanisms of acquired resistance are describes: (1) the overproduction of b-lactamases (cephalosporinase AmpC whose production can be 20 to 500 times higher than normal); (2) the overproduction of efflux systems (MexAB-OprM - example of mexR) and (3) the alteration of the porine OprD preventing carbapenem passage.

Moreover, it has been reported an increased number of resistant and multi-resistant strains for 20 years, in CF patients (Emerson et al. , 2010). The multi-drug resistant strains (MDR) are defined by their resistance to at least 3 out of the 4 principal antibiotic classes which are b-lactamins, carbapenems, aminosides and fluoroquinolones. Among CF patients, 10 to 20 % of the strains are MDR. In this case, the last treatment is colistin which acts by destabilizing the bacteria external membrane. However, certain strains of P. aeruginosa also develop resistances to this antibiotic (Barber & Wolf, 2010).

Hence, there is an important need in identifying new solutions for efficiently targeting and fighting AMR.

The present invention meets this need.

Recent studies relate to the improvement of formulations, antibiotic associations, or routes of administration. Another way is to explore the modulation of the immune response in order to facilitate the elimination of the bacteria. This approach includes the development of a vaccine against P. aeruginosa (Balls et al., 2011; Kipnis et al., 2006), the use of either human antimicrobial peptides such as the trappin-2 (Bellemare et al., 2010) or bacteriophages.

Previously, inventors showed that polycationic peptides had properties of interest for CF patients. Poly-L-Lysine (pLK) liquefies CF sputum by DNA condensation and exhibit antibacterial activity against P. aeruginosa. More recently, we revealed that pLK eliminates P. aeruginosa biofilm and altered its membrane. Based on these data, inventors evaluated the combination of pLK with antibiotics to counter P. aeruginosa resistance mechanisms.

SUMMARY OF THE INVENTION:

Therefore, the present invention relates to (i) a cationic polymer and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of Gram negative bacterial infections due to antibiotic-resistant bacteria wherein the cationic polymers have the following formula

(I): wherein

• R is NH2 or NH linked to a histidine residue or other molecules selected from charged amino acids, a gluconoyl residue, a glycosyl residue or a PEG moiety,

• i is the degree of polymerization comprised between 10 and 75, preferably between 10 and 50, and more preferably between 20 and 50, for example between 20 and 40 or between 30 and 50.

The present invention also relates to the cationic polymers of the invention, for use in a method for enhancing sensitivity to an antibiotic of a patient suffering from bacterial infection, especially from antibiotic-resistant bacterial infection.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors previously discovered that cationic polymers such as poly-aL-lysine act as mti-Pseudomonas aeruginosa biofilm agents (WO2017060489). The inventors have now found that cationic polymers such as poly-aL-lysine have the following properties: i/ they “permeabilize” the membrane of Gram-negative bacteria, such as P. aeruginosa strain (reference wild type strain PAOl and its mutant PAOl AoprD (for porin D2 alteration) (see figure 1) ii/ they act synergistically with different antibiotics (imipenem, ceftazidime, aztreonam, gentamicin) in vitro, ex vivo on various bacterial strains, not only sensitive “wild type” but also resistant bacterial strains (see figures 2 to 10).

The inventors demonstrated that cationic polymers in combination with antibiotic agents efficiently and rapidly eliminate bacteria strain even on resistant bacterial strains (with porin alteration or an overproduction of different active efflux systems as MexAB-OprM and MexXY/OprM).

Altogether these results provide new insights for treating Gram-negative bacterial infection, especially resistant and multi-resistant bacterial infection, using cationic polymers of the invention as main active principle ingredient in combination with antibiotic agent.

Combination and uses of the invention

A first aspect of the invention relates to a combination of (i) a cationic polymer according to the invention, and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of Gram-negative bacterial infections due to antibiotic-resistant bacteria.

The term "Gram-negative" bacterial infection refers to a local or systemic infection with Gram-negative bacteria. The proteobacteria are a major group of Gram-negative bacteria, including Escherichia coli (E. coli), Salmonella, Shigella, and other Enter obacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc. Other notable groups of gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria. Medically relevant Gram-negative cocci include the four types that cause a sexually transmitted disease ( Neisseria gonorrhoeae), a meningitis Neisseria meningitidis), and respiratory symptoms ( Moraxella catarrhalis, Haemophilus influenzae). Medically relevant Gram-negative bacilli include a multitude of species. Some of them cause primarily respiratory problems ( Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems {Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems {Helicobacter pylori, Salmonella Enteritidis, Salmonella Typhimurium). Gram-negative bacteria associated with hospital-acquired infection include Acinetobacter baumannii, which cause bacteremia, secondary meningitis, and ventilator- associated pneumonia in hospital intensive-care units.

In another particular embodiment of the invention, the Gram-negative bacteria according to the invention are selected from the group consisting of Escherichia coli, Pseudomonas spp, Salmonella spp, Klebsiella spp, Acinetobacter spp, E. corrodens, and Haemophilus influenza.

In a more particular embodiment of the invention, the Gram-negative bacteria according to the invention is Pseudomonas spp, Acinetobacter spp and Klebsiella spp.

The term ‘ Pseudomonas bacteria ” has its general meaning in the art and refers to bacteria that occur normally or pathogenically in lung of humans and other animals. The term ‘ Pseudomonas bacteria ” refers to but it is not limited to Gram-negative bacteria Pseudomonas, e.g ; a bacterium of the Pseudomonas aeruginosa group such as P. aeruginosa group, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea.

