WO2015078995A1 - Agents for treating cystic fibrosis - Google Patents

Abstract

The present invention relates to compositions comprising cationic polymers and their use in treating inflammatory lung diseases characterized by the recruitment of blood neutrophils in the airways favoring the formation of a thick, mucoid or mucopurulent sputum as found in cystic fibrosis.

Description

AGENTS FOR TREATING CYSTIC FIBROSIS

The present invention relates to novel cationic polymers and their use in treating cystic fibrosis, or other inflammatory lung disorders characterized by the recruitment of blood neutrophils in the airways favoring the formation of a thick, mucoid or mucopurulent sputum.

Cystic fibrosis and other lung disorders are characterized by obstruction of the airways caused by the accumulations of viscous secretions. In particular, cystic fibrosis (CF) owes its morbidity and mortality primarily to the devastating effects of chronic inflammation and infection within the pulmonary airway. The opportunistic bacteria P aeruginosa and S aureus are most often present in the lung secretions of patients with cystic fibrosis (CF), where they contribute to maintain chronic inflammation in the lung (1 ). The presence of thick and sticky sputum that characterizes CF secretions greatly impairs mucociliary clearance and thus the elimination of these pathogens. DNA is a major component of CF sputum that contributes to airways obstruction and to innate immune response failure observed during this disease (2). Thus it favors infection and colonization by these opportunistic bacteria.

Most of the DNA present in CF expectorations is released from dead neutrophils or is actively secreted as neutrophil extracellular traps (NETs) by activated neutrophils. NETs have been reported as antimicrobial weapons made of chromatin fibers covered with granular antimicrobial proteins and proteases that trap and kill pathogens (3). It was recently assumed that NETs formation would represent the major factor underlying the gel-like structure of CF sputum (4). The nebulization in CF airways of recombinant human Dornase (DNase) reduces the viscoelasticity of CF sputum and enhances the clearance of secretions, which illustrates the essential contribution of DNA to the physical properties of CF lung secretions. Though Dornase is the most widely used mucoactive agent for CF patients, sputum fluidization may also be obtained using hypertonic saline which increases the volume of airway surface liquid and restores mucus clearance (5, 6).

Another hallmark of cystic fibrosis is the presence in lung secretions of an uncontrolled proteolytic potential mainly due to neutrophil serine proteases (NSPs). Treatment of cystic fibrosis patients with exogenous protease inhibitors such as a1 -anti-trypsin, or secretory leukoprotease inhibitor have also been proposed (7). The inventors have recently shown that NSPs, i.e. neutrophil elastase, protease 3 and cathepsin G in CF sputum are active and lack sensitivity to exogenous protease inhibitors (8). Elastase activity in a CF sputum homogenate increases dramatically after Dornase treatment but also is far better controlled by exogenous protease inhibitors. The inventors hypothesized that this results from the release of DNA-bound elastase that gains in activity and/or becomes more accessible to protease inhibitors once solubilized. However, no such increase in activity is observed with the other two NSPs, probably because of their tighter binding to DNA or to their binding to other macromolecular components present in sputum.

Conventional treatments of CF include secretion clearance, antibiotic treatments, antiinflammatory treatments.

It was known in the Art that cationic polymers and in particular poly-L-lysine have the capacity to form electrostatic complexes with DNA and to induce its condensation as aggregates. Such cationic polymers are in development as gene therapy non-viral vectors (9) including for the treatment of cystic fibrosis (http://www.worldscibooks.conT/lifesci/7406.html). Cationic polymers have also been described as inhibitors of mucin secretion from airway goblet cells in vitro, and suggested to be used as a tool in the treatment of diseases associated with mucin hypersecretion (US 6,245,320). a-poly-L-lysine and positively charged amino acids have also been described in the art for their anti-microbial properties (10).

The inventors have now found that cationic polymers such as poly-L-lysine have the following properties: i/ they condensate DNA, and in particular extracellular DNA from neutrophil extracellular traps (NETs) in CF lung secretions and thus fluidize CF sputum as does recombinant human Dornase; ii/ they improve the control of extracellular proteases by exogenous inhibitors as a result of DNA condensation; i.e. they enhance anti-protease activity of protease inhibitor in cystic fibrosis sputa in vitro, iii/ they act as bactericidal agents because of their cationic character.

The inventors have further found that grafting histidine residues to the lysyl residues of polylysine cationic polymers, preferably poly-L-lysine with an average degree of polymerization comprised between 20 and 50, reduce the toxicity while maintaining the above advantageous properties of poly-L-lysine. Altogether these results provide new insights for treating cystic fibrosis, using cationic polymers either as main active principle ingredient or in combination with protease inhibitor treatment, as an adjuvant for in vivo stimulation of protease inhibition in secretions from patients suffering from an accumulation of thick, mucoid or mucopurulent sputum in the airways. Moreover, pretreatment with cationic polymers would facilitate accessibility of antibiotics and of viral and non viral vectors used for gene therapy strategies.

Summary of the invention

Therefore, in a first aspect, the present invention relates to a cationic polymer of the following formula (I) or their pharmaceutically acceptable salts,

wherein

• R is NH2 or NH linked to a histidine residue or other molecules including 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 40.

In specific embodiments, the percentage of lysyl derivatization by histidyl residue in a cationic polymer of formula (I) is at least 10%, 20%, 30%, 40%, preferably comprised between 15 and 60%, or between 10% and 55%, or between 10% and 40%, more preferably between 10% and 35%. In a more specific embodiment, the degree of polymerization is 36 and the percentage of lysyl derivatization is 54%. In another specific embodiment, the degree of polymerization is 30 and the percentage of lysyl derivatization is either 15% or 27%. The invention further relates to a pharmaceutical composition comprising a cationic polymer of the invention as defined above, and at least one pharmaceutically acceptable carrier.

In a specific embodiment, the pharmaceutical composition further comprises at least a protease inhibitor. Typically, such protease inhibitor may be selected from the group consisting of: i. serpins, e.g., a1 -antitrypsin, antichymotrypin, or serpin B1 ,

ii. non covalent canonical inhibitors, e.g. elafin, SLPI ecotin, or eglin C, iii. synthetic covalent inhibitors including acyl-enzyme inhibitors, transition state inhibitors, mechanism based inhibitors,

iv. synthetic non covalent inhibitors including substrate-like inhibitors, cyclic peptidyl inhibitors, heterocyclic inhibitors.

In a more specific embodiment, said cationic polymer is present in the pharmaceutical composition according to the present invention in an effective amount for enhancing anti- protease activity of said protease inhibitor in cystic fibrosis sputa in vitro. In one specific embodiment, the pharmaceutical composition of the invention is suitable for administration in the form of an aerosol.

In another specific embodiment, the pharmaceutical composition of the invention may not comprise any nucleic acid molecule. The pharmaceutical composition and cationic polymers of the present invention are useful in treating inflammatory lung disorders characterized by the recruitment of blood neutrophils in the airways favoring the formation of a thick, mucoid or mucopurulent sputum, for example cystic fibrosis.

Cationic polymers for use in the present invention The invention results from the discoveries of unexpected properties of certain cationic polymers to reduce the visco-elasticity of sputum present in the airways of patients suffering from disorders characterized by the recruitment of blood neutrophils in the airways favoring the formation of a thick, mucoid or mucopurulent sputum, such as cystic fibrosis. In particular, the inventors have found that grafting histidine to polylysine cationic polymers result in decreasing the risk of toxicity for use as a drug in mammals, while maintaining advantageous biological properties. As used herein the term "airway" refers to any part of the breathing system, including the lungs and the respiratory tract and nose.

The term "patient" refers to animals, non-human mammalian or human beings, having a pulmonary system. The cationic polymers of the present invention are polylysine where part of the lysine residues have been grafted at the ε-amino group of the lysyl residues with histidyl residues, resulting in histidinylated polylysine residues. The ratio of grafting, corresponding to the number of histidinylated polylysine residues divided by the degree of polymerisation of the cationic polymer is adapted to enable DNA condensation and/or to reduce in vitro the visco-elasticity of sputum from patients suffering from cystic fibrosis, while reducing the risk of toxicity for example as measured in a cellular cytotoxicity assay.

Typically, the percentage of lysyl derivatization by histidyl residue in the cationic polymers of the invention may be at least 10%, 20%, 30%, 40%, preferably comprised between 10% and 60%, or between 10% and 55%, or between 10% and 40%, for example between 10% and 35%.

The ratio of grafting of the cationic polymer in a composition may be preferably determined from 1 H-NMR spectrum analysis in D₂0 according to x = 6(h8.7 / hlys ).dp, where h8.7 is the value of the integration of the signal at 8.7 ppm corresponding to the proton of the imidazole ring (1 H C12), hlys is the range 1 .3-1 .9 ppm corresponding to the 6 methylene protons of lysine residues (C3, C4 and C5), and dp is the degree of polylysine polymerization.

More specifically, the cationic polymers that can be used in the present invention have the following formula (I):

wherein • R is NH2 or NH linked to a histidine residue or other molecules including 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 invention also relates to any pharmaceutically acceptable salts of the cationic polymers defined in the above formula (I).

