Osteocrin, Revestin, or ANP for destroying bacterial biofilms

Abstract

The present invention relates to a natriuretic peptide selected from the group consisting of (i) osteocrin, osteocrin propeptide, or osteocrin derivative, (ii) lebetin, lebetin fragment, or derivative of lebetin or lebetin fragment, and (iii) ANP peptide, ANP propeptide, or ANP peptide derivative, for use in a method for therapeutically treating a bacterial infection associated with a bacterial biofilm in a subject, wherein the natriuretic peptide disperses a bacterial biofilm. In certain embodiments, the natriuretic peptide is used in combination with an antibiotic.

Description

Technical Field

[0001] The present invention relates to the disruption of bacterial biofilms, particularly in the context of chronic bacterial infections.

Background Art

[0002] Bacterial resistance to antibiotic therapy largely results from the ability to switch from a planktonic life form to a communal life form called a biofilm in the tissues of an infected host. Biofilms enable bacteria to defend themselves from antimicrobial molecules and the host's immune defenses, thereby conferring resistance to various antibacterial molecules and allowing pathogens to survive in hostile environments. Furthermore, a small fraction of bacterial cells called "persister cells" are retained within biofilms exposed to antibiotics, temporarily becoming resistant and causing recurrent infections to start again.

[0003] The opportunistic pathogen Pseudomonas aeruginosa is a major cause of death in immunocompromised patients, particularly those suffering from cystic fibrosis.

[0004] The treatment of severe infections caused by P. aeruginosa involves several antibiotics, but due to the presence of nephrotoxicity and ototoxicity, the therapeutic indices of the most effective antibiotics (tobramycin, colistin, ciprofloxacin, gentamicin, netilmicin, and amikacin) are narrow. Furthermore, in the long term, these treatments cause the emergence of strains resistant to antibiotics.

[0005] This is why P. aeruginosa is classified as a member of the ESKAPE group of bacteria (Santajit and Indrawattana (2016) Biomed. Res. Int. 2016:2475067), and the ESKAPE group groups together the most important pathogens from the perspective of antibiotic resistance and requires the discovery of new molecules or treatment strategies. Very recently, the WHO classified P. aeruginosa among the top three bacteria with dangerous resistance to antibiotics (especially carbapenems).

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0007] The present invention describes meeting this need.

[0008] The present invention is the result of the unexpected discovery by the inventors that the hormone "atrial natriuretic peptide" (or ANP) can, at low doses (0.1 nM or more), very rapidly (within 30 minutes after administration), dose-dependently disrupt approximately 80% of a pre-established biofilm of P. aeruginosa. The inventors have also shown that other peptides belonging to the family of natriuretic peptides, more specifically osteocrin, leveptin 2α, and leveptin 1β, can similarly disrupt a pre-established biofilm of P. aeruginosa at low doses.

[0009] Natriuretic peptides form a family of hormones that were first identified for their osmoregulatory activity in the cardiovascular system in order to regulate blood pressure and renal sodium excretion (natriuresis, hence their name). The three main members of this family are ANP, "brain natriuretic peptide" or BNP, and "C-type natriuretic peptide" or CNP. The length of these peptides ranges from 22 to 38 amino acids, and the structure of most members is characterized by a disulfide bridge that forms a loop of 17 amino acids that gives this molecule its so-called "omega" shape.

[0010] ANP, BNP, and CNP have previously been shown to prevent the formation of biofilms of P. aeruginosa (Desriac et al., "The natriuretic peptide hormones prevent Pseudomonas aeruginosa biofilm formation through a specific bacterial target. 16th International conference on Pseudomonas", Sep 2017, Liverpool, United Kingdom; Rosay et al., (2015) MBio 6: 6; Desriac et al., (2018) Pathogens 7: 47).

[0011] However, the action of these peptides on established biofilms has not been demonstrated so far.

[0012] The inventors further show herein that the ability of ANP to disperse established biofilms is not related to or due to the ability of ANP to kill bacteria and does not affect the intrinsic toxicity of bacteria. This specific mechanism of action makes it possible to prevent the emergence of bacterial strains resistant to this molecule.

[0013] The inventors also showed that when low doses of ANP are used in combination with antibiotics such as tobramycin or ciprofloxacin, which are also used at low doses, more than 97% of established biofilms are disrupted.

[0014] Therefore, combining ANP and antibiotics has a synergistic effect on the disruption of bacterial biofilms.

[0015] Accordingly, the present invention relates to its use in a method for therapeutically treating bacterial infections associated with bacterial biofilms in a subject, particularly chronic bacterial infections, of (i) osteocrin, osteocrin propeptide, or an osteocrin derivative, (ii) lectin, a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) a natriuretic peptide selected from the group consisting of an ANP peptide, an ANP propeptide, or an ANP peptide derivative, wherein the natriuretic peptide, particularly the ANP peptide, the ANP propeptide, or the ANP peptide derivative, disperses the bacterial biofilm.

[0016] In one particular embodiment, the natriuretic peptide, particularly the ANP peptide, the ANP propeptide, or the ANP peptide derivative, is used in combination with an antibiotic.

[0017] A further object of the present invention relates to a pharmaceutical composition comprising (A) a natriuretic peptide selected from the group consisting of (i) osteocrin, osteocrin propeptide, or an osteocrin derivative, (ii) lectin, a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, and (B) an antibiotic.

[0018] A further object of the present invention relates to its use in the method of therapeutic treatment of bacterial infections associated with bacterial biofilms in a subject, particularly chronic bacterial infections, for (A) a natriuretic peptide selected from the group consisting of (i) osteocrin, osteocrin propeptide, or an osteocrin derivative, (ii) lectin, a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, and (B) a pharmaceutical composition comprising an antibiotic.

[0019] A further object of the present invention relates to a pharmaceutical antibacterial combination comprising (A) a natriuretic peptide selected from the group consisting of (i) osteocrin, osteocrin propeptide, or an osteocrin derivative, (ii) lectin, a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, and (B) an antibiotic for simultaneous, separate, or sequential use in the therapeutic treatment of bacterial infections associated with bacterial biofilms in a subject, particularly chronic bacterial infections.

[0020] The present invention also relates to the in vitro or ex vivo use of a natriuretic peptide selected from the group consisting of (A) (i) osteocrin, osteocrin propeptide, or an osteocrin derivative, (ii) lectin, a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative for dispersing bacterial biofilms.

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] Natriuretic peptide As used herein, the "natriuretic peptide" refers to a member of a family of eukaryotic and neurohormones composed of three major members: atrial natriuretic peptide (or ANP), brain natriuretic peptide (or BNP), and C-type natriuretic peptide (or CNP). These peptides are mainly synthesized and released by cardiomyocytes and endothelial cells. In addition to these three major members, the natriuretic peptide family also includes "natriuretic-like" peptides such as osteocrin and relaxin.

[0022] These various natriuretic peptides have some similarities in sequence. All except osteocrin and relaxin 1β have a 17-amino acid loop formed by disulfide bridges. Of the 17 amino acids forming this loop, 11 are common to ANP and relaxin 2α. Osteocrin also has a conserved characteristic sequence with 7 amino acids common to ANP in this region. This conserved region of the natriuretic peptide sequence enables this peptide to bind to human natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C). Furthermore, ANP, osteocrin, and relaxin 2α have been shown to bind with strong affinity to the AmiC sensor of P. aeruginosa (Rosay et al., (2015) mBio 25:e01033-15). On the other hand, many differences are observed in their C-terminal and N-terminal amino acid sequences, demonstrating that their physiological activities are different. It should be noted that in addition to the final active products listed below, the synthesis, maturation, and partial degradation of natriuretic peptides may cause the generation of numerous variants with physiological activity.

[0023] This family in humans and the animal kingdom contains many members: urodilatin, uroguanylin, guanylin, VNP, DNP, and KNP, which are described below.