In particular, the Pseudomonas according to the invention is Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common Gram-negative bacteria that can cause disease in animals, including humans. It is citrate, catalase, and oxidase positive. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many natural and artificial environments. It uses a wide range of organic material for food; in animals, its versatility enables the organism to infect damaged tissues or those with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis. If such colonizations occur in critical body organs, such as the lungs, the urinary tract, and kidneys, the results can be fatal (Balcht, et al., Informa Health Care, 1994). Because it thrives on moist surfaces, this bacterium is also found on and in medical equipment, including catheters, causing cross infections in hospitals and clinics.

The term ‘ lebsiella bacteria ” has its general meaning in the art and refers to bacteria that occur normally or pathogenically in lung of humans and other animals. The term ‘ lebsiella bacteria ” refers to but it is not limited to Gram-negative bacteria Klebsiella e.g, a bacterium of the Klebsiella pneumoniae group such as K. pneumoniae group, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella michiganensis, Klebsiella pneumoniae (species- type), Klebsiella pneumoniae subsp. Ozaenae, Klebsiella pneumoniae subsp. Pneumoniae, Klebsiella pneumoniae subsp. Rhinoscleromatis , Klebsiella quasipneumoniae, Klebsiella quasipneumoniae subsp. Quasipneumoniae, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella variicola.

In particular, the Klebsiella according to the invention is Klebsiella pneumoniae.

The term “ Acinetobacter bacteria ” has its general meaning in the art and refers to bacteria that occur normally or pathogenically in lung of humans and other animals. The term “ Acinetobacter bacteria ” refers to but it is not limited to gram-negative bacteria Acinetobacter, e.g; a bacterium of the Acinetobacter baumannii group such as A. baumannii group, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter genomospecies 3 and Acinetobacter genomospecies 13 (Ingela Tjemberg et Jan Ursing) grouped together in a group called the ' Acinetobacter calcoaceticus -baumannii complex

In particular, the Acinetobacter according to the invention is Acinetobacter baumannii.

The present invention aims in particular at fighting antimicrobial resistance, in particular antibiotic resistance.

Accordingly in a particular embodiment bacterial infections is due to antibiotic-multi resistant bacteria.

By “antimicrobial resistance” or “AMR” is meant herein the phenomenon that a microorganism does not exhibit decreased viability or inhibited growth or reproduction when exposed to concentrations of the antimicrobial agent that can be attained with normal therapeutic dosage regimes in patients. It implies that an infection caused by this microorganism cannot be successfully treated with this antimicrobial agent.

As used herein, the terms "antibiotic" and "antimicrobial compound" are used interchangeably and refer to a compound which decreases the viability of a microorganism, or which inhibits the growth or reproduction of a microorganism. The term “antibiotic agent” has its general meaning in the art and refers to antibacterial agent, such as described in US2013/0029981.

Suitable main class of antibiotic agents include, without limitation:

1. b-lactam antibiotic (beta-lactam antibiotic) are the antibiotic agents that contain a beta-lactam ring in their molecular structure and containing a beta-lactam functionality. This b- lactam antibiotics includes penicillin and derivatives (penams), cephalosporins (cephems), monobactams, carbapenems and carbacephems. Most b-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism and are the most widely used group of antibiotics (in 2003 more than half of all commercially available antibiotics in use were b- lactam compounds) By “cephalosporins” (cephems) is meant herein a subgroup of b-lactam antibiotics originally derived from the fungus Acremonium. Together with cephamycins, they constitute a subgroup of b-lactam antibiotics called cephems. Cephalosporins include ceftazidime.

By “monobactam” is meant herein a subgroup of b-lactam antibiotics, which are monocyclic and wherein the b-lactam ring is not fused to another ring. Monobactam include aztreonam.

By “carbapenems” is meant herein a subgroup of b-lactam antibiotics, which have a bactericide effect by binding to penicillin-binding proteins (CBPs) thus inhibiting bacterial cell wall synthesis This class of antibiotics is usually reserved for known or suspected multidrug- resistant (MDR) bacterial infections. Carbapenem include imipenem.

By “penicillin” and “penicylin derivatives” (penams) is meant herein a subgroup of b- lactam antibiotics, derived originally from common moulds known as Penicillium moulds; which includes penicillin G (intravenous use), penicillin V (use by mouth), procaine penicillin, and benzathine penicillin (intramuscular use). Penicillin antibiotics were among the first medications to be effective against many bacterial infections caused by staphylococci and streptococci. They are still widely used today, though many types of bacteria have developed resistance following extensive use. There are several enhanced penicillin families which are effective against additional bacteria; these include the antistaphylococcal penicillins, aminopenicillins and the antipseudomonal penicillins. They are derived from Penicillium fungi.

Example of Natural penicillin :Penicillin G, Penicillin K, Penicillin N, Penicillin O, Penicillin V.

Example of b-lactamase-resistant penicylin derivatives: Methicillin, Nafcillin, Oxacillin, Cloxacillin, Dicloxacillin, Flucloxacillin.

Example of Aminopenicillins: Ampicillin, Amoxicillin, Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin, Epicillin.

Example of Carboxypenicillins: Carbenicillin, Ticarcillin, Temocillin.

Example of Ureidopenicillins: Mezlocillin, Piperacillin, Azlocillin.