In one specific embodiment, said cationic polymers for use according to the invention comprise a sufficient amount of lysine residues (whether histidinylated or not) to enable DNA condensation and/or to reduce in vitro the visco-elasticity of sputum from patients suffering from cystic fibrosis.

In one more preferred 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-His19, pLK36-His8, pLK72-His31 , pLK72-His17, 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 assessing visco-elasticity of sputum sample from cystic fibrosis patients, as disclosed in the Examples below. At optimal concentration, the cationic polymers are capable of inducing the fractionation of the sputum in dense aggregates and a very fluid phase and/or reducing significantly the measured visco-elasticity of said sputum sample. Preferred cationic polymers for use according to the present invention include poly-L- lysine, with no more than 50% of the lysyl residue derivatized or substituted with histidine or neutral residue.

For example, derivatization of lysyl residues of poly-L-lysine with histidine is described in (Midoux, P. and Monsigny, M. (1999). Efficient gene transfer by histidylated polylysine/pDNA complexes. Bioconjugate Chem. 10, 406-41 1 ). Methods for synthesizing the cationic polymers of the present invention are also described in the Examples below.

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 of fluidizing sputum from cystic fibrosis patients. 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 (1 1 ). Alternatively, polycationic 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 (12-14). 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 (15). 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 ε-amino group of the lysyl residues of pLK (16). 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 (17); ii) ring opening polymerization of N rifluoroacetyl-L-lysine N-carboxyanhydride with the ω-ΝΗ₂ terminal group of a-methoxy-oo-amino PEG as described in (18). 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 (19, 20). Mannosyl-PEG, galactosyl- PEG or lactosyl-PEG may be grafted on poly-L-lysine as described in (21 ).

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 β- gluconolactone is described in (22).

Pharmaceutical Formulations and Modes of Administration

In another aspect the present invention provides a composition, e.g., a pharmaceutical composition, containing the cationic polymers as described above, formulated together with a pharmaceutically acceptable carrier or excipient. Pharmaceutical formulations comprising the cationic polymers of the invention may be prepared for storage by mixing the cationic polymers of the present invention, having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington: the Science and Practice of Pharmacy 20th edition (2000)), in the form of aqueous solutions, lyophilized or other dried formulations.

Therefore, the invention further relates to a lyophilized, dried or liquid formulations comprising at least cationic polymers of the invention as described in the previous paragraph.

One remarkable finding of the present invention is that the cationic polymers are useful as active principles, and not as vector for gene therapy or carrier (as previously disclosed in the prior art). Accordingly, the present invention provides composition essentially consisting of cationic polymers of the present invention as described above, as active principles, optionally formulated with pharmaceutically acceptable carrier or excipient or stabilizers. As used herein, 'pharmaceutically acceptable carrier' includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for inhalation, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The cationic polymers of the invention may include one or more pharmaceutically acceptable salts. A 'pharmaceutically acceptable salt' refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (23). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic hydroiodic phosphorous and the like as well as from nontoxic organic acids such as aliphatic mono- and di- carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

Sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

In a preferred embodiment, the compositions are formulated for their administration into the airways, e.g. by inhalation.

The compositions of the invention may thus be formulated as solution appropriate for inhalation. Any of the various means known in the art for administering therapeutically active agents by inhalation (pulmonary delivery) can be used in the methods of the present invention.

Such delivery methods are well-known in the art. Commercially available aerosolizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers may be used. For delivery in liquid form, liquid formulation can be directly aerosolized and lyophilized powder can be aerosolized after reconstitution. For delivery in dry powder form, the formulation may be prepared as a lyophilized and milled powder. In addition, formulations may be delivered using a fluorocarbon formulation or other propellant and a metered dose dispenser. In specific embodiments, for example, nebulizers, which convert liquids into aerosols of a size that can be inhaled into the lower respiratory tract, are used, either in conjunction with a mask or a mouthpiece. Other devices have been developed such as AERx (Aradigm, Hayward, CA) and Respimat (Boehringer, Germany) that generate an aerosol mechanically and vibrating mesh technologies such as AeroDose (Aerogen, Inc., Galway, Ireland), Eflow (Pari, Stanrberg, Germany) and MicroAir (Omron, Japan) nebulizers used to deliver proteins and peptide-based pharmaceuticals to the lungs. In every case the size of aerosolized particles should remain < 5μΜ to allow an adequate lung targeting.

In other embodiments, metered dose inhalers may be used. In yet other embodiments, dry powder delivery devices are also known and can be used. The amount of cationic polymers as active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

In one specific embodiment, the pharmaceutical composition of the invention comprises said cationic polymers in an amount therapeutically effective to reduce at least one of the following: (i) the visco-elasticity of thick, mucoid or mucopurulent sputum in the airways of a patient, (ii) pathogens infectivity, (iii) inflammation and (iv) protease activity and/or to enhance anti-protease activity in cystic fibrosis sputa in vitro.

The cationic polymers for use as a medicament

The cationic polymers according to the invention are predicted to be useful in (i) fluidizing lung secretions, (ii) protecting from infections and (iii) inflammation and (iv) stimulating activity of protease inhibitors, in patients suffering from accumulation of thick, mucoid or mucopurulent sputum in the airways.

Therefore, the cationic polymers according to the invention may be used as a medicament, in particular for the treatment of inflammatory lung disorders characterized by the accumulation of thick, mucoid or mucopurulent sputum in the airways. Disorders characterized by accumulation of thick, mucoid or mucopurulent sputum in the airways includes, without limitation, cystic fibrosis, chronic bronchitis, bronchiectasis, infectious pneumonia, chronic obstructive lung/pulmonary disease (COLD/COPD), asthma, tuberculosis, fungal infections, airways manifestations of mucopolysaccharidoses I, II, IMA, NIB, NIC, VI and VII and sinusitis. "Treatment" is herein defined as the application or administration of cationic polymers according to the invention, or a pharmaceutical composition comprising said cationic polymers, preferably as an aerosol, into the airways, to a subject, where the subject has a disorder or a symptom associated with accumulation of thick, mucoid or mucopurulent sputum in the airways, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve said inflammatory lung disorder, or any symptom associated to said disorder, in particular mucociliary clearance.

By "positive therapeutic response" with respect to said inflammatory lung disorders, such as cystic fibrosis, is intended an improvement in the disease in association with the mucus fluidizing activity of these molecules or compositions according to the invention, and/or their stimulatory effect on protease inhibitors, and/or an improvement in any of the symptoms associated with the disease.

The effect of mucus fluidizing activity can be measured by monitoring visco-elasticity of a sputum sample from the patient prior to and after treatment with the compositions according to the invention, using in vitro assays (viscosity measurement) as described in the Examples.

By "therapeutically effective dose or amount" or "effective amount" is intended to be an amount of cationic polymers of the invention, or a pharmaceutical composition according to the invention, that, when administered brings about a positive therapeutic response with respect to treatment of a subject with a lung disorder characterized by the recruitment of blood neutrophils in the airways favouring formation of a thick, mucoid or mucopurulent sputum (e.g. cystic fibrosis).

Since the cationic polymers of the invention have been shown to stimulate the in vivo inhibition of protease inhibitors in lung secretions of patients, in some embodiments of the invention, the cationic polymers of the present invention may be advantageously administered in combination with at least one protease inhibitor, wherein the cationic polymers and the other therapeutic agent(s) may be administered sequentially, in either order, or simultaneously (i.e., concurrently or within the same time frame). Examples of suitable protease inhibitors that can be administered in combination with the cationic polymers of the invention, include but are not limited to serpins (a1 -antitrypsin, antichymotrypin, serpin B1 ....), non covalent canonical inhibitors (for example elafin,

SLPI ecotin, eglin C ), synthetic covalent inhibitors (including acyl-enzyme inhibitors, transition state inhibitors, mechanism based inhibitors), and synthetic non covalent inhibitors (including substrate-like inhibitors, cyclic peptidyl inhibitors, heterocyclic inhibitors). Such non natural serine protease inhibitors are for example described in Groutas et al Expert Opin Ther Pat. 201 1 , 21 (3):339-54 and Epinette C et al Biochem Pharmacol 2012 788-796. Recombinant, and chemical inhibitors that inhibit one, two or the three neutrophil serine proteases, can be used alone or in combination for in vivo administration. Other a1 -antitrypsin includes plasma-derived, glycosylated, human a1 - antitrypsin, such as PROLASTIN®, ARALAST™, or ZAMAIRA™ Alternatively, compositions comprising recombinant a1 -antitrypsin or any functional derivatives may be used. In another specific embodiment, the cationic polymers are administered in combination with one or more additional active ingredients, especially, conventional active ingredients for the treatment of pulmonary disorders.

In specific embodiments, said additional active ingredients are selected from the group consisting of Dornase, antibiotics, N-acetylcysteine, trypsin, chymotrypsin, glucocorticosteroids, amiloride triphosphate, uridine triphosphate, hypertonic saline, secretory leukoprotease inhibitor, bronchodilators, anti-inflammatory agents, mucolyitics and a1 -antitrypsin.