[0024] Brain natriuretic peptide (VNP) was initially identified as a peptide having a physiological function similar to ANP in eels and later identified in many fish species (Kawakoshi et al., (2004) J. Mol. Endocrinol. 32: 547-555).

[0025] Mamba (dendroapsis) natriuretic peptide (DNP) was identified in the venom of the green mamba snake (Schweitz et al., (1992) J. Biol. Chem. 267: 13928-13932), and krait natriuretic peptide (KNP) was identified in the venom of the banded krait snake (Bungarus flaviceps) (Sridharan et al., (2015) Biochem. J. 469: 255-266).

[0026] Many other compounds similar to natriuretic peptides have been frequently identified from the venoms of venomous snakes such as lebetin isolated from the venom of the Levantine viper (Macrovipera lebetina), the venoms of large reptiles such as the Komodo dragon (natriuretic peptide toxin Var2) (Fry et al., (2009) Proc. Natl. Acad. Sci. USA 106: 8969-8974), and the venom of scorpions (Alves et al., (2013) Toxicon 74: 19-26). They are generally called peptides having natriuretic-like activity. Among these, preferred natriuretic peptides are L2α lebetin (Gly1-Gly38) and L1β (Asp2-Gly13). Finally, the presence of an enterotoxin called ST ("heat-stable enterotoxin") having a peptide structure similar to human guanylin and uroguanylin and having natriuretic-like activity has been described in Escherichia coli bacteria.

[0027] The natriuretic peptides used in the present invention are selected from the group consisting of (i) osteocalcin, osteocalcin propeptide, or an osteocalcin derivative, (ii) lectin, a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative.

[0028] As used herein, "ANP" means a peptide corresponding to the major mature form obtained by cleavage of the N-terminal fragment of the prohormone proANP.

[0029] Typically, human ANP is a 28-amino acid peptide obtained from cleavage of the N-terminal fragment of the prohormone proANP, which has 126 amino acids. Human ANP is formed from amino acids 99 to 126 of the prohormone proANP.

[0030] ANP is a peptide with a highly conserved amino acid sequence because this sequence is identical, for example, among humans, monkeys, gorillas, pigs, horses, and sheep (Takei et al., (2011) General and Comparative Endocrinology 171: 258-266). However, some differences in the sequence are observed in many animal species in which it is expressed. For example, rats, mice, and rabbits have a different amino acid at position 12 (isoleucine instead of human methionine). Similarly, elephant ANP is shortened to 27 amino acids and has two different amino acids compared to humans (Takei et al., (2011) General and Comparative Endocrinology 171: 258-266).

[0031] As used herein, "ANP propeptide" means any peptide obtained from the expression of the gene encoding preproANP, the maturation of preproANP, and optionally successive cleavage, and containing the peptide sequence of ANP.

[0032] Thus, the term "ANP propeptide" includes the prohormone proANP having ANP at its C-terminal portion (typically 126 amino acids in humans), and the preprohormone preproANP corresponding to the protein produced in humans by the NPPA gene (typically 151 amino acids in humans). The preprohormone preproANP has a peptide signal that is cleaved to form proANP.

[0033] In the context of the present invention, the term "ANP propeptide" also includes any peptide obtained from the maturation and cleavage of preproANP and proANP.

[0034] ANP typically consists of the amino acid sequence SLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 1), with a disulfide bridge located between amino acids 7 and 23.

[0035] ProANP typically consists of the amino acid sequence NPMYNAVSNADLMDFKNLLDHLEEKMPLEDEVVPPQVLSEPNEEAGAALSPLPEVPPWTGEVSPAQRDGGALGRGPWDSSDRSALLKSKLRALLTAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 3).

[0036] PreproANP typically consists of the amino acid sequence MSSFSTTTVSFLLLLAFQLLGQTRANPMYNAVSNADLMDFKNLLDHLEEKMPLEDEVVPPQVLSEPNEEAGAALSPLPEVPPWTGEVSPAQRDGGALGRGPWDSSDRSALLKSKLRALLTAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 4).

[0037] As used herein, the term "ANP derivative" means a natural or synthetic derivative of ANP.

[0038] As used herein, the term "ANP derivative" means an ANP peptide as defined above, in which one or more amino acid residues are chemically modified, for example, through alkylation, acylation, amidation, methoxylation, ester formation, amide formation, insertion of a lipophilic substituent, addition of polyethylene glycol (PEG), or addition of a carbohydrate chain.

[0039] As used herein, the term "ANP derivative" means a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full-length of ANP, an antiparallel dimer of ANP that is a polymer composed of ANP monomers, particularly a form having the structure of a homodimeric β-ANP.

[0040] As used herein, the term "ANP derivative" also means a peptide variant of ANP, that is, an intermediate form obtained during the synthesis, maturation, and partial degradation of ANP as defined above, and an ANP peptide in which 1 to 12, particularly 1 to 10, particularly 1 to 5, particularly 1 to 4, particularly 1 to 3, and more particularly 1 or 2 amino acids are deleted, substituted, or added while maintaining the activity of the wild-type ANP.

[0041] In one particular embodiment, the ANP derivative is an ANP peptide in which the phenylalanine (Phe) amino acid is substituted at position 8 of the loop (e.g., substituted by an alanine amino acid).

[0042] In another particular embodiment, the ANP derivative is an ANP peptide in which the amino acid phenylalanine (Phe) is substituted at position 26 or the amino acid serine is substituted at position 25, and the two amino acids are involved in the degradation of wild-type ANP (Ichiki and Burnett (2017) Circ J. 81: 913-919).

[0043] In one particular embodiment, the ANP derivative is an extended form of ANP having a disulfide bridge between amino acids 11 and 27, and is urodilatin having the sequence TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 2). This peptide corresponds to ANP with a 4-amino acid extension at the N-terminal side.

[0044] In another particular embodiment, the ANP derivative is a form of ANP extended by 12 amino acids, for example, a form of ANP extended by 12 amino acids at the C-terminal side, and is fsANP discovered in a human having a mutation in the gene encoding ANP (Dickey et al., (2009) J Biol Chem 284:19196-19202).

[0045] In another particular embodiment, the ANP derivative is a linear form of ANP in which a disulfide bridge (originally located between amino acids 7 and 23) is cleaved.

[0046] Typically, the ANP derivative that can be used in the present invention is preferably modified as compared to ANP so as to have a longer half-life than ANP and / or to have a weaker affinity for the human endogenous receptor of ANP than ANP.

[0047] As used herein, "osteocrin" means a hormone that acts as a regulator of dendritic growth in the cerebral cortex that develops in response to sensory experience.

[0048] Typically, human osteocrin is a 50-amino acid peptide obtained from the cleavage of the N-terminal fragment of the prohormone proosteocrin having 106 amino acids. Human osteocrin is formed from amino acids 83 to 132 of the prohormone proosteocrin.

[0049] As used herein, the term "osteocrin propeptide" means prohormone pro-osteocrin having 106 amino acids and osteocrin at its C-terminal portion, and / or preprohormone prepro-osteocrin having 132 amino acids corresponding to the protein produced by the OSTN gene in humans. Prepro-osteocrin has a peptide signal that is cleaved to form pro-osteocrin having 106 amino acids.

[0050] Osteocrin typically consists of the amino acid sequence SFSGFGSPLDRLSAGSVDHKGKQRKVVDHPKRRFGIPMDRIGRNRLSNSR (SEQ ID NO: 5).

[0051] Pro-osteocrin typically consists of the amino acid sequence VDVTTTEAFDSGVIDVQSTPTVREEKSATDLTAKLLLLDELVSLENDVIETKKKRSFSGFGSPLDRLSAGSVDHKGKQRKVVDHPKRRFGIPMDRIGRNRLSNSRG (SEQ ID NO: 6).