Example of b-lactamase inhibitors penicylin derivatives: Clavulanic acid, Sulbactam, Tazobactam.

2. Aminoglycoside are the antibiotic agents directed to Gram negative bacteria that inhibit protein synthesis (targeting the small ribosome sub-unit of (30 Svedberg)) and contain as a portion of the molecule an amino-modified glycoside (Mingeot-Leclercq MP, et al (1999). Antimicrob. Agents Chemother. 43 (4): 727-37). The term “Aminoglycoside” can also refer more generally to any organic molecule that contains amino sugar substructures. Aminoglycoside antibiotics display bactericidal activity against Gram-negative aerobes and some anaerobic bacilli where resistance has not yet arisen but generally not against Gram positive and anaerobic Gram-negative bacteria.

Streptomycin is the first-in-class aminoglycoside antibiotic. It is derived from Streptomyces griseus and is the earliest modem agent used against tuberculosis. Streptomycin lacks the common 2-deoxystreptamine moiety present in most other members of this class. Other examples of aminoglycosides include the deoxystreptamine-containing agents, kanamycin, tobramycin, gentamicin, and neomycin.

3. Antibiotic agents which inhibit acid nucleic synthesis

Antibiotic agent which block DNA gyrase (topoisomerase specific to bacteria) : aminocoumarines, and quinolones.

Antibiotic agents which block the bacterial RNA polymerase: rifampicine.

4. Antibiotics which inhibit protein synthesis (other than Aminoglycoside)

Antibiotic agents which block the formation of the peptide bond: amphenicols (examples: chloramphenicol, thiamphenicol azidamfenicol and florfenicol)

Antibiotic agents which block elongation of the polypeptide chain: Tetracyclins (examples: tetracycline, doxycycline, aureomycine, eravacycline, sarecycline ,omadacycline) macrolides (examples : erythromycin, azithromycin) and ketolides (examples : telithromycin, cethromycin and solithromycin).

4. Antibiotics which inhibit folate metabolism

Sulfonamides also called sulphonamides, sulfa drugs or sulpha drugs (examples: Sulfamethoxazole) andsulfanilamides.

5. New classes of antibiotics compounds

Four new classes of antibiotics have been brought into clinical use in the late 2000s and early 2010s: cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), and lipiarmycins (such as fidaxomicin)

In a particular embodiment, the combination according to the invention, and pharmaceutical compositions of the invention aims at fighting bacterial resistance against cephalosporin (i.e. ceftazidime), monobactam (i.e. aztreozam), carbapenem (i.e. imipenem) and Aminoglycoside (i.e- gentamicin).

In a particular embodiment, the combination according to the invention, and pharmaceutical compositions of the invention aims at fighting multi-resistance bacterial infection. Bacteria are said to be multidrug-resistant (MDR) to antibiotics when, due to the accumulation of acquired resistance to several families of antibiotics, they are only sensitive to a small number of antibiotics usable in therapy (resistance to more than 3 different families).

In a particular embodiment, the cationic polymers used in the present invention have the following formula (I): wherein

• R is NH2 or NH linked to a histidine residue or other molecules selected from charged amino acids, a gluconoyl residue, a glycosyl residue or a PEG moiety,

• i is the degree of polymerization comprised between 10 and 75, preferably between 10 and 50, and more preferably between 20 and 50, for example between 20 and 40 or between 30 and 50.

In particular embodiment, the cationic polymer having the formula (I) is a alpha- cationic polymer.

As used herein the term “alpha-cationic polymer” has its general meaning in the art and refers to a cationic polymer where each unit/monomer is linked through NH2 residue in position alpha on lysine.

In one specific embodiment, said cationic polymers according to the combination of the invention comprise a sufficient amount of lysine residues (whether histidinylated or not) permeabilize the Gram-negative bacteria membrane and/or to act synergistically with antibiotics as antibacterial agents.

By "amount" or "sufficient amount" “or dosage level” is intended to be an amount of cationic polymers of the invention, that, when applied brings about a positive response with respect to permeabilize the membrane of Gram negative bacteria and/or to act as ti-bacterial agents. Actual dosage levels of the cationic polymer of the present invention may be varied so as to obtain an amount of the cationic polymer which is effective to achieve the desired permeabilization of the membrane of Gram negative bacteria. Typically, the dosage when cationic polymers of the invention are administered in solution is between 0.5 to 5 mM, preferably between 1 to 2mM.

The effect of permeabilization of the Gram negative bacteria membrane can be measured by monitoring cytoplasmic membrane depolarization activity prior to and after application with the compositions according to the invention, using in vitro assays (see experimental section : Membrane permeabilization assay) by measuring the fluorescence using the membrane potential-sensitive dye diSC3(5) (Sims P.J. et al. 1974). Briefly, this fluorescence probe is taken up by bacteria according to the magnitude of the electrical gradient of the cytoplasmic membrane and becomes concentrated in the cytoplasmic membrane, where it self- quenches its own fluorescence. Any compound that alters the permeability of the cytoplasmic membrane and thus induces depolarization will lead to the release of DiSC3(5) and a consequent increase in fluorescence.

In one more embodiment, the cationic polymers of formula (I) have a ratio of grafting of at least 10%, 20%, 30%, 40%, 50%, or 60%, preferably comprised between 10% and 60%, or between 10% and 55%, or between 10% and 40%, more preferably between 10% and 35%.