The invention also relates to a method for reducing, in patients in need thereof, the visco- elasticity of thick, mucoid or mucopurulent sputum present in the airways of said patients, the method comprising the step of administering to the patient, at least the cationic polymers of the invention, or the pharmaceutical compositions, as defined above, in an amount therapeutically effective to reduce the visco-elasticity of said thick, mucoid or mucopurulent sputum.

The following examples are offered by way of illustration and not by way of limitation. LEGENDS OF THE FIGURES

Figure 1 : (A) variations of the apparent cell count in sputum homogenate after Dornase and pLK treatment (results are expressed as a percentage of control; median ± interquartiles). (B) Quantification of extracellular DNA in CF sputum effect after treatment with Dornase and pLK (results are expressed as a percentage of control; median ± interquartiles). pLK is pLK, HBr of 40,000 - 50,000 Da molecular weight.

Figure 2: (A) Quantification of proteases activities in whole CF sputa before and after treatment by Dornase, pLK or Dornase + pLK showing the increase of elastase activity but not that of the other two proteases (median ± interquartiles). (B) Inhibition of proteases activities by a1 -Pi (HNE and Pr3) and ACT (CG) before and after treatment by Dornase, pLK or Dornase + pLK. pLK is pLK, HBr of 40,000 - 50,000 Da molecular weight. Figure 3: (A) Extracellular DNA quantification in untreated or pLK- or PEG-g-pLK- treated sputum homogenates. (B) Quantification of proteases activities in whole CF sputa before and after treatment by pLK or by PEG-g-pLK) (median ± interquartiles). (C) Inhibition of proteases activities by a1 -Pi (HNE and Pr3) and ACT (CG) before and after treatment by pLK or PEG-g-pLK. pLK is pLK, HBr of 40,000 - 50,000 Da molecular weight. PEG-g-pLK is pLK, HBr of 40,000 - 50,000 Da molecular weight grafted with one mPEG molecule of 5,000 Da.

Figure 4: (A) Influence of Dornase and pLK on bacterial proliferation and wall permeabilization as assessed by DNA staining with a non-cell permeant fluorophore. (B) Scanning electron microscopy of bacteria after Dornase and pLK treatment showing the spiculated surface of pLK-treated P aeruginosa indicating cell pemeabilization as described by (24). pLK is pLK, HBr of 40,000 - 50,000 Da molecular weight.

Figure 5: Cytotoxic effect of pLK=36 (pLK=36 is pLK, HBr of 5,000 - 10,000 Da molecular weight) on transformed human bronchial epithelial cells (BEAS-2B). Figure 6: H1 -NMR spectrum of pLK=36-His19 in D₂0. This shows the NMR spectrum at 300 MHz in D₂0 of polylysine derivatized by histidyl residues:

(a) 1.28 to 1.88 ppm: 6 protons of carbons 3, 4 and 5 of substituted or unsubstituted lysines.

(b) 2.39 ppm: protons of the CH3 group of p-toluenesulphonate

(c) 2.75 ppm: DMSO plot

(d) 2.99 ppm: 2 protons of carbon 6 of an unsubstituted lysyl residue

(e) 3.15 ppm: 2 protons of carbon 6 of a substituted lysyl residue

(f) 3.35 ppm: 2 protons of carbon 9 of a histidyl residue

(g) 4.36 ppm: 2 protons of carbons 2 and 8

(h) 4.78 ppm: peak of water

(i) 7.36 ppm: 2 protons (doublet, ortho-coupling constant=7.97 Hz) of the protons of carbons 2 and 6 of the aromatic ring of p-toluenesulphonate

(j) 7.42 ppm: 1 proton of carbon 11 of a histidyl residue

(k)7.71 ppm: 2 protons (doublet, ortho-coupling constant=8.01 Hz) of the protons of carbons 3 and 5 of the aromatic ring of p-toluenesulphonate

(I) 8.7 ppm: 1 proton of carbon 12 of a histidyl residue

Figure 7: H¹-NMR spectrum of pLK=36-His8 in D₂Q Figure 8: H¹-NMR spectrum of pLK=72-His17 in D₂0

Figure 9: H¹-NMR spectrum of pLK=72-His31 in D₂0

Figure 10: H¹-NMR spectrum of pLK=36-GlcA16 in D₂0.

(a) 1.28 to 1.88 ppm: 6H of carbons 3, 4 and 4 of lysine

(b) 2.41 ppm: CH.sub.3 group of p-toluene sulfonate

(c) 2.75 ppm: trace of DMSO

d) 2.97 ppm: 2H of carbon 6 of non-gluconoylated lysine residue

(e) 3.26 ppm: 2H of carbon 6 of a gluconoylated lysine residue

(f) 3.68 to 3.88 ppm: 4H of a gluconoyle residue (see spectrum number 1,856B in: The Aldrich Library of .sup.13 C an .sup.1 H FTNMR Spectra, Ed I, C. J. Pouchert and J.

Behnke, Vol. 1)

(g) 4.3 ppm: 1 H of carbon 8 of a gluconoyle residue and 1H of carbon 2 of a lysine residue

(h) 4.78 ppm: peak of water

(i) 7.36 ppm: 2 protons (doublet, ortho-coupling constant=7.97 Hz) of the protons of carbons 2 and 6 of the aromatic ring of p-toluenesulphonate

(j)7.71 ppm: 2 protons (doublet, ortho-coupling constant=8.01 Hz) of the protons of carbons 3 and 5 of the aromatic ring of p-toluenesulphonate

Figure 11 : H¹-NMR spectrum of pLK=72-GlcA31 in D₂0 Figure 12: Free DNA after DNA condensation by pLK=36 (pLK=36 is pLK, HBr of 5,000 - 10,000 Da molecular weight), pLK=72 (pLK=72 is pLK, HBr 10,000 - 20,000 Da molecular weight) or their derivatives

Figure 13: Sputum HNE activity after treatment by pLK=36 and pLK=72 derivatives

Figure 14: Sputum CG activity after treatment by pLK=36 and pLK=72 derivatives Figure 15: Sputum Pr3 activity after treatment by pLK=36 and pLK=72 derivatives

Figure 16: Inhibition of neutrophil serine protease activities before (control) or after treatment by pLK=36 or pLK=36-His19.

Figure 17: Effect of pLK=36 and pLK=72 derivatives against Pseudomonas aeruginosa. pLK=36 and pLK=72 derivatives were tested at 1 or 10 μΜ against PA01 strain (A) and at 140 μΜ against a Strain isolated from CF sputum (B). Figure 18: Effect of pLK=36 and pLK=72 derivatives against Staphylococcus aureus. pLK=36 and pLK=72 derivatives were tested at 10 μΜ against laboratory strain (A) and at 140 μΜ against a strain isolated from CF sputum (B).

Figure 19: Cytotoxic effect of pLK=36 and pLK=72 derivatives on transformed human bronchial epithelial cells (BEAS-2B).

Figure 20: H¹-NMR spectrum of pLK30-His4.5 in D₂0

Figure 21 : H¹-NMR spectrum of pLK30-His8 in D₂0

Figure 22: Free DNA after DNA condensation by pLK30, compared to pLK=36 and pLK=36-His19. Figure 23: Effect of pLK30 against Pseudomonas aeruginosa, compared to pLK=36 and pLK=36-His19. Molecules were tested at 1 μΜ against laboratory strain (PA01 ).

Figure 24: Free DNA after DNA condensation by pLK30 and pLK30 derivatives

Figure 25: Sputum HNE activity after treatment by pLK30 and pLK30 derivatives

Figure 26: Sputum CG activity after treatment by pLK30 and pLK30 derivatives Figure 27: Sputum Pr3 activity after treatment by pLK30 and pLK30 derivatives

Figure 28: Inhibition of proteases activities by a1 -Pi (HNE and Pr3) and ACT (CG) before (control) and after treatment by pLK30 or pLK30 derivatives.

Figure 29: Effect of pLK30 and pLK30 derivatives against Pseudomonas aeruginosa. pLK30 and pLK30 derivatives were tested at 1 or 10 μΜ against PA01 strain (A) and at 140 μΜ against strains isolated from CF sputum (B).

Figure 30: Effect of pLK30 and pLK30 derivatives against Staphylococcus aureus. pLK30 and pLK30 derivatives were tested at 1 or 10 μΜ against laboratory strain (A) and at 140 μΜ against strains isolated from CF sputum (B).

Figure 31 : Cytotoxic effect of pLK30 and pLK30 derivatives on transformed human bronchial epithelial cells (BEAS-2B). Figure 32: Effect of pLK30-His8 on P. aeo/g/^'nosa-i nfected mice. 2 groups of 10 infected mice (PAK-Lux Apscf) were intranasally administered 50 μΙ_ of physiological serum (vehicle) or pLK30-His8 (10 mg/kg) 90 min post-infection, then killed 6 hours later. Bacterial load in mouse BALF was evaluated in Unit Forming Colony (UFC) per 50 μΙ_ of BALF.

EXAMPLES

EXAMPLE 1 : Polylysine polymers pLK Materials and methods

All reagents were purchased from Sigma (St. Quentin Fallavier, France) unless otherwise stated.