[0052] Prepro-osteocrin typically consists of the amino acid sequence MLDWRLASAHFILAVTLTLWSSGKVLSVDVTTTEAFDSGVIDVQSTPTVREEKSATDLTAKLLLLDELVSLENDVIETKKKRSFSGFGSPLDRLSAGSVDHKGKQRKVVDHPKRRFGIPMDRIGRNRLSNSRG (SEQ ID NO: 7).

[0053] In the context of the present invention, the term "osteocrin propeptide" also includes any peptide obtained from the maturation and cleavage of prepro-osteocrin and pro-osteocrin.

[0054] As used herein, the term "osteocrin derivative" means a natural or synthetic derivative of osteocrin.

[0055] As used herein, the term "osteocrin derivative" means osteocrin as defined above, in which one or more amino acid residues are chemically modified, for example, through alkylation, acylation, amidation, methoxylation, ester formation, amide formation, insertion of a lipophilic substituent, addition of polyethylene glycol (PEG), or addition of a carbohydrate chain.

[0056] The term "osteocrin derivative" also means a peptide variant of osteocrin, i.e., an intermediate obtained during the synthesis, maturation, and partial degradation of osteocrin as defined above.

[0057] As used herein, the term "lecthin" means a peptide obtained from the venom of the eastern green mamba snake.

[0058] Lectin as defined above is preferably lecthin 2α or lecthin 1β.

[0059] As used herein, the term "lecthin 2α" means a peptide produced by the eastern green mamba snake as described by Barbouche et al. (1996) FEBS Lett. 392: 6-10.

[0060] Typically, lecthin 2α is a 38-amino acid peptide.

[0061] Lectin 2α typically consists of the amino acid sequence GDNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG (SEQ ID NO: 8), which has a disulfide bridge between amino acids 14 and 30.

[0062] As used herein, the term "lecthin 1β" means a peptide produced by the eastern green mamba snake as described by Barbouche et al. (1998) Toxicon 36(12): 1939-47.

[0063] Typically, lecthin 1β is a 12-amino acid peptide.

[0064] Levetin 1β typically consists of the amino acid sequence DNKPPKKGPPNG (SEQ ID NO: 9).

[0065] As used herein, the term "levetin fragment" means at least a 10 - amino acid fragment of the above - defined levetin, preferably levetin 2α as defined above or levetin 1β as defined above.

[0066] The levetin fragment preferably contains at least 10 amino acids, preferably at least 12 amino acids, preferably at least 14 amino acids, preferably at least 16 amino acids, and / or 30 amino acids or less, preferably 25 amino acids or less, preferably 20 amino acids or less.

[0067] A fragment of levetin 2α contains, for example, amino acids 4 to 10 of the sequence of SEQ ID NO: 8, preferably amino acids 2 to 12 of the sequence of SEQ ID NO: 8.

[0068] A fragment of levetin 2α consists of, for example, amino acids 1 to 13 of the sequence of SEQ ID NO: 8.

[0069] Levetin 1β of the sequence of SEQ ID NO: 9 is also a fragment of levetin 2α consisting of amino acids 2 to 13 of the sequence of SEQ ID NO: 8.

[0070] As used herein, the term "derivative of levetin or levetin fragment" means a natural or synthetic derivative of levetin or levetin fragment.

[0071] As used herein, the term "derivative of levetin or levetin fragment" means, for example, a levetin or levetin fragment as defined above, wherein one or more amino acid residues are chemically modified through alkylation, acylation, amidation, methoxylation, ester formation, amide formation, insertion of lipophilic substituents, addition of polyethylene glycol (PEG), addition of a carbohydrate chain.

[0072] "Derivative of levetin or levetin fragment" also means a peptide variant of levetin or levetin fragment, i.e., an intermediate obtained during the synthesis, maturation and partial degradation of levetin or levetin fragment as defined above.

[0073] "Other natriuretic peptides" means, in this specification, natriuretic peptides that are not ANP, ANP propeptide, or ANP derivatives (such as urodilatin).

[0074] Bacterial biofilms and bacterial infections "Bacterial infection" means, in this specification, an infection caused by one or more bacterial species, particularly gram-negative bacteria and / or gram-positive bacteria.

[0075] In one particular embodiment, the bacterial infection is a chronic bacterial infection.

[0076] In one particular embodiment, the bacterial infection is an infection caused by gram-negative bacteria.

[0077] In one particular embodiment, the bacterial infection is an infection caused by bacteria of the genus Pseudomonas, more particularly the species Pseudomonas aeruginosa.

[0078] In another particular embodiment, the bacterial infection is an infection caused by gram-positive bacteria.

[0079] In one particular embodiment, the bacterial infection is an infection caused by bacteria of the genus Staphylococcus, more particularly the species Staphylococcus aureus.

[0080] "Bacterial biofilm" means, in this specification, a community of bacteria that adhere together and attach to a surface. The formation of biofilms can bind to many types of surfaces and is characterized by the secretion of a matrix that enables the aggregation and protection of the bacteria that form this structure.

[0081] In the present invention, the bacterial biofilm contains at least one or more bacterial species that cause the infectious disease to be treated.

[0082] In the present invention, the natriuretic peptide, particularly the ANP peptide, ANP propeptide, or ANP peptide derivative, disperses the bacterial biofilm.

[0083] As used herein, "dispersion of bacterial biofilm" or "destruction of bacterial biofilm" means that the bacteria forming the constituent community of the bacterial biofilm separate from each other and from the surface, and thus the biofilm is reduced and even disappears, typically making the bacteria more accessible to antibiotics.

[0084] In one particular embodiment, the bacterial biofilm is dispersed by at least 50%, particularly at least 55%, particularly at least 60%, particularly at least 65%, particularly at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0085] Advantageously, the natriuretic peptide, particularly the ANP peptide, ANP propeptide, or ANP peptide derivative, disperses the bacterial biofilm without killing the constituent bacteria of the biofilm.

[0086] However, it should be noted that when the natriuretic peptide, particularly the ANP peptide, ANP propeptide, or ANP peptide derivative is used in combination with an antibiotic, particularly when the constituent bacteria of the biofilm are further released to the dispersion effect of the natriuretic peptide, particularly ANP, ANP propeptide, or ANP peptide derivative, they may be killed by the antibiotic.

[0087] Therapeutic applications The present invention relates to the use of a natriuretic peptide selected from the group consisting of (i) osteocrin, osteocrin propeptide, or an osteocrin derivative, (ii) rebeccin (preferably rebeccin 2α or rebeccin 1β), a rebeccin fragment, or a derivative of rebeccin or a rebeccin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the foregoing section "Natriuretic Peptide", for its use in a method for therapeutically treating a bacterial infection associated with a bacterial biofilm as defined in the foregoing section "Bacterial Biofilm and Bacterial Infections" in a subject, wherein the natriuretic peptide disperses the bacterial biofilm.

[0088] The present invention also relates to the use of a natriuretic peptide selected from the group consisting of (i) osteocrin, osteocrin propeptide, or an osteocrin derivative, (ii) rebeccin (preferably rebeccin 2α or rebeccin 1β), a rebeccin fragment, or a derivative of rebeccin or a rebeccin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the foregoing section "Natriuretic Peptide", for the manufacture of a medicament for therapeutically treating a bacterial infection associated with a bacterial biofilm as defined in the foregoing section "Bacterial Biofilm and Bacterial Infections" in a subject, wherein the natriuretic peptide disperses the bacterial biofilm.

[0089] The present invention also relates to a method for the therapeutic treatment of a bacterial infection associated with a bacterial biofilm as defined in the preceding section "Bacterial biofilms and bacterial infections" in a subject suffering from a bacterial infection, particularly a chronic bacterial infection, comprising administering to the subject a therapeutically effective amount of a natriuretic peptide selected from the group consisting of (i) osteocalcin, osteocalcin propeptide, or an osteocalcin derivative, (ii) lectin (preferably lectin 2α or lectin 1β), a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the preceding section "Natriuretic peptides", wherein the natriuretic peptide disperses the bacterial biofilm.