For example, the cationic polymers of the present invention have a ratio of grafting of between at least 10% and 60%, and a degree of polymerization i between 20 and 50 or between 20 and 40.

More specifically, the cationic polymers of the present invention may have a ratio of grafting of between at least 10% and 60%, or between 10% and 55%, or between 10% and 40%, and a degree of polymerization i between 30 and 40.

For ease of reading, specific cationic polymers of formula (I) according to the invention will be named hereafter as pLK(XX)His(YY), wherein XX refers to the average degree of polymerisation of the cationic polymers and YY refers to the average number of histidine grafted to lysine residues.

In specific embodiments, the cationic polymers of the present invention are pLK36- Hisl9, pLK36-His8, pLK72-His31, pLK72-Hisl7, pLK30-His4.5 and pLK30-His8. Lysyl (non-histidinylated) residues of the cationic polymers of the present invention may optionally be further at least partially replaced by other known positively charged amino acids, including without limitation histidine, arginine or ornithine. As used herein, the term “positively charged” refers to the side chain of the amino acids which has a net positive charge at a pH of 7.0.

A cationic polymer according to the invention typically may include at least 50% (per monomeric unit), 60% or at least 70% of positively charged amino acid residues, preferably at least 50%, 60%, 70% (per monomeric unit) of lysine residues, for example between 50% and 90% (per monomeric unit) of lysine residues.

Parameters such as the number of monomers (e.g. number of amino acids), the type of monomeric units (e.g. type of amino acids) and the percentage of positively charged monomeric units (e.g percentage of positively charged amino acids in a polyaminoacid) may be optimized by measuring the efficacy of the final structure in an in vitro assay for permeabilization of the membrane of Gram negative bacteria, as disclosed in the Examples below (see experimental section The Membrane permeabilization assay .

In particular cationic polymers for use according to the present invention include poly- L-lysine, without derivatized or substituted with histidine or neutral residue.

In particular embodiment, the cationic polymer having the formula (I) is an alpha- polylysine (a-Polylysine).

For example, derivatization of lysyl residues of poly-L-lysine with histidine is described in Midoux, P. and Monsigny, M. (Efficient gene transfer by histidylated polylysine/pDNA complexes. Bioconjugate Chem. 1999, 10, 406-411). Methods for synthesizing the cationic polymers of the present invention are also described in WO2015078995.

Equivalent amino acids may also be used in the cationic polymers of the invention, including amino acids having side chain modifications or substitutions, the final polymer retaining its advantageous property on permeabilization of the membrane of Gram negative bacteria.

In particular, (D) or (L) amino acids may be used, or chemically modified amino acids, including amino acid analogs such as penicillamine (3-mercapto-D-valine), naturally occurring non-proteogenic amino acids and chemically synthesized compounds that have properties known in the art to be characteristic of an amino acid.

Cationic polymers useful for this invention can be produced using technique well known in the Art, including either chemical synthesis or recombinant DNA techniques. Cationic polypeptides can be synthesized using Solid Phase Peptide Synthesis techniques with tBoc or Fmoc protected alpha-amino acids (Scholz, C.et al, J Control Release, 2011). Alternatively, poly cationic polypeptides can be produced using recombinant DNA techniques (See Coligan et al. , Current Protocols in Immunology, Wiley Intersciences, 1991, Unit 9; US Pat. No. 5,593,866).

In specific embodiments, the cationic polymers may be PEGylated. PEGylation is the process of covalent attachment of polyethylene glycol polymer chains to another molecule. Polyethylene glycol (PEG) molecules may be added onto cationic polymers in order to limit DNA complexes aggregation, adsorption of proteins and to lower aggregate as well as polymer cytotoxicity (Ogris, M.et al., P. Gene Ther, 1999; Toncheva, V. et al., Biochim Biophys Acta, 1998 ; Choi, Y. et al., 1999). The covalent attachment of PEG to cationic polymers may facilitate and does not compromise administration of said cationic polymers into the airways in the form of an aerosol (Dailey, L. A., et al., J Control Release, 2004).

The covalent attachment of PEG moiety onto cationic polymer can be performed by two ways leading either to a PEG-grafted-polymer or a block copolymer.

For example, PEG-grafted polylysine (PEG-g-pLK) is prepared by reaction of the N- hydroxysuccinimide derivative of the methoxypolyethylene glycol (mPEG)propionic acid (for instance of 5000 Da)with the e-amino group of the lysyl residues of pLK (Mockey, M., et al., Cancer Gene Ther, 2007).

For example, PEG-pLK(XX)His(YY) block copolymer can be prepared either by :

-(i) reaction between equal molar ratios of pLK(XX)His(YY) containing a cysteinyl residue at its C-terminal end with methoxy-PEG-maleimide as described in Ziady, A.G .et al. (Mol Ther., 2003);

- (ii) ring opening polymerization of N ^£ -trifluoroacetyl-L-lysine N-carboxyanhydride with the co-NFh terminal group of a-methoxy-co-amino PEG as described in Itaka, K., et al.(J Control Release, 2010).

In other specific embodiments, the cationic polymers in accordance with the present invention are glycosylated. For example, derivatization of lysyl residues of poly-L-lysine with mannose, galactose or lactose is described in (Erbacher, P. et al (Bioconjug Chem,1995); Erbacher, P., et al. (Hum Gene Ther, 1996).Mannosyl-PEG, galactosyl-PEG or lactosyl-PEG may be grafted on poly-L-lysine as described in (Sagara, K. et al. (J Control Release, 2002).