Patients: CF patients of the "Centre de Ressources et de Competences de la Mucoviscidose" (CRCM) of Tours were included in the study and gave written informed consent. The inclusion criteria were a stable pulmonary disease, as defined by the clinical profile, and no hospitalization or change in their antibiotic and anti-inflammatory regimen during the month prior to inclusion. The research was carried out in accordance with the Helsinki Declaration (2000) of the World Medical Association and was approved by the local Ethical Committee (# 2007-R17).

Sputum processing- CF sputum was collected into 50 ml Falcon^® tubes by physiotherapy and processed immediately. Sputum was diluted with 3 volumes of PBS per gram and homogenized to obtain a crude homogenate that was kept on ice. An aliquot of each homogenate was incubated for 2 h in low-binding microtubes with 400 μg ml Dornase or with 1 .5 mg/ml of poly-L-Lysine (pLK) or PEGylated pLK (PEG-g-pLK) under gentle stirring at room temperature, then layered on a glass slide for cell counting by trypan blue exclusion and visual aspect under the optical microscope. Different sizes of pLK were tested (pLK, HBr of either 500-2,000 Da, 1000-5,000 Da, 4,000-15,000 Da and 40,000- 50,000 Da molecular weight). PEG-g-pLK was pLK of 40,000 - 50,000 Da molecular weight grafted with one mPEG molecule of 5,000 Da.

Viscosity measurements

The viscoelastic properties of the mucus were analyzed by measuring the ciliary beat frequency of human epithelia covered either by untreated or pLK-treated, or Dornase- treated CF sputum homogenates. Epithelia were reconstituted from endobronchial biopsies of healthy subjects and beat frequency was recorded by videomicroscopy (Gras D, et al . J Allergy Clin Immunol 2012;129:1259-1266).

Quantification of extracellular DNA

Extracellular DNA is quantified with the non-cell-permeant fluorochrome EvaGreen™ dsDNA reagent (λ_βχ = 488 nm and _em = 520 nm).

Measurement of peptidase activities

Proteases activities were measured as described in (26) with the specific FRET (fluorescence resonance energy transfer) substrates of HNE, Pr3 and CG: Abz- APEEIMRRQ-YN02, Abz-VADnVADQ-EDDnp and Abz-TPFSGQ-YN02 respectively (λ_βχ = 320 nm and _em = 420 nm). The concentrations of active proteases in biological samples were determined by comparing the rates of hydrolysis of their specific substrates with those of commercial titrated proteases under the same experimental conditions. HNE and Pr3 were titrated as described in (27) and CG was titrated with HNE-titrated recombinant human secretory leukocyte protease inhibitor. Sputum proteases inhibition by exogenous inhibitors

Aliquots of sputum homogenates containing peptidases in the 10 nanomolar range, were incubated for 30 min at room temperature with increasing amounts of inhibitors up to a (l)/(E) molar ratio of about 40. The low Mr recombinant inhibitor EPI-hNE4 or a1 - proteinase inhibitor (a1 -Pi) were used to inhibit HNE, while a1 -antichymotrypsin (ACT) was used to inhibit CG; Pr3 was inhibited with a1 -Pi after samples had been incubated with a 1000-fold molar excess of EPI-hNE4 to avoid the a1 -Pi interacting with HNE (8).

Culture of bacteria

S. aureus (strain CIP 10381 1 ) and P. aeruginosa (strain PA01 ) were grown to exponential phase in brain heart infusion medium with aeration, collected by centrifugation at 10,000 x g for 10 min at 20°C, washed and suspended in PBS. The bacterial count was determined by the measure of OD₆oonm-

Antimicrobial tests

6 x 10⁶ bacteria were incubated in low-binding 96-wells microplates 3h in 150 μΙ PBS containing 1 10 pg of Dornase or 2 μg of pLK (MW 40,000-50,000 Da). Aliquots of the media were then collected and serial dilutions in PBS were laid on agar plates. CFUs were numbered after incubation at 37°C. Extracellular DNA in the remaining media was quantified as previously described as a marker of bacterial wall permeability.

Confocal microscopy

Aliquots of Dornase-treated, pLK-rhodamine-treated (pLK of 40,000-50,000 Da) or untreated sputum homogenate were seeded onto Superfrost slides (CML, Nemours, France). Dry samples were washed in PBS to remove the excess of pLK-rhodamine and were then fixed by incubation in 4% (v/v) formaldehyde in PBS for 30 min at room temperature. After washing, dsDNA was detected by incubating the samples with 10nM of the cell-permeant fluorochrome DRAQ5™ in PBS for 30 min. Finally, samples were washed, mounted with Fluoromount and examined under an Olympus FV 500 confocal microscope.

Scanning electron microscopy

6 x 10⁶ bacteria were settled on polylysine-coated glass slides and treated (or not) with Dornase or pLK (pLK of 40,000-50,000 Da). They were then fixed with 1 % (v/v) glutaraldehyde and 4% (v/v) paraformaldehyde in 0.1 M PBS, pH 7.4, post-fixed in 2 % (v/v) osmium tetroxide, dehydrated in a graded acetone series, dried to the critical point using carbon dioxide, and sputter coated with platinum. Fixed cells were examined with a Zeiss Gemini 982 scanning electron microscope.

Results pLK fluidizes CF sputum:

The increased viscosity of mucus in the CF lung leads to lung obstruction and subsequent decrease in lung function. Because DNA is a major component of CF lung secretions that contributes to their viscosity, rhDornase has been used for long by CF patients as a liquefying expectorant (28). A possible other mean to fluidize lung secretions could be through the condensation of extracellular DNA by positively charged polymers. We checked this hypothesis using poly-L-lysine (pLK) and comparing the rheological properties of CF sputa before and after treatment by pLK. Then we compared the results with those obtained using Dornase in the same conditions. We observed no morphological change of the sputum after Dornase treatment whereas pLK induced the fractionation of the sputum in dense aggregates and a very fluid phase (data not shown). Most of sputum neutrophils were found in these aggregates that explain an apparent decrease in cell counting (Fig. 1A). We observed an important decrease in the amount of extracellular DNA after Dornase treatment and also after incubation with pLK (Fig. 1 B). But DNA was actually not degraded in pLK-treated sputum, since its extracellular content was restored after addition of dextran sulfate (Data not shown). It is well known that DNA condensation into dense aggregates impairs dye intercalation as ethydium bromide, propidium bromide including DRAQ5™ and visualization by confocal microscopy (Data not shown). This is supported by the observation of a more intense DNA labeling at the periphery of aggregates where DNA condensation is lower.

The formation of dense aggregates results in the formation of a liquid and almost transparent phase. Cilia beating of bronchial epithelium, reconstituted from biopsies of healthy individuals were measured. CF sputum homogenates with or without Dornase or pLK were layered on the epithelium and ciliary beating was recorded by videomicroscopy.

As expected, in presence of untreated sputum, cilia beat frequency significantly decreased (data not shown). Cilia beat frequency returned to normal when CF sputum homogenates after pLK treatement, but not after Dornase one, suggesting that sputum liquefaction favors mucociliary clearance and that DNA aggregates would not be deleterious. pLK helps improving neutrophil serine proteases inhibition in sputum:

CF-associated chronic lung inflammation depends in part on a proteases/antiproteases imbalance resulting from the recruitment of blood neutrophils in the airways. Thus, a protease inhibitor-based therapeutic treatment could potentially help combating protease- dependant inflammation. But we previously showed that proteases resisted inhibition in CF sputum due to their binding to DNA and other negatively charged macromolecular components (8). Dornase treatment of whole sputum induces a dramatic increase in elastase activity but this can be completely and stoichiometrically inhibited by elastase inhibitors (Fig. 2). We obtained the same result after compaction of DNA by pLK: elastase activity was stoichiometrically inhibited by a1 -Pi after pLK treatment in spite of a dramatic increase in its activity in whole sputum (Fig. 2). Neither pLK nor Dornase treatments significantly affected the activity and inhibition of PR3 by a1 -Pi but pLK, unlike Dornase, significantly improved CG inhibition by ACT (Fig. 2). This is due to the absence of DNA fragmentation after pLK treatment since the DNA fragments generated by Dornase counteract interaction of CG with its inhibitors (8, 29); Using a mixture of pLK and Dornase gave the same result as pLK alone which demonstrates that DNA compaction occurs more rapidly than DNA fragmentation and that compacted DNA resist degradation by Dornase. Thus the combined use of pLK and inhibitors of HNE and CG could help limiting uncontrolled proteolysis in the lung and the resulting inflammation.

Testing pLK of different sizes showed that pLK with a MW in the 4,000-15,000 Da range gave the same results as those with higher MW, whereas those with lower MW (500- 2,000 Da and 1 ,000-5,000 Da) were far less efficient. Experiments are currently done to determine the optimal size of pLK combining good DNA condensation and low toxicity.

Because of the possible difficulty to remove large aggregates by mucociliary clearance, macrophages uptake or expectoration we tested a PEGylated pLK that decreases DNA/polycations complexes aggregation, interaction with plasma proteins, and cytotoxicity (30). Preliminary results show that the aggregates generated by PEG-g-pLK are less dense and viscous, and they regulate proteases activities in the same way as crude pLK (Fig. 3).