[0090] As used herein, "subject" means an animal such as a human or non-human mammal, bird, or fish.

[0091] In one particular embodiment, the subject suffers from a chronic respiratory lesion such as cystic fibrosis, chronic obstructive pulmonary disease, or bronchiectasis. Preferably, the subject suffers from cystic fibrosis.

[0092] In another particular embodiment, the subject suffers from severe burns (victims of severe burns) or a superficial infection.

[0093] In another embodiment, the subject has a graft and / or a prosthesis.

[0094] In one particular embodiment, the natriuretic peptide, particularly the ANP peptide, ANP propeptide, or ANP peptide derivative, is used in combination with an antibiotic.

[0095] As used herein, "antibiotic" means a natural or synthetic substance that destroys or inhibits the growth of bacteria.

[0096] Antibiotics include - antibiotics that inhibit bacterial cell wall synthesis, among others - Beta-lactams, in particular, - Penicillins of Group A such as amoxicillin, benzathine benzylpenicillin, benzathine penicillin, benzathine phenoxymethylpenicillin, penicillin G, and penicillin V, penicillins of Groups G and V such as benzathine benzylpenicillin, benzathine penicillin, benzathine phenoxymethylpenicillin, penicillin G, and penicillin V, penicillins of Group M such as cloxacillin and oxacillin, carboxypenicillins such as ticarcillin, ureidopenicillins such as piperacillin, aminidopenicillins such as pivmecillinam, and penicillins including temocillin, - Carbapenems such as ertapenem, imipenem, and meropenem, - Monobactams such as aztreonam, - Cephalosporins including first-generation cephalosporins such as cefaclor, cefadroxil, cephalexin, cephalothin, cefazolin, and cefradine, second-generation cephalosporins such as cefamandole, cefoxitin, cefuroxime sodium, and cefuroxime axetil, and third-generation cephalosporins such as cefixime, cefpodoxime proxetil, cefotiam hexetil, cefepime, cefotaxime, cefpirome, ceftazidime, and ceftriaxone, - Phosphomycins such as phosphomycin and phosphomycin trometamol, - Glycopeptides such as teicoplanin and vancomycin, - Lipopeptides such as daptomycin, and - Polymyxins such as polymyxin B and polymyxin E or colistin, - Antibiotics that inhibit protein synthesis, among others, - Aminoglycosides such as amikacin sulfate, gentamicin, neomycin, netilmicin, spectinomycin, streptomycin, and tobramycin, - Macrolides, etc., in particular, - True macrolides such as amphotericin B, azithromycin, clarithromycin, erythromycin, josamycin, midecamycin, and roxithromycin, - Lincomycins such as clindamycin and lincomycin, - Ketolides such as telithromycin, and - Synergisticins such as pristinamycin, - Phenicols such as thiamphenicol, - Cyclins such as chlorotetracycline, doxycycline, lymecycline, methacycline, minocycline, and tigecycline, - Fusidic acids such as fusidic acid, and - Oxazolidinones such as linezolid and tedizolid, - Antibiotics that inhibit nucleic acid synthesis, especially - Quinolones such as pipemidic acid, flumequine, enoxacin, lomefloxacin, norfloxacin, ciprofloxacin, ofloxacin, pefloxacin, levofloxacin, and moxifloxacin, - Quinolines such as hydroxyquinoline, - Mupirocin, and - Rifamycin, - Antibiotics that inhibit folic acid synthesis, including sulfonamides such as sulfadiazine, sulfamethizole, sulfafurazole, and sulfamethoxazole, and - Antibiotics with complex or unknown mechanisms, especially - Nitrogen products, especially - Nitrofurans such as nitrofurantoin and nifroxide, and - Nitroimidazoles such as metronidazole, omidazole, and tinidazole, and - Include ethambutol, isoniazid, pyrazinamide, rifabutin, and rifampicin.

[0097] In one particular embodiment, the antibiotic is an antibiotic that inhibits protein synthesis, in particular an aminoglycoside such as tobramycin, an antibiotic that inhibits nucleic acid synthesis, in particular a quinolone such as ciprofloxacin, an antibiotic that inhibits bacterial cell wall synthesis, in particular a beta-lactam, more particularly a carbapenem such as imipenem, doripenem, or meropenem, or an antibiotic that acts on the structure of the bacterial cell wall, in particular a polymyxin such as polymyxin B or colistin.

[0098] In one particular embodiment, the antibiotic is an aminoglycoside antibiotic, a quinolone, a carbapenem, or a polymyxin. More particularly, the antibiotic may be an aminoglycoside or a quinolone.

[0099] In another particular embodiment, the antibiotic is tobramycin, ciprofloxacin, imipenem, and / or colistin.

[0100] More particularly, the antibiotic may be tobramycin and / or ciprofloxacin.

[0101] The inventors have shown that the combined use of ANP peptides and these antibiotics has a synergistic effect on the destruction of biofilms. Without being bound by a particular theory, it is believed that ANP peptides make these bacteria more accessible to antibiotics by dispersing the constituent bacteria of the biofilm and are therefore more effective.

[0102] In another particular embodiment, the natriuretic peptide is used in combination with at least one other natriuretic peptide as defined in the section "Natriuretic Peptides".

[0103] Thus, in one particular embodiment, when the natriuretic peptide is ANP, an ANP propeptide, or an ANP peptide derivative, it can be used in combination with osteocalcin, an osteocalcin propeptide, or an osteocalcin derivative and / or lectin (preferably lectin 2α or lectin 1β), a lectin fragment, or a derivative of lectin or a lectin fragment.

[0104] Alternatively, in another particular embodiment, when the natriuretic peptide is osteocalcin, an osteocalcin propeptide, or an osteocalcin derivative, it can be used in combination with ANP, an ANP propeptide, or an ANP derivative, and / or lectin (preferably lectin 2α or lectin 1β), a lectin fragment, or a derivative of lectin or a lectin fragment.

[0105] Alternatively, in another particular embodiment, when the natriuretic peptide is lectin (preferably lectin 2α or lectin 1β), a lectin fragment, or a derivative of lectin or a lectin fragment, it can be used in combination with ANP, an ANP propeptide, or an ANP peptide derivative, and / or osteocalcin, an osteocalcin propeptide, or an osteocalcin derivative.

[0106] The combined use of several natriuretic peptides has the advantage that the respective amounts of the compounds used can be made smaller, thereby more effectively limiting the possible side effects.

[0107] In this particular embodiment, the natriuretic peptide can also be used in combination with an antibiotic, optionally sequentially (first the natriuretic peptide, then the antibiotic) as described above.

[0108] In one particular embodiment, the natriuretic peptide, in particular the ANP peptide, ANP propeptide, or ANP peptide derivative, can be used optionally sequentially (first the natriuretic peptide, then the compatible substance) in combination with any other substance adapted for treating a subject suffering from a bacterial infection as described above.

[0109] In particular, when the subject suffers from a chronic respiratory lesion, the natriuretic peptide, in particular the ANP peptide, ANP propeptide, or ANP peptide derivative, is preferably used optionally sequentially (first the natriuretic peptide, then the compatible substance) in combination with a substance adapted for treating this chronic respiratory lesion.

[0110] Thus, in embodiments where the subject suffers from cystic fibrosis, the natriuretic peptide, in particular the ANP peptide, ANP propeptide, or ANP peptide derivative, is preferably used optionally sequentially (first the substance for mucociliary clearance, then the natriuretic peptide) in combination with a substance for mucociliary clearance such as DNase (e.g., Pulmozyme®) and / or a bronchodilator.

[0111] In these particular embodiments, the natriuretic peptide can also be used in combination with an antibiotic as described above and / or another natriuretic peptide as described above, preferably in combination with an antibiotic as described above.