In other specific embodiments, the cationic polymers in accordance with the present invention are gluconoylated in order to decrease the number of positive charges and the cytotoxicity. For example, derivatization of lysyl residues of poly-L-lysine with b- gluconolactone is described in (Erbacher, P. et al(Biochim Biophys Acta, 1997).

As used herein, the terms "combination" refers to a "kit-of-parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combined preparation can vary. The combination partners can be administered by the same route or by different routes. When the administration is sequential, the first partner may be for instance administered 1, 2, 3, 4, 5, 6, 7, days before the second partner.

The present invention also provides the cationic polymers according to the invention, for use in a method for enhancing sensitivity to an antiobitic of a patient suffering from antibiotic-resistant bacterial infection.

Pharmaceutical compositions according to the invention

The present invention also provides a pharmaceutical composition comprising: i. a cationic polymer (as defined here above), ii. an antiobiotic agent (as defined here above); and iii. a pharmaceutically acceptable carrier. for use in the prevention or the treatment of antibiotic-resistant bacterial infection in a patient in need thereof.

Pharmaceutical compositions formulated in a manner suitable for administration to humans are known to the skilled in the art. The pharmaceutical composition of the invention may further comprise stabilizers, buffers, etc.

The compositions of the present invention may, for example, be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for administration by injection.

The choice of the formulation ultimately depends on the intended way of administration, such as e.g. an intravenous, intraperitoneal, subcutaneous or oral way of administration, or a local administration.

The pharmaceutical composition according to the invention may be a solution or suspension, e.g. an injectable solution or suspension. It may for example be packaged in dosage unit form.

In a preferred embodiment, the cationic polymers and an antiobiotic agent of the invention is preferably administered by the oral route or intravenous route.

Typically medicaments according to the invention comprise a pharmaceutically- acceptable carrier. A person skilled in the art will be aware of suitable carriers. Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known text such as Remington; The Science and Practice of Pharmacy. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Effect of pLK on the fluorescence intensity changes of reference P. aeruginosa PAOl strain, incubated with DiSC3(5). The experiments are performed at 1, 10 and 100 mM Results are expressed in relative intensity fluorescence observed at 670 nm as compared to negative control (PBS). Values are mean ± SD of three determinations..

Figure 2: Synergistic effect in vitro of combination pLK and imipenem against reference P. aeruginosa PAOl strain. Bacteria (1.107 /mL in exponential phase) were incubated at 37°C in Mueller Hinton medium in presence or absence of 2 mM poly-L-Lysine (pLK), 1 pg/mL imipenem (IMP) or the combination. (A, B) Bacterial growth was measured using the optical density at 600 nm after 24 h-incubation. (C) Determination of colony forming unit (CFU)/mL was evaluated after 24 hour-incubation. Values are mean ± SD of three independent experiments.

Figure 3: Synergistic effect in vitro of combination pLK and imipenem against imipenem-sensitive clinical strain. Bacteria (1.105/mL in exponential phase) were incubated at 37°C in Mueller Hinton medium in presence or absence of 1 pM poly-L-Lysine (pLK), 1 pg/mL imipenem (IMP) or the combination. (A) Bacterial growth was measured using the optical density at 600 nm after 24 h-incubation. (B) Determination of colony forming unit (CFU)/mL was evaluated after 24 hour-incubation. Values are mean ± SD of three independent experiments.

Figure 4: Synergistic effect in vitro of combination pLK and imipenem against PAO UoprD. Bacteria (1.10 ⁷ /mL in exponential phase) were incubated at 37°C in Mueller Hinton medium in presence or absence of 1 pM poly-L-Lysine (pLK), 1 pg/mL imipenem (IMP) or the combination. (A and B) Bacterial growth was measured using the optical density at 600 nm after 24 h-incubation. (C) Determination of colony forming unit (CFU)/mL was evaluated after 24 hour-incubation. Values are mean ± SD of three independent experiments

Figure 5: Synergistic effect in vitro of combination pLK and imipenem against imipenem-resistant clinical strain. (A) Bacteria (1.10 ⁵ /mL in exponential phase) were incubated at 37°C in Mueller Hinton medium in presence or absence of 1 pM poly-L-Lysine (pLK), 1 pg/mL imipenem (IMP) or the combination. Bacterial growth was measured using the optical density at 600 nm after 24 h-incubation. (B) Determination of colony forming unit (CFU)/mL was evaluated after 24 hour-incubation. Values are mean ± SD of three independent experiments.

Figure 6: Synergistic effect ex vivo of combination pLK and imipenem against imipenem-resistant clinical strain. Human primary bronchial epithelial cells maintained in air-liquid interface were infected with PAOlAOprD strain and then treated one hour post infection either with PBS, 2 mM pLK, 2 pg/mL imipenem or the combination imipenem and pLK. Determination of colony forming unit (CFU)/mL was evaluated after 24 hours post infection. Values are mean ± SD of three independent experiments.

Figure 7: Synergistic effect in vitro of combination pLK and ceftazidime against MexAB-OprM. (A) Bacteria (1.10 ⁵ /mL in exponential phase) were incubated at 37°C in Mueller Hinton medium in presence or absence of 1 or 2 pM poly-L-Lysine (pLK), 1 pg/mL ceftazidime (IMP) or the combination. Bacterial growth was measured using the optical density at 600 nm after 24 h-incubation. (B) Determination of colony forming unit (CFU)/mL was evaluated after 24 hour-incubation. Values are mean ± SD of five independent experiments.