Microbicidal properties ofpLK towards S. aureus and P. aeruginosa:

CF is characterized by persistent lung infections, especially by Staphylococcus aureus and Pseudomonas aeruginosa (1 ). Resistant strains of these bacteria that colonize the thick CF mucus impair antibiotics access and thus compromise their elimination from the lungs of contaminated patients. Because natural antimicrobial peptides and proteins act through their cationic charge (31 ), we looked at whether pLK possessed antimicrobial properties against bacterial cultures of S. aureus and P. aeruginosa. Indeed pLK displayed a significant bactericidal effect toward the two pathogens (Fig. 4A). This associates a significant modification of the morphology of P. aeruginosa as visualized by a spiculated cell surface (Fig. 4) and a disruption of the bacterial wall as quantified by the measure of extracellular DNA with a non-cell permeant fluorophore (Fig. 4A). Morphological changes were less marked and no wall permeabilization was observed with S. aureus (Fig. 4B). Unlike pLK, Dornase showed no antimicrobial properties nor it affected the morphology of the bacteria (Fig. 4). We conclude that in addition to its fluidizing properties pLK may control bacterial colonization of CF lungs by gram negative and gram-positive bacteria.

Toxicological studies The objective of the study was to identify the nature and the dose of polycations to be aerosolized safely in control mice. In vivo assessment of polycations toxicity has been done by cytologic analysis of bronchoalveolar lavage fluids (BALFs), quantification of proinflammatory cytokines (IL6 and KC) and lung anatomopathology.

Aerosol administration of different doses of pLK was done using a MicroSprayer® Aerosolizer - Model IA-1 C - connected to a FMJ-250 High Pressure Syringe (Penn Century, Philadelphia, PA). Mice were killed at day 2 and day 5 and bronchoalveolar lavage fluids (BALF) were obtained with instillation of 5 x 0.5 mL of sterile PBS into the lungs. Lungs were perfused with 4% formaldehyde for histological studies. Cells were collected after centrifugation of BALFs, suspended in PBS, and analyzed by flow cytometry. The supernatant was used for cytokine quantification using commercially available ELISA kits.

Remarkably, we observed no significant differences in BALF cellularity, and cytokines quantification between control and treated mice (Mann-Whitney test) in response to aerosol administration of 2.4 mg/kg pLK (4-15 kDa) or pLK(4-15kDa)-PEG (66 pLK in 50 L corresponding to a concentration of about 130 μΜ) for a mouse of 27 - 30 g. The histological study confirmed these observations by showing a minimal broncho-interstitial inflammation at day 2 both in treated and in control groups, but no lung lesion was observed at day 2 nor at day 5.

However, a cellular toxicity of pLK(4-15 kDa) or pLK(4-15kDa)-PEG can be observed using in vitro cultures of Beas-2B cells. Interestingly, the in vitro toxicity threshold was lowered by a factor 20 (5 μΜ versus 100 μΜ) using pLK=36-His19 (His 53%) whereas the antibacterial properties of pLK=36-His19 against P. aeruginosa remained unchanged.

Example 2: Polylysine derivatives (pLK-His)

1/ Material and methods

1.1 Synthesis of polylysine derivatives Material

All reagents were purchased from Sigma (St. Quentin Fallavier, France) or from Bachem Feinchemikalien, (Bubendorf, Switzerland) unless otherwise stated.

Methods Polylysine p -toluene sulfonate salt: pLK, pLK=36 and pLK=72.

100 mg pLK (Poly- L -lysine, HBr 4,000 - 15, 000 from Sigma) (average molecular weight of 9500 Da ; average degree of polymerization = 45; Sigma), pLK=36 (Poly- L - lysine, HBr 5,000 - 10,000 from Bachem Feinchemikalien, Bubendorf, Switzerland) (average molecular weight of 7500 Da; average degree of polymerization = 36) or pLK=72 (Poly- L -lysine, HBr 10,000 - 20,000 from Bachem Feinchemikalien, Bubendorf, Switzerland) (average molecular weight of 15,000; average degree of polymerization = 72) is dissolved in 50 mL water and the solution is passed through an anion-exchange column (Dowex 2 x 8, -OH form, 20-50 mesh). The eluate is neutralized with a 10% p - toluenesulfonic acid solution water and freeze-dried.

Histidinylated polylysines: pLK=36-His8 & pLK=36-His19.

pLK36 (100 mg; 0.013 mmol) is dissolved in 5 mL DMSO in a 50 mL pyrex balloon with ground-glass joint and N,N -diisopropylethylamine (67 μί; 0.48 mmol) is added under agitation. Then 56 mg or 134 mg (0.12 or 0.28 mmol) of Boc-His(1 -Boc)-OSu dissolved in 3 mL DMSO is added. The reaction is kept for 24 h at room temperature under agitation. The N-protected groups are removed by acidic treatment by addition of 20 ml of a cold solution of H₂0/TFA (1 :1 ; v/v) overnight at room temperature under agitation. TFA and water are evaporated under reduced pressure. The polymer is precipitated into 150 ml of ethyl acetate. The precipitate is collected by centrifugation at 1 ,800 ^χ g for 15 min, solubilized in 20 mL distilled water and freeze-dry.

Histidinylated polylysines: pLK=72His17 & pLK=72-His31.

pLK72 (100 mg; 0.0067 mmol) is dissolved 5 mL DMSO in a 50 mL pyrex balloon with ground-glass joint and N,N -diisopropylethylamine (67μί; 0.48 mmol) is added under agitation. Then 62 mg (0.137 mmol) or 1 12 mg (0.250 mmol) of Boc-His(1 -Boc)-OSu dissolved in 3 mL DMSO is added. The reaction is kept for 24 h at room temperature under agitation. The N-protected groups are removed by acidic treatment by addition of 20 ml of a cold solution of H₂0/TFA (1 :1 ; v/v) overnight at room temperature under agitation. TFA and water are evaporated under reduced pressure. The polymer is precipitated into 150 ml of ethyl acetate. The precipitate is collected by centrifugation at 1 ,800 x g for 15 min, solubilized in 20 mL distilled water and freeze-dry.

Histidinylated polylysines: pl_K30Hisn

Several pLK30 derivatized with different numbers of histidinyl residues are prepared by reaction with Boc-His(1 -Boc)-OSu as followed: 250 mg (0.034 mmol) pLK30 (Poly- L - lysine, TFA; PLKF30; Alamanda Polymers, Huntsville, AL, USA) (molecular weight of 7300 Da; degree of polymerization = 30) are dissolved in 8 mL DMSO in a 50 mL pyrex balloon with ground-glass joint and N,N -diisopropylethylamine (500μΙ_; 3.58 mmol) is added under agitation. Then various quantities (150 mg; 0.33mmol to 300 mg; 0.66 mmol) of Boc-His(1 -Boc)-OSu (Bachem Feinchemikalien, Bubendorf, Switzerland) dissolved in 2 mL DMSO are added.

The reaction is kept for 24 h at room temperature under agitation. The N-protected groups are removed by acidic treatment by addition of 20 ml of a cold solution of H₂0/TFA (1 :1 ; v/v) overnight at room temperature under agitation. TFA and water are evaporated under reduced pressure. The polymers are precipitated into 150 ml of ethyl ether. The supernatants are discarded, the precipitates washed with ethyl ether, solubilized in distilled water and freeze-dry.

The number of His residues per polymer molecule is determined from the 1 H-NMR spectrum in D₂0 according to x = 6(h8.7 / hlys ).dp, where h8.7 is the value of the integration of the signal at 8.7 ppm corresponding to the proton of the imidazole ring (1 H C12), hlys is the range 1.3-1 .9 ppm corresponding to the 6 methylene protons of lysine residues (C3, C4 and C5), and dp is the degree of polylysine polymerization.

Gluconoylated polylysine: pLK=36GlcA16.

pLK36 (100 mg; 0.013 mmol) is dissolved 6 mL DMSO in a 50 mL pyrex balloon with ground-glass joint and N,N -diisopropylethylamine (196 μί; 1 .4 mmol) and 60 μί H₂0 (1 %) are added under agitation. Then 62 mg (0.346 mmol) of D-gluconic acid δ-lactone (δ-gluconolactone) dissolved in 3 mL DMSO is added. The reaction is stirred for 24 h at 60°C. The polymer is precipitated in 10 volumes isopropanol and spun down by centrifugation at 1 ,800 ^χ g for 15 min, solubilized in distilled water and freeze-dried.

Gluconoylated polylysine: pLK=72GlcA31.

pLK72 (100 mg; 0.0067 mmol) is dissolved 6 mL DMSO in a 50 mL pyrex balloon with ground-glass joint and N,N -diisopropylethylamine (196 μί; 1 .4 mmol) and 60 μί H20 (1 %) are added under agitation. Then 62 mg (0.346 mmol) of D-gluconic acid δ-lactone (δ-gluconolactone) dissolved in 3 mL DMSO is added. The reaction is stirred for 24 h at 60°C. The polymer is precipitated in 10 volumes isopropanol and spun down by centrifugation at 1 ,800 ^χ g for 15 min, solubilized in distilled water and freeze-dried.