[0112] In the context of the present invention, the term "treatment" or "treating" means reversing, alleviating, or inhibiting the progression of the disease to which this term applies, or one or more symptoms of this disease.

[0113] As used herein, "therapeutically effective amount" means, in the context of treating a bacterial infection, an amount of a compound of interest sufficient to disperse a bacterial biofilm having a reasonable risk-benefit ratio applicable to any medical treatment. However, it should be clearly understood that the total daily dosage of the compounds used in the present invention will be determined by a physician based on a medical evaluation. The therapeutically effective dosage level specific to a particular subject will depend on a number of factors including the disease being treated and its severity, the activity of the specific compound being used, age, body weight, general health, sex and diet, time of administration, route of administration and rate of excretion of the specific compound being administered, length of treatment, drug therapies used in combination with or concurrently with the specific compound being administered, and like factors well known in the medical arts. For example, in this field it is well known to start with a dosage of the compound at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is obtained.

[0114] The compounds used in the context of the present invention can be administered in the form of a pharmaceutical composition comprising a pharmaceutically acceptable excipient for forming a therapeutic composition and, optionally, a sustained-release matrix such as a biodegradable polymer.

[0115] As used herein, "pharmaceutical" or "pharmaceutically acceptable" means molecular entities and compositions that do not produce allergy or other untoward secondary reactions when administered to a mammal, particularly a human. A pharmaceutically acceptable vehicle or excipient refers to a solid, semi-solid, or liquid non-toxic filler, diluent, encapsulating material, or formulation aid of any type.

[0116] The form and route of administration of a pharmaceutical composition comprising the compounds used in the context of the present invention will clearly depend on the disease being treated, the severity of the disease, the age, body weight, and sex of the patient, and the like.

[0117] The compounds used in the context of the present invention can be formulated, for example, for administration via the respiratory tract or by inhalation (particularly by nebulization), orally, parenterally, intranasally, intravenously, intramuscularly, topically, subcutaneously, or by the intraocular route.

[0118] In one particular embodiment, the compounds used in the context of the present invention are administered via the airway route or by inhalation (in particular by nebulization).

[0119] In one particular embodiment, the natriuretic peptides used in the context of the present invention, in particular ANP peptides (or propeptides or derivatives), are administered at a daily dose of from 0.3 ng / ml to 3,000 ng / ml.

[0120] In another particular embodiment, the antibiotics used in combination are administered at a maximum daily dose of 600 mg (300 mg twice a day).

[0121] Pharmaceutical compositions and pharmaceutical combinations The present invention also relates to a pharmaceutical composition comprising (A) a natriuretic peptide selected from the group consisting of (i) osteocalcin, osteocalcin propeptide, or an osteocalcin derivative, (ii) reelin (preferably reelin 2α or reelin 1β), a reelin fragment, or a derivative of reelin or a reelin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the section "Natriuretic peptides" above, and (B) an antibiotic as defined in the section "Therapeutic applications" above.

[0122] In one particular embodiment, the composition of the present invention further comprises a pharmaceutically acceptable vehicle or excipient as defined above.

[0123] In another particular embodiment, the composition of the present invention further comprises another natriuretic peptide as defined in the section "Natriuretic peptides" above.

[0124] The present invention also relates to an antibacterial pharmaceutical combination for the simultaneous, separate, or sequential use in the therapeutic treatment of a subject as defined above for a bacterial infection associated with a bacterial biofilm as defined in the section "Bacterial biofilms and bacterial infections" above, comprising (A) a natriuretic peptide selected from the group consisting of (i) osteocalcin, osteocalcin propeptide, or an osteocalcin derivative, (ii) rexin (preferably rexin 2α or rexin 1β), a rexin fragment, or a derivative of rexin or a rexin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the section "Natriuretic peptides" above, and (B) an antibiotic as defined in the section "Therapeutic applications" above.

[0125] The present invention also relates to the use of (A) a natriuretic peptide selected from the group consisting of (i) osteocalcin, osteocalcin propeptide, or an osteocalcin derivative, (ii) rexin (preferably rexin 2α or rexin 1β), a rexin fragment, or a derivative of rexin or a rexin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the section "Natriuretic peptides" above, and (B) an antibiotic as defined in the section "Therapeutic applications" above, for the manufacture of an antibacterial pharmaceutical combination for the simultaneous, separate, or sequential administration in the therapeutic treatment of a subject as defined above for a bacterial infection associated with a bacterial biofilm as defined in the section "Bacterial biofilms and bacterial infections" above.

[0126] A further object of the present invention is to simultaneously, separately, or sequentially administer to a subject in need thereof a therapeutically effective amount of an antibiotic and a natriuretic peptide selected from the group consisting of: (A) (i) osteocalcin, osteocalcin propeptide, or an osteocalcin derivative, (ii) lectin (preferably lectin 2α or lectin 1β), a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the section "Natriuretic Peptide" above, for the therapeutic treatment of a bacterial infection associated with a bacterial biofilm as defined in the section "Bacterial Biofilm and Bacterial Infections" above.

[0127] In the context of the present invention, the term "combination" or "pharmaceutical combination" defines either a fixed combination in a single unit dosage form or a kit for combined administration in which the natriuretic peptide and the antibiotic can be administered simultaneously or separately at time intervals such that the combination partners can exert a synergistic effect.

[0128] Accordingly, the combination compounds of the present invention can be formulated into one or two separate pharmaceutical combinations, and each composition can be administered via the same or different routes of administration.

[0129] In one particular embodiment, the pharmaceutical combination of the present invention also comprises (C) another natriuretic peptide as defined in the section "Natriuretic Peptide", and / or (D) another substance suitable for the treatment of a subject suffering from a bacterial infection as defined in the section "Therapeutic Applications" above.

[0130] In this particular embodiment, the combination may be a fixed combination of single unit dosage forms, or a kit for combination administration in which a natriuretic peptide, an antibiotic, other natriuretic peptides, and / or other substances suitable for the treatment of a subject are administered simultaneously or separately at time intervals such that the combination partners can exert a synergistic effect.

[0131] Thus, in this embodiment, the compounds for the combination can be formulated into a combination of one, two, three, or four separate medicaments, and each composition is for the same or different routes of administration.

[0132] To prepare the pharmaceutical compounds used in the present invention, an effective amount of the compounds used in the present invention can be dissolved or dispersed in a pharmaceutically acceptable vehicle or aqueous medium.

[0133] Dosage forms suitable for administration by injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions, for example in micronized form. In each case, they must be sterile and in a fluid form so as to be readily usable with a syringe. They must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria, viruses, or fungi.

[0134] The vehicle may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, or others), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the particle size necessary for dispersion, and by using surfactants, stabilizers, cryoprotectants, or antioxidants. Defense against the action of microorganisms can be provided by antibacterial and antifungal agents. In many cases, it will be preferable to include isotonic agents such as sugars or sodium chloride.

[0135] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount, along with several other ingredients, into a suitable solvent and subsequently filtering and sterilizing. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary ingredients. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, by which powders of the active ingredient and any desired additional ingredients are produced from solutions that have been previously sterilized by filtration.

[0136] For example, in the case of parenteral administration by aqueous solution, the solution is preferably buffered appropriately if necessary, and the liquid diluent is made isotonic with sufficient saline or glucose solution. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration.

[0137] Formulations of pharmaceutical compositions for administration via inhalation are well known to those skilled in the art. Generally, the active ingredient is delivered in the form of an aerosol spray from a pressurized metered-dose inhaler using a suitable propellant gas such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, in the form of a powder administered by a dry powder inhaler, or in the form of an aqueous liquid aerosol using a nebulizer. Nebulizers for delivering liquid aerosols can be classified into jet nebulizers that apply a pressurized air stream using a portable compressor or central air supply in a hospital, ultrasonic nebulizers incorporating a piezoelectric crystal that provides the energy necessary to cause aerosolization, and electronic nebulizers based on the principle of a perforated vibrating membrane.