Figure 8: Synergistic effect in vitro of combination pLK and aztreonam against MexAB-OprM. (A) Bacteria (1.10 ⁵ /mL in exponential phase) were incubated at 37°C in Mueller Hinton medium in presence or absence of 1 or 2 pM poly-L-Lysine (pLK), 1 pg/mL ceftazidime or the combination. Bacterial growth was measured using the optical density at 600 nm after 24 h-incubation. (B) Determination of colony forming unit (CFU)/mL was evaluated after 24 hour-incubation. Values are mean ± SD of three independent experiments.

Figure 9: Synergistic effect in vitro of combination pLK and ceftazidime against MexXY/OprM. (A) Bacteria (1.10 ⁵ /mL in exponential phase) were incubated at 37°C in Mueller Hinton medium in presence or absence of 1 or 2 pM poly-L-Lysine (pLK), 1 pg/mL ceftazidime or the combination. Bacterial growth was measured using the optical density at 600 nm after 24 h-incubation. (B) Determination of colony forming unit (CFU)/mL was evaluated after 24 hour-incubation. Values are mean ± SD of five independent experiments.

Figure 10. Synergistic effect in vitro of combination pLK and gentamycin against MexXY/OprM. Bacteria (1.10 ⁵ /mL in exponential phase) were incubated at 37°C in Mueller Hinton medium in presence or absence of 2 pM poly-L-Lysine (pLK), 1 or 2 pg/mL gentamycin or the combination. Determination of colony forming unit (CFU)/mL was evaluated after 24 hour-incubation. Values are recorded from one independent experiment.

EXAMPLE:

Material & Methods 1. Material

Poly-L-Lysine was purchased by Sigma-Aldrich (Sant-Quentin Fallavier, France) and reconstituted at 1 mM in PBS (. Phosphate Buffered Saline [137 mM NaCl, 2.7 mM KC1, 8.1 mM Na2HP04, 1.5 mM KH2PO4, pH 7, 2-7, 4, 0.2 pm filtered]. Ceftazidime, imipenem/cilastatine and gentamicin sulfate salt were purchased from Sigma Aldrich (Sant- Quentin Fallavier, France). 3,3' Dipropyl thiadicarbocyanine iodide (DiSC3(5)) was obtained from Molecular probes, Inc. (Eugene, OR).

For this study, different strains ofP. aeruginosa strains were selected exhibiting natural or identified acquired mechanism of resistance : reference wild type strain PAOl, its mutant PAOl AoprD (for porin D2 alteration), and clinical strains from CF and non CF patients (with porin alteration or an overproduction of different active efflux systems as MexAB-OprM and MexXY/OprM). These strains were kindly provided by Dr. Katy Jeannot (Centre National de Reference de la Resistance aux Antibiotiques, Besancon. France). Lauryl Broth (LB) medium was purchased from Sigma Aldrich (Sant-Quentin Fallavier, France) and Mueller-Hinton (MH) medium was purchased from Biorad (Roanne, France). Tryptic Soy Agar (TSA) plates were purchased from Biomerieux (Craponne, France).

Air-liquid interface (ALI) culture of human Primary Bronchial Epithelial Cells (PBEC) was used for ex vivo experiment. ALI-PBEC were kindly provided by Pr Pieter Hiemstra (Laboratoire de Biologie Cellulaire et dTmmunologie du Centre medical, Leiden University, Netherlands). PneumaCult-Ex and PneumaCult-ALI media were purchased from Stemcell (Grenoble, France). Penicillin-Streptomycin solution was purchased from Pan BioTech (Germany). Phosphate buffered saline (PBS), Keratinocyte Serum-Free Medium (KSFM), human recombinant Epithelial Growth Factor (EGF), Bovine Pituitary Extract (BPE), Bovine Serum Albumin (BSA) and trypsin-EDTA were purchased from Gibco (Grenoble, France). Transwell® culture inserts (6.5 mm diameter, 0.4 mM pore size) were purchased from Coming Costar (Thermo Fisher, France). PureCol was purchased from Advanced BioMatrix (San Diego, CA), human fibronectin stabilized solution from Clinisciences (Alfa Aesar), and human interleukin-6 and -8 (IL-6 and IL-8) DuoSet ELISA were purchased from Fisher Scientific (Illkirch-Graffenstaden, France).

2. Methods

The Membrane permeabilization assay. The cytoplasmic membrane depolarization activities of pLK were determined with the membrane potential-sensitive dye diSC3(5) (Sims P.J. et al. 1974) and different strains of P. aeruginosa. Briefly, overnight cultures of P. aeruginosa were diluted in LB medium and allowed to grow to the mid-logarithmic phase. Bacteria were collected by centrifugation, washed 3 times with buffer (5 mM HEPES, pH 7.8) and resuspended in the same buffer to an optical density at 600 nm (Oϋboo) of 0.05. The cells were treated with 0.2 mM EDTA (pH 8.0) in order to permeabilize the outer membrane to allow dye uptake. Then, the cell suspension was incubated during 20 min at 37 °C under shaking (150 rpm) with 0.4 mM DiSC3(5) until dye uptake was maximal and KC1 was added to the cell suspension to a final concentration of 100 mM to equilibrate the cytoplasmic and external K+ concentrations. The desired concentration of pLK was then added and the fluorescence was monitored under shaking (150 rpm) at 37 °C at an excitation wavelength of 622 nm and an emission wavelength of 670 nm after 15, 30, 60 and 90 min (TEC AN Infinite 200, Lyon, France). A blank with only bacteria and the dye was used as background.