The schematic structure of partially gluconoylated poly-L-lysine (pLKiGlcAx) is shown in formule (II) below:

wherein R is a gluconyl (GIcA) moiety or a NH₂ group; and i is the degree of polymerization of polylysine; X is the number of gluconyl residues.

The number of GIcA residues bound per polymer molecule (x) is determined from the 1 H-NMR spectrum in D20 according to x = 3/2. (hGIcA/ hlys) . dp, where hGIcA is the value of the integration of the signal in the range from 3.6 to 3.0 ppm corresponding to the 4 protons (1 H C10, 1 H C1 1 ,and 2H C12) of GIcA, hlys that in the range from 1 .3-1 .9 ppm corresponding to the 6 methylene protons (C3, C4 and C5) of lysine residues, and dp is the degree of polylysine polymerization.

1.2 Effects of pLK derivatives

Materials Patients

Sputum samples were collected from adult patients with CF of the Centre de Ressources et de Competences de la Mucoviscidose of Tours who gave written informed consent.

Sputum Processing

CF sputum was collected into 50-ml tubes after chest physiotherapy and immediately processed. Sputum was diluted with 2 ml of phosphatebuffered saline (PBS) per gram and homogenized to obtain a crude homogenate. An aliquot of each homogenate was incubated for 2 hours in low-binding microtubes with 140 μΜ of the different pLK or their derivatives under gentle stirring at room temperature. Bacteria and cells

S. aureus (strain CIP 10381 1 ) and P. aeruginosa (strain PA01 ) were a kind gift from the Laboratoire de Bacteriologie-virologie of the Bretonneau hospital of Tours, France. Bronchial immortalized epithelial cells BEAS-2B (CRL-9609) were from ATCC (ATCC, Rockville, MD, USA).

Reagents

Poly-L-Lysine was from Sigma-Aldrich (Saint-Quentin Fallavier, France), PBS was from Invitrogen (Cergy Pontoise, France), EvaGreen™ dsDNA reagent from Interchim (Montlugon, France). F12-K nutrient mixture, fetal calf serum, L-glutamine, Hepes, streptomycin and penicillin were from Gibco (Invitrogen, Cergy Pontoise, France) and Brain Heart Infusion medium, and tryptic soy, Cetrimide and Baird Parker agar plates were from Biomerieux (Craponne, France). Alpha 1 -proteinase inhibitor (Alpha 1 -Pi) and Alpha 1 -antichymotrypsin (ACT) were purchased at Biocentrum (Krakow, Poland) and EPI-hNE4 was a kind gift from Debiopharm (Lausanne, Switzerland). The DNA extraction kit came from Stratagene (Agilent, Les Ulis, France). Low-binding 96-wells plates were from Corning (Chorges, France) and MTS assay was from Promega (Lyon, France).

Methods

Quantification of extracellular DNA

Extracellular DNA was quantified using the non-cell-permeant fluorochrome EvaGreen™ dsDNA reagent (λ_βχ = 488 nm and X_em = 520 nm).

Measurement of peptidase activities

Peptidase activities were measured by spectrofluorometry using selective FRET substrates developed in our laboratory. Protease activity was measured in low-binding 96-wells plates with 13 μΜ (final concentration) of the specific FRET (fluorescence resonance energy transfer) substrates of human HNE, Pr3 and CG: Abz-APEEIMRRQ- EDDnp, Abz-VADnVADQ-EDDnp and Abz-TPFSGQ-YN0₂ respectively (λ_βχ = 320 nm and = 420 nm). The concentrations of active proteases in biological samples were determined by comparing the rates of hydrolysis of their specific substrates with those of commercial titrated proteases under the same experimental conditions. Proteases were titrated as described in (1 ). Sputum proteases inhibition by exogenous inhibitors

Aliquots of sputum homogenates were incubated with increasing amounts of inhibitors. Briefly, aliquots of sputum homogenates containing proteases in the 10 nM range, were incubated for 30 min at room temperature with increasing amounts of inhibitors up to a [lnhibitor]/[Enzyme] molar ratio of about 40. The inhibitor a1 -proteinase inhibitor (a1 -Pi) was used to inhibit HNE, while a1 -antichymotrypsin (ACT) was used to inhibit CG; Pr3 was inhibited with a1 -Pi after samples had been incubated with a 1000-fold molar excess of the HNE inhibitor EPI-hNE4 (3) to avoid the a1 -Pi interacting with HNE.

Culture of bacteria S. aureus (strain CIP 10381 1 ) and P. aeruginosa (strain PA01 and PAK-Lux Apscf) were grown to exponential phase in brain heart infusion medium in aerobic conditions, collected by centrifugation at 10,000 x g for 10 min at 20°C, washed and suspended in PBS. The bacterial suspension was adjusted by OD₆oo measurements to give the desired concentration. Antimicrobial tests

Bacteria were incubated with increasing doses of the different pLK or their derivatives. Briefly, 6 x 10⁶ bacteria were incubated in low-binding 96-wells microplates for 3h in 150 μΙ_ PBS containing the different pLK or their derivatives at 0.1 , 1 or 10 μΜ. Aliquots were then collected and serial dilutions in PBS were plated on agar plates. CFUs were counted after incubation at 37°C. Extracellular DNA in the remaining medium was quantified as described above as a marker of bacterial wall permeability. For the ex vivo experiments, CF sputum was processed and treated with the different pLK or their derivatives as described above. An aliquot of sputum was collected after 5 hours incubation time, diluted and plated on Cetrimide or Baird Parker agar plates that are selective for Pseudomonas aeruginosa and Staphylococcus aureus respectively. Tryptic soy (TS) agar plates were also used for the patients colonized by only one of the two pathogens. Agar plates were then incubated at 37°C for 24 hours and 48 hours for the Baird Parker and the Cetrimide plates respectively. CFUs were counted and the plates were scanned.

Cell Culture BEAS-2B, transformed human bronchial epithelial cells (CRL-9609) were maintained in F12-K nutrient mixture, supplemented with 10 % heat-inactivated foetal calf serum, L- glutamine 1 %, Hepes 10 mM, 0.1 mg/mL streptomycin, and 100 units/mL penicillin, at 37°C in a humidified atmosphere containing 5 % C02. For the experiments, cells were seeded in 96 well plates (10,000 cells/well). After 48 hours of culture, medium was removed and cells were incubated with different concentrations of the different pLK or their derivatives (from 0 to 100 μΜ), during 48 hours.

MTS Cell Viability Assay

Cellular viability was determined by MTS assay (Promega, Lyon, France), which looks at the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS) to formazan in viable cells. After 1 hour, formazan absorbance was measured at 490 nm, according manufacturer's instructions. The amount of colored product formed was proportional to the number of live cells in culture. Mean absorbance of cells incubated without pLK derivatives, served as the reference value for calculating 100% cellular viability.

Tolerance in Mice

Balb/c mice were housed and handled according to the guidelines from the European Animal Care and Use Committee (agreement 2012-12-7). Mice were anesthetized and received 50 μΙ_ of physiological serum (vehicle), or pLK30His4.5 (10 mg/kg) or pLK30His8 (10 mg/kg) via an Aerosolizer MicroSprayer® Model IA-1 C (Penn Century) (6 mice per group). Mice were killed at Day 5 and bronchoalveolar lavage fluids (BALFs) were collected following instillation of 4 x 0.5 mL of sterile PBS into the lungs. BALFs were analyzed for their cell content by flow cytometry, and for their content in inflammatory cytokines (mouse IL-6 and KC ) by ELISA (R&D). Lungs were perfused with 4% formaldehyde for histologic studies (LeNet Pathology).

In Vivo Antimicrobial Activity

Balb/c mice were housed and handled according to the guidelines from the European Animal Care and Use Committee (agreement 02144.01 ). The mice were infected with a freshly prepared inoculum of Pseudomonas aeruginosa (strain PAK-Lux Apscf). Mice were anaesthetized (Isoflurane) and were administered 40 μί of the bacterial solution (1 .10⁷ bacteria) intranasally using an ultrafine pipette tip. They were reanesthetized 90 minutes post-infection and were intranasally administered 50 μΙ_ of either physiological serum (vehicle) or pLK30His8 (10 mg/kg) (10 mice per group). Mice were killed 6 hours post infection. BALFs were collected following instillation of 4 x 0.5 mL sterile PBS into the lungs and counted for their bacterial content (serial dilutions in PBS were plated on agar plates and CFUs were counted after a 24 hours incubation at 37°C). Immune cells were counted by flow cytometry, and the inflammatory cytokines IL-6 and KC quantified by ELISA (R&D). Lungs were perfused with 4% formaldehyde for histologic studies (LeNet Pathology).

Histological analyses were performed by Le Net Pathology Consulting (Amboise, France). Histological sections of approximately 4 μηη were stained with hematoxylin and eosin and mounted on glass slides, then examined by light microscopy on a Leica Diaplan microscope, with full knowledge of the dosage group to which individuals had been assigned. All histopathological findings were graded in severity using a five point system of minimal, slight, moderate, marked or severe. 21 Results

2.1 ρΙ_Κ¾36, pLK¾72 and their derivatives pLK~36 cellular cytotoxicity Poly-L-lysine, pLK=36 allows CF sputum DNA compaction, the control of neutrophil elastase and cathepsin G by their natural inhibitors and exhibits antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at 10 μΜ. However, pLK=36 was tested for its cytotoxicity in transformed human bronchial epithelial cells (BEAS-2B) and results showed no effect on cell viability at 1 .56 μΜ, but a significant cytotoxicity from 6.25 μΜ (figure 5).