[0138] Non-therapeutic uses The present invention also relates to the in vitro or ex vivo use of a natriuretic peptide selected from the group consisting of (i) osteocalcin, osteocalcin propeptide, or an osteocalcin derivative, (ii) lectin (preferably lectin 2α or lectin 1β), a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the above section "Natriuretic peptide", for dispersing bacterial biofilms.

[0139] Thus, natriuretic peptides, particularly ANP, an ANP propeptide, or an ANP peptide derivative, can generally be used, for example, in antifouling applications on ship hulls or pipelines.

[0140] The present invention also relates to an in vitro or ex vivo method for dispersing bacterial biofilms on a surface, which includes applying to the surface a composition containing a natriuretic peptide selected from the group consisting of (i) osteocalcin, osteocalcin propeptide, or an osteocalcin derivative, (ii) lectin (preferably lectin 2α or lectin 1β), a lectin fragment, or a derivative of lectin or a lectin fragment, and (iii) an ANP peptide, an ANP propeptide, or an ANP peptide derivative, as defined in the above section "Natriuretic peptide".

[0141] The surface may typically be a pipe surface, a ship surface, or the surface of a transplant device before transplantation.

[0142] The application of the composition to the surface can be carried out using any suitable technique depending on the formulation of the composition.

[0143] The present invention will be described in more detail in the following examples and figures.

[0144] Brief description of the sequences

[0145] [Table 1]

Brief Description of the Drawings

[0146]

Figure 1

Examples

[0147] (Example 1) The effect of ANP alone (0.1 μM) on the growth of P. aeruginosa, on biofilms being formed or pre-formed biofilms of P. aeruginosa This example shows the effect of ANP on pre-formed biofilms of P. aeruginosa.

[0148] Materials and Methods Tested substances, bacterial strains, and bacterial cultures The P. aeruginosa wild-type strain PA14 used was provided by Harvard Medical School (Boston, MA) (Liberati et al., (2006) Proc. Natl. Acad. Sci. USA 103: 2833 - 2838).

[0149] The bacterial strain was cultured at 37°C in Luria Bertani medium (LB) with stirring.

[0150] Formation of P. aeruginosa biofilms under dynamic conditions After pre-culturing at 37°C for 3 hours in LB medium, P. aeruginosa was inoculated into LB medium at OD600 = 0.08 and subcultured for 2 hours.

[0151] ANP (Calbiochem Merck) was added 2 hours after the start of culture, which is the time corresponding to the middle of the exponential growth phase of the bacteria. The final bacterial density and contamination prevention were controlled by seeding.

[0152] Biofilms were formed at 37°C in a 3-channel flow chamber under dynamic conditions as described by Bazire et al. (2010) J. Bacteriol. 192: 3001-3010. Briefly, from an 18-hour preculture, a bacterial suspension with an OD600 = 0.08 prepared in a sterile physiological saline aqueous solution was injected into each channel of the flow chamber. The bacteria were left at 37°C for 2 hours under static conditions (no flow) to adhere to the slide glass. Next, the study of biofilm formation was carried out at 37°C for 24 hours under the flow of LB medium (2.5 ml / h).

[0153] Destruction of P. aeruginosa biofilms under dynamic conditions To study the effect of ANP on biofilms preformed for 24 hours, 300 μl of ANP (0.1 μM) or 300 μl of sterile ultrapure water (control condition) was injected into various channels of the flow chamber. The treatment of the preformed biofilms was carried out at 37°C for 2 hours under static conditions. After 2 hours, the flow of the medium was applied again for 15 minutes to remove any cells that might have detached from the biofilm under the action of the treatment.

[0154] Biofilms were stained with 5 μM of Syto9 green fluorescent dye (Invitrogen) for 15 minutes and then observed with a confocal laser scanning microscope (Zeiss LSM710 (Zeiss)). Images were taken at various layers of the biofilm to enable 3D reconstruction, and at least 3 images were taken at different points in one identical channel of the flow chamber. Next, the files of each image were analyzed with COMSTAT software (Heydorn et al. (2000) Microbiology 146: 2395-2407), and 3 images from at least 3 independent experiments were analyzed to enable the determination of the average and maximum thickness of the biofilm, as well as the biovolume of the bacteria.

[0155] Cell culture The IIA549 type (ATCC-CCL185TM, ATCC Manassas, VA) human lung epithelial cell line was cultured at 37 °C in a 5% CO2 atmosphere in DMEM medium (Lonza) supplemented with 10% fetal bovine serum (Lonza) and 1% penicillin and streptomycin (Penistrep, Lonza). In normal cell culture, the cells were seeded in 25 ml flasks and used at 80% confluence.

[0156] For the cytotoxicity test, the cells were seeded in 24-well plates at a final density of 3×10 5 cells per well and cultured for 48 hours before use. Antibiotics and fetal bovine serum were removed from the cells by adding fresh serum-free medium at least 24 hours before the infection test.

[0157] Measurement of the release of cytoplasmic lactate dehydrogenase (LDH) by A549 cells LDH is a stable cytoplasmic enzyme that is released into the medium after cell lysis and is therefore a marker of cell death. The amount of LDH released by eukaryotic cells in the presence of bacteria was determined using the Cytotox96 enzyme assay (Promega, Charbonnieres, France), regardless of whether they were exposed to ANP (concentrations from 1 μM to 10 nM). A549 cells were incubated for 6 hours with a control (untreated) or P. aeruginosa PA14 (pre-treated with ANP) at a multiplicity of infection of 10. A lysis buffer consisting of Triton X-100 solution (9% in water) was used to determine the maximum LDH (100% LDH release) that could be released by A549 cells under the experimental conditions. To exclude the involvement of the medium, the level of background noise was determined using only the medium and defined as 0% LDH release. Next, the percentage of LDH release in the cell population was calculated using the following formula:

[0158]

Equation

[0159] This test had sufficient sensitivity to measure LDH concentrations equivalent to 1% lysis of the cell population.

[0160] Results Effect of ANP on pre - formed biofilms of P. aeruginosa The inventors investigated the potential antibiofilm activity of ANP against established biofilms of P. aeruginosa. From this perspective, 24 hours after forming P. aeruginosa biofilms in a dynamic flow system, the inventors exposed the bacteria to 0.1 μM ANP for 2 hours. Under these conditions, the inventors observed the dispersion of the biofilm disrupted after exposure to ANP and the absence of the mushroom - like structures and P. aeruginosa biofilm characteristics observed under control conditions. The bacterial biomass was reduced by 81.4 ± 4.1% (P < 0.001) after exposure to 0.1 μM ANP for 2 hours compared to the biomass present under control conditions. In parallel, the inventors observed a significant reduction in the biofilm thickness after exposure to ANP for 2 hours.

[0161] Similar results were obtained even when the pre - formed biofilms were exposed to ANP for 30 minutes. Under these conditions, the inventors observed that the biofilm was strongly dispersed by ANP and the mushroom - like structures and P. aeruginosa biofilm characteristics observed under control conditions were absent after exposure to ANP. The remaining bacterial biomass was reduced by 80.2 ± 2.0% (P < 0.05) after exposure to ANP (30 minutes) compared to the biomass present under control conditions.

[0162] Effect of ANP on the growth of P. aeruginosa To determine the potential direct effect of ANP on the growth of P. aeruginosa, the inventors studied the influence of 1 μM and 0.1 μM ANP on the growth of P. aeruginosa P14 in a liquid medium. As shown in Figure 1, none of the ANP concentrations tested affected the growth of the bacteria.

[0163] Therefore, the effect of ANP on the pre-formed biofilm is unlikely to be due to its action on bacterial growth.