This probe is taken up by bacteria according to the magnitude of the electrical gradient of the cytoplasmic membrane and becomes concentrated in the cytoplasmic membrane, where it self-quenches its own fluorescence. Any compound that alters the permeability of the cytoplasmic membrane and thus induces depolarization will lead to the release of DiSC3(5) and a consequent increase in fluorescence.

Susceptibility testing. Broth microdilution method was used for the determination of minimal inhibitory and bactericidal concentrations (MIC and MBC). MICs were determined, in accordance with the guidelines of the Clinical and Laboratory Standards Institute. Briefly, Pseudomonas aeruginosa strains were cultured on TSA plates overnight. Three isolated colonies were suspended in 3 mL of LB medium and grown overnight at 37°C, under agitation (200 rpm). Then several dilutions of this fresh suspension were prepared and incubated at 37°C during 4 hours, under agitation (200 rpm), to Oϋboo of 0.3-0.6, representing the logarithmic phase. The suspension with Oϋboo between 0.3 and 0.6 was centrifuged 10 minutes at 3000 g. Bacteria were suspended in MH medium to obtain approximately 2.10 ⁵ CFU/mL (CFU for colony forming unit). The inoculum size was verified by plating 5-fold dilutions on TSA plates and incubating overnight at 37°C for CFU counts.

Hundred microliter/ well of the bacterial suspension was inoculated into 96-well microtiter plate and 100 pL/well of MH (control) or antibiotic or/and pLK was added in duplicate for each condition. The microtiter plate was incubated in a plate reader (TECAN Infinite 200, Lyon, France) for 24 h at 37°C in ambient air. The absorbance at Oϋboo was read at 30-min intervals. After incubation, the entire volume (100 pL) of each well were spread across the centre of a blood agar plate and a sterile spreading rod was used to evenly disperse the inoculum over the entire surface of the plate, which was then incubated at 37°C for 24 h. The MBC was recorded as the lowest dilution that produced a reduction of growth > 99.99% (> 4-logio reduction in CFU/mL) in comparison to the control growth.

Cell Culture. Human primary bronchial epithelial cells (PBEC, Passage #1) were thawed at 37°C water bath and amplified in 75 cm ² flask in KSFM Medium with human recombinant EGF at 2.5 pg/mL, BPE at 25 pg/mL and antibiotics (Penicillin 10000 U/mL and Streptomycin 10 mg/mL).

Then, PBEC (passage # 2) were transferred on porous membrane Transwell™ insert in 24-well plate (6.5 mm diameter with 0.4 pm pore) at a density of 7*10 ⁴ cells/mL and cultured with PneumaCult-Ex medium, at 37°C, 5% CCh incubator during 4 to 6 days. Transwells were coated with a mixture of 30 pg/mL PureCol, 0.75 mg/mL BSA and 5 pg/mL human fibronectin stabilized solution. Then, PBEC cells were maintained in air-liquid interface (ALI), using PneumaCult-ALI medium only in the basolateral chamber (500 pL/well), during 21 days.

Ex vivo determination of antibacterial activity. Differentiated ALI-PBEC were infected with PAO 1 Aoprl) (in logarithmic phase) at a multiplicity of infection (MOI) of 0.01, in the apical chamber. One hour post-infection, the cells were washed once from the apical side using warm PBS, followed by addition of 100 pL PBS or pLK with/or imipenem. Then, cells were incubated at 37°C for 20 h, in 5% CO2 incubator. After incubation, apical and basal medium were removed, centrifuged 5 min at 500 g. Supernatants were stored at -20°C for measuring human interleukin 6 and 8 (hIL-6 and hIL-8), by enzyme-linked immunosorbent assay (DuoSet ELISA - R & D Systems). Ice-cold water (300 pL) was used to remove cells and bacteria from the inserts. Hundred microliters of each insert were spread across the centre of a blood agar plate and a sterile spreading rod was used to evenly disperse the inoculum over the entire surface of the plate, which was then incubated at 37°C for 24 h. The MBC was recorded as the lowest dilution that produced a reduction of growth > 99.99% (> 4-logio reduction in CFU/mL) in comparison to the control growth.

TransEpithelial Electrical Resistance Measurement

During the culture of ALI-PBEC in inserts, the development of tight junctions of the cell layers were followed up by measuring the TransEpithelial Electrical Resistance (TEER) values using Epithelial Volt/Ohm Meter or EVOM2™ (World Precision Instruments, Sarasota, Florida, USA). The basolateral chambers were filled with fresh medium before the measurement. The inserts with the cells were equilibrated in IX PBS at 37°C, humidified CO2 incubator for 10 min. TEER values were corrected by subtracting the background TEER values measured in inserts without the cells, only with the media in the both chambers and the area of membranes was considered in the TEER units (W ah ² ). Results