Thus different modifications of pLK=36 were done with different sizes of pLK (pLK=72 or pLK=36) and different degree of grafting like Histidine or Gluconoyle, in the aim to decrease the cytotoxic effect while preserving the others (DNA compaction, protease inhibition, antibacterial activities). Synthesis of LK~36 and pLK~72 derivatives

The different synthesis of pLK derivatives are summarized in the following table with for each pLK derivative, its NMR spectrum (Figures 6-1 1 ). Table 1 : Average molecular weight and percentage of lysyl derivatization of the various polylysine derivatives.

Effects of LK~36 and pLK~72 derivatives

DNA condensation

Most of pLK derivatives were able to condense DNA in CF sputum and resulted in the liquefaction of the sputum. pLK or pLK derivative treatment (140 μΜ) of CF sputum samples resulted in an 80-90% apparent, excepted for pLK=36-GlcA16 and pLK=72- GlcA31 (Figure 12). The apparent decrease in extracellular DNA obtained after treatment is caused by the compaction of DNA that reduces the volume of the pellet fraction obtained after sputum centrifugation by about 50%, and impairs intercalation of the dye. Condensation of DNA by pLK or pLK derivatives induced the formation of clearly visible dense aggregates floating in a liquid phase.

Protease activities

The activities of the main neutrophil serine proteases present in CF sputa, (Human Neutrophil Elastase (HNE), Cathepsin G (CG) and proteinase 3 (Pr3)) were measured before and after pLK treatment using specific FRET substrates (Dubois et al., 2013). HNE activity increased dramatically after pLK treatment. But almost no change was observed measuring Pr3 and CG activities. pLK derivatives also increased HNE activity but this increase was dependent on the nature and the degree of derivatization (figure 13). pLK derivatives behaved as pLK towards CG and PR3 (figures 14-15).

We previously observed that proteolytic activities in CF sputa strongly resist inhibition by protease inhibitors. We observed here that after pLK treatment, HNE and CG activities were totally inhibited by a small molar excess of a1 -proteinase inhibitor and antichymotrypsin respectively in spite of the recorded increase in total proteolytic activity. Pr3 however resisted inhibition after pLK treatment, suggesting it was more firmly bound to extracellular components than the other two neutrophil serine proteases. We obtained similar results using pl_K=36-His19 (figure 16).

Antibacterial effects ofpLK derivatives

pLK exhibits antimicrobial activities against laboratory strains of Staphylococcus aureus and Pseudomonas aeruginosa in laboratory media (PBS) at a concentration of 10 μΜ, and in CF sputum at a concentration of 140 μΜ. Antibacterial effects of pLK derivatives against the same strains depended on their degree of derivatization (figures 17-18). Except for pLK=36-His8 and pLK with GlcA substitutes, the other pLK derivatives exhibited also anti-P. aeruginosa (figure 17) and ant\-S.aureus activities (figure 18).

Cellular cytotoxicity

pLK and pLK derivatives were tested for their cytotoxicity in transformed human bronchial epithelial cells (BEAS-2B). Results showed no effect on cell viability at 1 μΜ; a significant cytotoxicity was observed using 10 μΜ pLK. All pLK and pLK derivatives when used at 100 μΜ, exhibited a cytotoxic effect with 0 % of viability, except for pLK=36-His19 and pLK=36-GlcA16 (figure 19).

Conclusion

pLK=36-His19 appears as the best candidate among all molecules tested, regarding its in vitro cytotoxicity (95 % of cellular viability at 10 μΜ), DNA compaction and antibacterial activities. Moreover, preliminary in vivo results showed that pLK=36-His19 was tolerated at 10 mg/kg in mice.

2.2. pLK30 and pLK30 derivatives Synthesis of pLK30 and pLK30 derivatives

pLK=36 is a mixture of polylysyl residues of different molar mass (between 5,000 and 10,000 g/mol). We synthesized a peptide containing exactly 30 lysine residues (pLK30) that served as template to synthesize pLK30-histidyl derivatives with 15 to 27 % derivatization (see below). We thus obtained His derivatives (Table 2, Figures 20-21 ). Table 2: Average molecular weight and percentage of lysyl derivatization of the various polylysine derivatives determined from 1 H-NMR spectra.

% of Lysyl

Polymers Average MW ( Da)

derivatization

pLK30 7.26 0

pLK30-His4.5 7.876 15 pLK30-His8 8.356 27

Characterization of LK30

Results showed that pLK30 possessed exactly the same effects that those observed with pl_K=36, e.g. DNA compaction, protease inhibition, and antibacterial activities, at the same concentration (figures 22 and 23).

Characterization of pLK30 derivatives

Characterization of pLK30 derivatives with different histidine grafting percentage is in progress, but some experiments have already been validated (see below).

DNA condensation

As pLK30, pLK30 derivatives treatment (140 μΜ) resulted in the liquefaction of the sputum, with DNA condensation and formation of clearly visible dense aggregates floating in a liquid phase (Figure 24).

Protease activities

The activities of HNE, CG and Pr3 were measured before and after pLK30 derivatives treatment using specific FRET substrates (Dubois et al., 2013). We observed an increase of HNE activity (figure 25). No changes were observed measuring Pr3 and CG activities (figures 26-27). Concerning inhibition of protease activity by inhibitors, results obtained with pLK30 or pLK30 derivatives were similar to those with pLK=36 or pLK=36 His19. Indeed, HNE and CG activities were totally inhibited by a small molar excess of inhibitors, excepted for pLK30-His8 for which we observed an inhibition around 85 % of CG activity, instead 100 % for the others molecules (figure 28).

Antibacterial effects ofpLK derivatives

pLK30 derivatives exhibit antimicrobial activities against strains of Pseudomonas aeruginosa (figure 29) and Staphylococcus aureus (figure 30), isolated from CF sputum in laboratory media (PBS), at a concentration of 140 μΜ. Experiments concerning antimicrobial activities against laboratory strains are in progress.

Cellular cytotoxicity

No effect on the viability of transformed human bronchial epithelial cells (BEAS-2B) was observed using pLK30 derivatives at 1 μΜ. A significant cytotoxicity was observed with pLK30 at 10 μΜ, but not with pLK30-His4.5 or pLK30-His8. For the two latter, cytotoxicity was observed at 100 μΜ (figure 31 ). pLK30-His4.5 and pLK30-His8 tolerance in Balb/c mice Aerosolized pLK30-His4.5 or pLK30-His8 at 10 mg/kg induced no respiratory distress or behavioral changes in Balb/c mice. Analysis of the BALFs inflammatory markers i.e. neutrophils percentage, IL-6, and keratinocyte chemoattractant (KC), showed no significant difference between treated and control mice. A low bronchointerstitial inflammation was observed both in treated and control groups most probably due to the Microsprayer® instillation but no lung lesions have been detected by histologic studies.

In vivo antibacterial properties of pLK30-His8 in P. aeruginosa-infected mice

Treatment of P. aeo/g/^'nosa-i nfected mice by pLK30-His8 6 hours post-infection significantly reduces the BALFs bacterial load (figure 32). At that time however, we observed no decrease in the inflammatory cytokines IL-6 and KC the concentration of which was strongly increased in response to infection. The histologic analysis confirmed that inflammation may continue or slightly increase over this 6-hours period. But one should remind that the mouse model is not perfectly representative of the infection/inflammation observed during CF, in particular because of the absence of free DNA, the target of pLK30-His8 and related molecules).

Conclusion

Because of their low cytotoxicity, their low pro-inflammatory properties and their resistance to an in vivo administration, the cationic polymers of the invention, such as pLK30-His4.5 and pLK30-His8, appear as promising candidates for reducing the viscoelasticity of CF sputum, improve the control of proteolytic enzymes in CF lung secretions and fight the bacteria that chronically infect the lung secretions of CF patients and resist antibiotics. References

1 . Hauser, A. R., Jain, M., Bar-Meir, M., and McColley, S. A. (201 1 ) Clinical significance of microbial infection and adaptation in cystic fibrosis. Clin Microbiol Rev 24, 29-70

2. Voynow, J. A., and Rubin, B. K. (2009) Mucins, mucus, and sputum. Chest 135, 505-512

3. Brinkmann, V., and Zychlinsky, A. (2007) Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 5, 577-582 4. Manzenreiter, R., Kienberger, F., Marcos, V., Schilcher, K., Krautgartner, W. D., Obermayer, A., Huml, M., Stoiber, W., Hector, A., Griese, M., Hannig, M., Studnicka, M., Vitkov, L, and Hartl, D. (201 1 ) Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy. J Cyst Fibros 5. Suri, R. (2005) The use of human deoxyribonuclease (rhDNase) in the management of cystic fibrosis. BioDrugs 19, 135-144