[0164] Effect of ANP on the cytotoxicity of bacteria in cultured human lung cells The cytotoxic activity of P. aeruginosa exposed to various concentrations of ANP (from 1 μM to 10 nM) was studied in A549 lung cells. The inventors observed that, as shown in the following table, ANP did not affect the cytotoxic activity of P. aeruginosa in lung cells at any concentration.

[0165] [Table 2]

[0166] Conclusion Therefore, the inventors have shown in this example that ANP used at a concentration of 0.1 μM completely prevents the formation of P. aeruginosa biofilm and strongly disrupts the pre-formed biofilm of P. aeruginosa.

[0167] For a long time, research on preventing biofilm formation has focused on the initial stages of biofilm formation, including biofilm attachment and maturation. The concentration of drugs required to inhibit biofilm formation is generally much lower than the concentration required to disrupt or disturb pre-formed biofilms.

[0168] The main advantage of ANP demonstrated herein by the inventors is that the use of ANP at 0.1 μM for 2 hours, and even for 30 minutes, is sufficient to disperse approximately 80% of the biofilm structure. This is particularly interesting from the perspective of its synergistic use with antibiotics.

[0169] This effect of ANP on P. aeruginosa biofilm is greater than the inhibitory effect on formation previously observed with CNP, which is even more advantageous because CNP slightly increased the bacterial toxicity and cytotoxicity by activating the bacterial quorum sensing system.

[0170] The inventors have shown herein that, despite its strong antibiofilm action, ANP does not kill bacteria and does not increase the cytotoxicity of P. aeruginosa in cultured human lung cells. The fact that bacteria are not killed is particularly interesting because this prevents the emergence of resistant strains.

[0171] (Example 2) Dose-dependent effect of ANP on pre-formed biofilm of P. aeruginosa This example shows that the action of ANP alone on pre-formed biofilm of P. aeruginosa is dependent on the dose used and can be observed at 0.3 ng / ml or higher.

[0172] Materials and methods The materials and methods used in this example are the same as those described in Example 1 (destruction of P. aeruginosa biofilm under dynamic conditions).

[0173] Results The inventors studied the potential antibiofilm activity of ANP against established biofilms of P. aeruginosa after exposure for 2 hours or 30 minutes at different ANP concentrations: 0.3 ng / ml, 3 ng / ml, and 30 ng / ml, compared to the activity obtained in Example 1 ([ANP] = 300 ng / ml or 0.1 μM).

[0174] The inventors observed that the biofilm was strongly dispersed by ANP immediately after 30 minutes of exposure at a minimum concentration of 0.3 ng / ml or higher.

[0175] The results obtained are listed in the following table.

[0176]

Table 3

[0177] Therefore, this example verifies the advantage of ANP that it is active from an exposure time of 30 minutes at concentrations above very low concentrations.

[0178] (Example 3) Effect of osteocalcin on pre-formed biofilm of P. aeruginosa This example shows the effect of osteocalcin alone at a dose of 10 -8 M on pre-formed biofilm of P. aeruginosa.

[0179] Materials and methods The materials and methods used in this example are the same as those described above in Example 1 (destruction of P. aeruginosa biofilm under dynamic conditions).

[0180] Results The inventors studied the potential anti-biofilm activity of osteocalcin against established P. aeruginosa biofilm after exposure for 2 hours at a concentration of 10 -8 M.

[0181] The inventors observed that the biofilm was strongly and significantly dispersed by osteocalcin.

[0182]

Table 4

[0183] (Example 4) Effect of leveticin 2α peptide on pre-formed biofilm of P. aeruginosa This example shows the effect of leveticin L2α peptide alone at a dose of 10 -8 M on pre-formed biofilm of P. aeruginosa.

[0184] Materials and Methods The materials and methods used in this example are the same as those described in Example 1 (Destruction of P. aeruginosa biofilm under dynamic conditions).

[0185] Results The inventors studied the potential antibiofilm activity of the levantin L2α peptide against an established biofilm of P. aeruginosa after exposure at a concentration of 10 -8 M for 2 hours.

[0186] The inventors observed that the biofilm was strongly and significantly dispersed by the levantin L2α peptide.

[0187] [Table 5]

[0188] (Example 5) Effect of the levantin 1β peptide on a pre-formed biofilm of P. aeruginosa This example shows the effect of the levantin L1 beta peptide alone at a dose of 10 -8 M on a pre-formed biofilm of P. aeruginosa.

[0189] Materials and Methods The materials and methods used in this example are the same as those described in Example 1 (Destruction of P. aeruginosa biofilm under dynamic conditions).

[0190] Results The inventors studied the potential antibiofilm activity of the levantin L1 beta peptide against an established biofilm of P. aeruginosa after exposure at a concentration of 10 -8 M for 2 hours.

[0191] The inventors observed that the biofilm was strongly dispersed by the levantin L1 beta peptide.

[0192]

Table 6

[0193] (Example 6) Effect of the combination of ANP and tobramycin on pre - formed biofilms of P. aeruginosa This example demonstrates the synergistic effect of the combination of ANP + tobramycin on pre - formed biofilms of P. aeruginosa.

[0194] Materials and methods The materials and methods used in this example were the same as those described in Example 1, with the following specified.

[0195] To study the effect of ANP combined with tobramycin on 24 - hour pre - formed biofilms (as described in Example 1), a mixture of 300 μl of ANP (1 nM) and tobramycin (50 μg / ml) or 300 μl of tobramycin alone (control condition) was injected into various channels of the flow chamber. The treatment of the pre - formed biofilms was carried out at 37 °C for 2 hours under static conditions. After 2 hours, the flow of the medium was applied again for 15 minutes to remove any cells that might have been detached from the biofilm under the action of the treatment.

[0196] Biofilms were observed under a confocal laser scanning microscope as described in Example 1. To test the integrity of the bacterial membrane, a mixture of SYTO9 Green 5 μM and propidium iodide (PI) 0.3 μM was used (Live / Dead BacLight Bacterial Viability Kit, Invitrogen).

[0197] Results The inventors studied the potential antibiofilm activity of the combination of ANP (10 -9 M; 3 ng / ml) + tobramycin (50 μg / ml or 10 μg / ml) against established biofilms of P. aeruginosa.

[0198] Determine the biovolume of the biofilm and list the results in the following table.

[0199] [Table 7]

[0200] [Table 8]

[0201] Under these conditions, the inventors observed that the combination of ANP (1 nM) + tobramycin (50 μg / ml) acted synergistically to result in 96.3% destruction of the biofilm and strong dispersion of the biofilm. Similarly, the combination of ANP (1 nM) + tobramycin (10 μg / ml) acts synergistically to act to result in 94.8% destruction of the biofilm.

[0202] (Example 7) Effect of the combination of ANP and ciprofloxacin on pre-formed biofilms of P. aeruginosa This example shows the synergistic effect of the combination of ANP + ciprofloxacin on pre-formed biofilms of P. aeruginosa.

[0203] Materials and methods To study the effect of ANP combined with ciprofloxacin on 24-hour pre-formed biofilms (as described in Example 1), 300 μl of a mixture of ANP (10 nM) and ciprofloxacin (0.01 μg / ml or 0.04 μg / ml) or 300 μl of ciprofloxacin alone (control condition) was injected into various channels of the flow chamber. The treatment of the pre-formed biofilm was carried out at 37 °C for 2 hours under static conditions. After 2 hours, the flow of the medium was applied again for 15 minutes to remove any cells that might have detached from the biofilm under the action of the treatment.

[0204] As described in Example 1, the biofilm was stained with 5 μM Syto9 green fluorescent dye (Invitrogen) for 15 minutes and then observed under a confocal laser scanning microscope.

[0205] Results The inventors studied the potential antibiofilm activity of the combination of ANP (10 -8 M; 30 ng / ml) + ciprofloxacin (0.04 μg / ml or 0.01 μg / ml) against established biofilms of P. aeruginosa.