Permeabilization of P. aeruginosa membrane by pLK

We investigated the ability of pLK to depolarize the bacterial membranes of P. aeruginosa (PAOl), using DiSC3(5), a membrane potential-dependent probe. Upon membrane permeabilization, the membrane potential is dissipated, and DiSC3(5) is released into the medium leading to a consequent increase in fluorescence. The pLK was tested at 3 concentrations (1, 10 and 100 mM) in PBS, during 90 minutes, against PAOl. As expected, negative control (PBS) showed no release of relative fluorescence (Figure 1). The pLK tested at 1 mM, induced a small effect, with an increase of relative fluorescence around 2.5-fold after 15, 30, 60 or 90 minutes (Figure 1). The pLK at 10 pM induced a membrane depolarization increasing over time and reached a 10-fold increase in fluorescence after 90 minute-incubation. Results obtained with 100 pM pLK also showed a time-dependent increase of relative fluorescence with a maximum around 12.5-fold after 90 minutes (Figure 1). Altogether, these results indicated the ability of pLK to permeabilize the bacteria membrane of P. aeruginosa.

Synergistic effect in vitro of the association of pLK and imipenem against wild-type PAOl strain and clinical strains imipenem-sensitive.

Different combinations of pLK and imipenem were evaluated against reference wild type strain, PAOl and clinical strains which are imipenem-sensitive. First, concerning PAOl strain, MICs were determined for pLK at 5 pM (50 mg/L) and for imipenem at 7,5 pM (2 mg/L). The results showed that the synergistic effect was observed against PAOl strain, with pLK 2 pM and imipenem 1 pg/mL (Figures 2A and B). Moreover, a bactericidal effect was also demonstrated with this combination (Figure 2B). Indeed, we determined a reduction of bacterial growth of 7-logio in comparison to the control (1.10 ⁷ CFU/mL at the beginning of the experiment)

Almost same results were obtained against three imipenem-sensitive clinical strains, showing a synergistic effect with the association of 1 pM pLK (CMI/5) and 1 pg/mL imipenem (CMI/4) (Figure 3A) and also a bactericidal effect, showing a reduction of bacterial growth of 4-loglO in comparison to the control (1.105 CFU/mL at the beginning of the experiment) (Figure 3B).

Synergistic effect in vitro and in vivo of the association of pLK and imipenem against PAOl AoprD strain and imipenem-resistant clinical strains.

PAOl AoprD strain presents an OprD porin modification, making it resistant to imipenem. MICs were determined at 5 pM for pLK (same as PAOl strain) and at a concentration greater than 16 pg/mL for imipenem (expected with this imipenem-resistant strain). Our results showed a synergistic effect of the association of 2 mM pLK and 4 pg/mL imipenem (Figure 4A and B), as well as a bactericidal effect showing a reduction of bacterial growth of 7-logio in comparison to the control (1.10 ⁷ CFU/mL at the beginning of the experiment) (Figure 4B).

Then, we also tested these combinations on clinical isolates presenting an imipenem- resistance and we obtained a synergistic effect with 1 mM pLK and 8 pg/mL imipenem with a bactericidal effect showing a reduction of bacterial growth of 5-logl0 in comparison to the control (1.105 CFU/mL at the beginning of the experiment) (Figure 5).

Next, we evaluated ex vivo effect of the pLK/imipenem combination against VAOlAOprD strain, using a culture of human primary bronchial epithelial cells (PBEC) maintained in air-liquid interface (ALI). This cell culture model partially reconstitutes the environment of human bronchial epithelium. Infection with the VAOlAOprD strain was realized at a MOI of 0.01. The tested conditions are the following: PBS, 2 pM pLK, 2 pg/mL imipenem and the combination at same concentrations. One hour post-infection, each treatment is deposited on the epithelium surface. Twenty-four hour post-infection, the bacterial count is realized for each condition. Results revealed a synergistic effect of the combination 2mM pLK and 2 pg/mL imipenem (Figure 6), with a reduction of bacterial growth of 3-logio in comparison to the control (1.10 ⁵ CFU/mL at the beginning of the experiment).

In conclusion, the combination of pLK with imipenem could contribute to counteract P. aeruginosa “OprD resistance”.

Synergistic effect in vitro of the association of pLK and ceftazidime or aztreonam against MexAB-OprM clinical strain.

MexAB-OprM clinical strain presents an overproduction of its efflux pump. MICs were determined at 2 mM for pLK, at 4 pg/mL for ceftazidime and at a concentration greater than 8 pg/mL for aztreonam. Our results showed a synergistic effect of the association of 1 pM pLK and 1 pg/mL ceftazidime, and a reduction of bacterial growth of 2-logio in comparison to the control (1.10 ⁵ CFU/mL at the beginning of the experiment) (Figure 7A and B).

The combination pLK and aztreonam showed also a synergistic effect with 1 mM pLK and 4 pg/mL aztreonam, and a reduction of bacterial growth of 2-logio in comparison to the control (1.10 ⁵ CFU/mL at the beginning of the experiment) (Figures 8 A and B).

In vitro analysis of the synergistic effect of the association of pLK and ceftazidime against MexXY/OprM clinical strain.

MexXY/OprM clinical strain presents an overproduction of its efflux pump. MICs were determined at 2 mM for pLK, and at 4 pg/mL for ceftazidime. Our results showed a synergistic effect of the association of 1 mM or 2mM pLK with 1 mg/mL ceftazidime, and a reduction of bacterial growth of 4-logio or 5-logio respectively, in comparison to the control (1.10 ⁵ CFU/mL at the beginning of the experiment) (Figure 9A and B).

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