6. Donaldson, S. H., Bennett, W. D., Zeman, K. L, Knowles, M. R., Tarran, R., and Boucher, R. C. (2006) Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 354, 241 -250 7. Griese, M., Kappler, M., Gaggar, A., and Hartl, D. (2008) Inhibition of airway proteases in cystic fibrosis lung disease. Eur Respir J 32, 783-795

8. Dubois, A. V., Gauthier, A., Brea, D., Varaigne, F., Diot, P., Gauthier, F., and Attucci, S. (2012) Influence of DNA on the activities and inhibition of neutrophil serine proteases in cystic fibrosis sputum. Am J Respir Cell Mol Biol 9. Park, T. G., Jeong, J. H., and Kim, S. W. (2006) Current status of polymeric gene delivery systems. Adv Drug Deliv Rev 58, 467-486

10. Shima, S., Matsuoka, H., Iwamoto, T., and Sakai, H. (1984) Antimicrobial action of epsilon-poly-L-lysine. J Antibiot (Tokyo) 37, 1449-1455

1 1 . Scholz, C, and Wagner, E. (201 1 ) Therapeutic plasmid DNA versus siRNA delivery: Common and different tasks for synthetic carriers. J Control Release

12. Ogris, M., Brunner, S., Schuller, S., Kircheis, R., and Wagner, E. (1999) PEGylated DNA transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther 6, 595- 605 13. Toncheva, V., Wolfert, M. A., Dash, P. R., Oupicky, D., Ulbrich, K., Seymour, L. W., and Schacht, E. H. (1998) Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers. Biochim Biophys Acta. 1380, 14. Choi, Y. H., Liu, F., Choi, J. S., Kim, S. W., and Park, J. S. (1999) Characterization of a targeted gene carrier, lactose-polyethylene glycol-grafted poly-L- lysine and its complex with plasmid DNA. Hum Gene Ther. 10, 2657-2665

15. Dailey, L. A., Kleemann, E., Merdan, T., Petersen, H., Schmehl, T., Gessler, T., Hanze, J., Seeger, W., and Kissel, T. (2004) Modified polyethylenimines as non viral gene delivery systems for aerosol therapy: effects of nebulization on cellular uptake and transfection efficiency. J Control Release 100, 425-436

16. Mockey, M., Bourseau, E., Chandrashekhar, V., Chaudhuri, A., Lafosse, S., Le Cam, E., Quesniaux, V. F., Ryffel, B., Pichon, C, and Midoux, P. (2007) mRNA-based cancer vaccine: prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes. Cancer Gene Ther 14, 802-814

17. Ziady, A. G., Gedeon, C. R., Miller, T., Quan, W., Payne, J. M., Hyatt, S. L, Fink, T. L, Muhammad, O., Oette, S., Kowalczyk, T., Pasumarthy, M. K., Moen, R. C, Cooper, M. J., and Davis, P. B. (2003) Transfection of airway epithelium by stable PEGylated poly-L-lysine DNA nanoparticles in vivo. Mol Ther 8, 936-947

18. Itaka, K., Osada, K., Morii, K., Kim, P., Yun, S. H., and Kataoka, K. (2010) Polyplex nanomicelle promotes hydrodynamic gene introduction to skeletal muscle. J Control Release 143, 1 12-1 19

19. Erbacher, P., Roche, A. C, Monsigny, M., and Midoux, P. (1995) Glycosylated polylysine/DNA complexes: gene transfer efficiency in relation with the size and the sugar substitution level of glycosylated polylysines and with the plasmid size. Bioconjug Chem 6, 401 -410

20. Erbacher, P., Bousser, M. T., Raimond, J., Monsigny, M., Midoux, P., and Roche, A. C. (1996) Gene transfer by DNA/glycosylated polylysine complexes into human blood monocyte-derived macrophages. Hum Gene Ther 7, 721 -729

21 . Sagara, K., and Kim, S. W. (2002) A new synthesis of galactose-poly(ethylene glycol)-polyethylenimine for gene delivery to hepatocytes. J Control Release 79, 271 -281

22. Erbacher, P., Roche, A. C, Monsigny, M., and Midoux, P. (1997) The reduction of the positive charges of polylysine by partial gluconoylation increases the transfection efficiency of polylysine/DNA complexes. Biochim Biophys Acta 1324, 27-36 23. Berge, S. M., Bighley, L. D., and Monkhouse, D. C. (1977) Pharmaceutical salts. J Pharm Sci 66, 1 -19

24. Hilpert, K., Elliott, M., Jenssen, H., Kindrachuk, J., Fjell, C. D., Korner, J., Winkler, D. F., Weaver, L. L, Henklein, P., Ulrich, A. S., Chiang, S. H., Farmer, S. W., Pante, N., Volkmer, R., and Hancock, R. E. (2009) Screening and characterization of surface- tethered cationic peptides for antimicrobial activity. Chem Biol 16, 58-69

25. Zahm, J. M., Girod de Bentzmann, S., Deneuville, E., Perrot-Minnot, C, Dabadie, A., Pennaforte, F., Roussey, M., Shak, S., and Puchelle, E. (1995) Dose-dependent in vitro effect of recombinant human DNase on rheological and transport properties of cystic fibrosis respiratory mucus. Eur Respir J 8, 381 -386

26. Korkmaz, B., Attucci, S., Juliano, M. A., Kalupov, T., Jourdan, M. L, Juliano, L, and Gauthier, F. (2008) Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates. Nat Protoc 3, 991 -1000 27. Korkmaz, B., Poutrain, P., Hazouard, E., de Monte, M., Attucci, S., and Gauthier, F. L. (2005) Competition between elastase and related proteases from human neutrophil for binding to alphal -protease inhibitor. Am J Respir Cell Mol Biol 32, 553-559

28. Hodson, M. E., and Shah, P. L. (1995) DNase trials in cystic fibrosis. Eur Respir J 8, 1786-1791 29. Duranton, J., Boudier, C, Belorgey, D., Mellet, P., and Bieth, J. G. (2000) DNA strongly impairs the inhibition of cathepsin G by alpha(1 )-antichymotrypsin and alpha(1 )- proteinase inhibitor. J Biol Chem 275, 3787-3792

30. Lv, H., Zhang, S., Wang, B., Cui, S., and Yan, J. (2006) Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release 1 14, 100-109 31 . Hancock, R. E., and Diamond, G. (2000) The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol 8, 402-410

Claims

CLAIMS A cationic polymer of the following formula (I) or their pharmaceutically acceptable salts,

wherein

R is NH2 or NH linked to a histidine residue or other molecules including 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.

The cationic polymer of Claim 1 , wherein the percentage of lysyl derivatization by histidyl residue is at least 10%, 20%, 30%, 40%, preferably comprised between 10% and 60%, more preferably between 10% and 35%.

The cationic polymer, wherein i is 30 and the percentage of lysyl derivatization is either 15% or 27%.

A pharmaceutical composition comprising a cationic polymer of any one of Claims 1 -3, and at least one pharmaceutically acceptable carrier.

The pharmaceutical composition of Claim 4, in combination with at least a protease inhibitor.

The pharmaceutical composition of Claim 5, wherein said protease inhibitor is selected from the group consisting of: serpins, e.g., a1 -antitrypsin, antichymotrypin, or serpin B1 ,

non covalent canonical inhibitors, e.g. elafin, SLPI ecotin, or eglin C, synthetic covalent inhibitors including acyl-enzyme inhibitors, transition state inhibitors, mechanism based inhibitors, iv. and synthetic non covalent inhibitors including substrate-like inhibitors, cyclic peptidyl inhibitors, heterocyclic inhibitors.

7. The pharmaceutical composition of any one of Claims 4-6, wherein said cationic polymer is present in an effective amount for enhancing anti-protease activity of said protease inhibitor in cystic fibrosis sputa in vitro.

8. The pharmaceutical composition of any one of Claims 4-7, suitable for administration in the form of an aerosol.

9. The pharmaceutical composition according to any one of Claims 4-8, wherein said composition does not comprise any nucleic acid molecule.

10. The pharmaceutical composition according to any one of Claims 4-9, for use in treating inflammatory lung disorders characterized by the recruitment of blood neutrophils in the airways favoring the formation of a thick, mucoid or mucopurulent sputum, for example cystic fibrosis.

1 1 . The pharmaceutical composition for use according to Claim 10, wherein said inflammatory lung disorders are selected among the group consisting of: cystic fibrosis, chronic bronchitis, bronchiectasis, infectious pneumonia, chronic obstructive lung/pulmonary disease (COLD/COPD), asthma, tuberculosis, fungal infections, airways manifestations of mucopolysaccharidoses I, II, IIIA, NIB, NIC, VI and VII and sinusitis.

12. The cationic polymer of any one of Claims 1 -3, for use in treating inflammatory lung disorders characterized by the recruitment of blood neutrophils in the airways favoring the formation of a thick, mucoid or mucopurulent sputum.

13. The cationic polymer for use of Claim 12, wherein said inflammatory lung disorders are selected among the group consisting of: cystic fibrosis, chronic bronchitis, bronchiectasis, infectious pneumonia, chronic obstructive lung/pulmonary disease (COLD/COPD), asthma, tuberculosis, fungal infections, airways manifestations of mucopolysaccharidoses I, II, IIIA, NIB, NIC, VI and VII and sinusitis.