[0206] The biovolume of the biofilm was determined and the results are listed in the following table.

[0207] [Table 9]

[0208] [Table 10]

[0209] Under these conditions, the inventors observed that the biofilm was more strongly dispersed by the combination of ANP + ciprofloxacin compared to treatment with ciprofloxacin antibiotic alone.

[0210] Therefore, the combination with ANP can result in 97% destruction of the biofilm when combined with a ciprofloxacin concentration of 0.01 μg / ml.

[0211] (Example 8) Effect of the combination of ANP and colistin on pre-formed biofilms of P. aeruginosa This example shows the synergistic effect of the combination of ANP + colistin against pre-formed biofilms of P. aeruginosa.

[0212] Materials and Methods To study the effect of ANP in combination with colistin on 24-hour pre-formed biofilms (as described in Example 1), 300 μl of a mixture of ANP (10 nM) and colistin (1 μg / ml) or 300 μl of colistin alone (control condition) was injected into various channels of the flow chamber. The treatment of the pre-formed biofilm was carried out at 37 °C for 2 hours under static conditions. After 2 hours, the flow of the medium was applied again for 15 minutes to remove any cells that might have detached from the biofilm under the action of the treatment.

[0213] As described in Example 1, the biofilm was stained with 5 μM of Syto9 green fluorescent dye (Invitrogen) for 15 minutes and then observed under a confocal laser scanning microscope.

[0214] Results We studied the potential antibiofilm activity of the combination of ANP (10 -8 M; 30 ng / ml) + colistin (1 μg / ml) against established biofilms of P. aeruginosa.

[0215] The biovolume of the biofilm was determined and the results are listed in the following table.

[0216] [Table 11]

[0217] Under these conditions, we observed that the biofilm was more strongly dispersed by the combination of ANP + colistin (1 μg / ml) compared to treatment with colistin antibiotic alone or ANP alone.

[0218] Thus, the combination with ANP can result in 97.3% destruction of the biofilm when combined with a colistin concentration of 1 μg / ml.

[0219] (Example 9) Effect of the combination of ANP and imipenem on pre-formed biofilms of P. aeruginosa This example demonstrates the synergistic effect of the combination of ANP and imipenem on pre-formed biofilms of P. aeruginosa.

[0220] Materials and methods To study the effect of ANP combined with imipenem on 24-hour pre-formed biofilms (as described in Example 1), 300 μl of a mixture of ANP (10 nM) and imipenem (0.5 μg / ml) or 300 μl of imipenem alone (control condition) was injected into various channels of a flow chamber. The treatment of the pre-formed biofilms was carried out at 37 °C for 2 hours under static conditions. After 2 hours, the flow of the medium was applied again for 15 minutes to remove any cells that might have been detached from the biofilm under the action of the treatment.

[0221] As described in Example 1, the biofilms were stained with 5 μM Syto9 green fluorescent dye (Invitrogen) for 15 minutes and then observed under a confocal laser scanning microscope.

[0222] Results We studied the potential antibiofilm activity of the combination of ANP (10 -8 M; 30 ng / ml) + imipenem (0.5 μg / ml) against established biofilms of P. aeruginosa.

[0223] The biovolume of the biofilms was determined and the results are listed in the following table.

[0224] [Table 12]

[0225] Under these conditions, the inventors observed that the biofilm was more strongly dispersed by the combination of ANP + imipenem (0.5 μg / ml) compared to treatment with imipenem antibiotic alone or ANP treatment alone.

[0226] Therefore, the combination with ANP can result in 97.9% destruction of the biofilm when combined with an imipenem concentration of 0.5 μg / ml.

[0227] (Example 10) Effect of the combination of ANP and polymyxin B on pre - formed biofilms of P. aeruginosa This example demonstrates the synergistic effect of the combination of ANP + polymyxin B on pre - formed biofilms of P. aeruginosa.

[0228] Materials and methods To study the effect of ANP combined with polymyxin B on 24 - hour pre - formed biofilms (as described in Example 1), a mixture of 300 μl of ANP (10 nM) and polymyxin B (4 μg / ml) or 300 μl of polymyxin B alone (control condition) was injected into various channels of the flow chamber. The treatment of the pre - formed biofilm was carried out at 37 °C for 2 hours under static conditions. After 2 hours, the flow of the medium was applied again for 15 minutes to remove any cells that might have detached from the biofilm under the action of the treatment.

[0229] As described in Example 1, the biofilm was stained with 5 μM Syto9 green fluorescent dye (Invitrogen) for 15 minutes and then observed under a confocal laser scanning microscope.

[0230] Results The inventors studied the potential antibiofilm activity of the combination of ANP (10 -8 nM; 30 ng / ml) + polymyxin B (4 μg / ml) against established biofilms of P. aeruginosa.

[0231] Determine the biovolume of the biofilm and list the results in the following table.

[0232] [Table 13]

[0233] Under these conditions, the inventors observed that the biofilm was more strongly dispersed by the combination of ANP + polymyxin B (4 μg / ml) compared to treatment with polymyxin B antibiotic alone or ANP treatment alone.

[0234] Therefore, the combination with ANP can result in the destruction of 83.5% of the biofilm when combined with a polymyxin B concentration of 4 μg / ml.

[0235] (Example 11) Effect of consecutive treatment with tobramycin following ANP on pre-formed biofilms of P. aeruginosa In this example, the continuous action of treatment with tobramycin following treatment with ANP on pre-formed biofilms of P. aeruginosa is shown.

[0236] Materials and methods The materials and methods used in this example are the same as those described above in Example 1, with the following specified.

[0237] To study the effect of sequential exposure of ANP followed by tobramycin on 24-hour pre-formed biofilms (as described in Example 1), 300 μl of ANP (1 nM) (control condition) was injected into two channels of the flow chamber. The biofilm treatment was carried out at 37 °C for 2 hours under static conditions. After 2 hours, the flow of the medium was applied again for 15 minutes to remove any cells that might have been detached from the biofilm under the action of the treatment. Next, 300 μl of tobramycin (50 μg / ml) or 300 μl of milliQ sterilized water (control condition) was injected into two channels of the flow chamber pre-exposed to ANP. The second treatment of the pre-formed biofilm was carried out again at 37 °C for 2 hours under static conditions. After 2 hours, the flow of the medium was applied again for 15 minutes to remove any cells that might have been detached from the biofilm under the action of the treatment.

[0238] As described in Example 1, the biofilm was stained with 5 μM of Syto9 green fluorescent dye (Invitrogen) for 15 minutes and then observed under a confocal laser scanning microscope.

[0239] Results We studied the potential antibiofilm activity of sequential exposure of ANP (10 -9 M; 3 ng / ml) followed by tobramycin (50 μg / ml) on established biofilms of P. aeruginosa.

[0240] The biovolume of the biofilm was determined and the results are listed in the following table.

[0241] [Table 14]

[0242] Under these conditions, we observed that the biofilm was more strongly dispersed by two consecutive treatments of ANP (10 -9 M) (2 hours) followed by tobramycin (50 μg / ml) (2 hours) compared to treatment with ANP alone.

[0243] Therefore, continuous treatment with ANP (2 hours) followed by tobramycin (2 hours) can result in the destruction of 94.7% of the biofilm.

Claims

A pharmaceutical composition for the therapeutic treatment of Pseudomonas aeruginosa infection, comprising the ANP peptide of SEQ ID NO: 1 and an antibiotic, wherein the strain of Pseudomonas aeruginosa is sensitive to the antibiotic. Claim 2 (A) the ANP peptide of SEQ ID NO: 1, and (B) an antibiotic, for simultaneous, separate, or sequential use in the therapeutic treatment of Pseudomonas aeruginosa infection in a subject by a strain of Pseudomonas aeruginosa that is sensitive to the antibiotic.

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