OSTEOCRIN, LEBETIN OR ANP FOR THE DESTRUCTION OF BACTERIAL BIOFILMS

Description

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

[0002] Antibiotic resistance in bacteria is largely attributed to their ability to transition from a planktonic to a communal lifestyle, known as a biofilm, within the tissues of an infected host. The biofilm allows bacteria to protect themselves against antimicrobial molecules and the host's immune defenses, thus enabling pathogens to survive in hostile environments by conferring resistance to various antibacterial agents. Furthermore, a small percentage of bacterial cells, referred to as "persistent cells," remain within a biofilm exposed to antibiotics, thereby becoming temporarily resistant and responsible for the recurrence of infections.

[0003] The opportunistic pathogen Pseudomonas aeruginosais a major cause of mortality in immunocompromised patients, particularly in patients with cystic fibrosis.

[0004] The treatment of severe infections with P. aeruginosa This treatment is carried out using several antibiotics, the most effective of which (tobramycin, colistin, ciprofloxacin, gentamicin, netilmicin, and amikacin) have a narrow therapeutic index due to nephrotoxicity and ototoxicity. Furthermore, these treatments can lead to the development of antibiotic-resistant strains over time.

[0005] That is why P. aeruginosa has been classified in the ESKAPE group of bacteria (Santajit and Indrawattana (2016) Biomed. Res. Int. 2016:2475067), which includes the most critical pathogens in terms of antibiotic resistance and requiring the discovery of new molecules or therapeutic strategies. More recently, the WHO has classified P. aeruginosain the top 3 priorities in terms of critical bacteria for their antibiotic resistance (to carbapenems in particular).

[0006] Lesouhaitier et al. (2019, J. Innate Immun, 11:227-241) describe peptide hormones capable of modifying bacterial biofilm formation, as well as their mechanisms of action.

[0007] The present invention aims to address this need.

[0008] The present invention resulted from the unexpected discovery by the inventors that the hormone " Atrial Natriuretic Peptide or ANP (“Atrial Natriuretic Peptide” (in English) is capable, at low doses (from 0.1 nM), and very rapidly (from 30 min after administration), of destroying approximately 80% of biofilms P. aeruginosapre-established and in a dose-dependent manner. The inventors also demonstrated that other peptides belonging to the natriuretic peptide family, particularly osteocrine, lebetin 2α, and lebetin 1β, were also capable, at low doses, of destroying biofilms of P. aeruginosa pre-established.

[0009] Natriuretic peptides are a family of hormones initially identified for their cardiovascular, osmoregulatory, blood pressure regulation, and renal sodium excretion (natriuresis, hence their name) activities. The three main members of this family are ANP, the peptide " Brain Natriuretic Peptide or BNP and the peptide " C-type Natriuretic Peptide or CNP. The length of these peptides is between 22 and 38 amino acids and their structure is characterized, for most members, by a disulfide bridge forming a loop of 17 amino acids giving this molecule a so-called "omega" shape.

[0010] It has previously been shown that ANP, BNP, and CNP prevented biofilm formation. 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, no action of these peptides on already established biofilms has been shown to date.

[0012] The inventors have further demonstrated that ANP's ability to disperse already established biofilms is not associated with, or due to, ANP's ability to kill bacteria and has no effect on the intrinsic virulence of the bacteria. This particular mode of action prevents the emergence of bacterial strains resistant to this molecule.

[0013] Furthermore, the inventors have shown that when ANP, at low doses, is used in combination with an antibiotic, such as tobramycin or ciprofloxacin, also used at low doses, more than 97% of the established biofilm is destroyed.

[0014] Thus, ANP and antibiotics have a synergistic effect on the destruction of bacterial biofilms.

[0015] The invention is as defined in the set of claims.

[0016] The present invention therefore relates to a natriuretic peptide selected from the group consisting of (i) osteocrine, an osteocrine propeptide or an osteocrine derivative, (ii) lebetin, a lebetin fragment or a lebetin derivative or a lebetin fragment, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative, for its use in a method of therapeutic treatment of a bacterial infection, in particular a chronic bacterial infection, associated with a bacterial biofilm in a subject, in which said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, disperses said bacterial biofilm, in which the osteocrine derivative is an osteocrine in which one or more amino acid residues have been chemically modified or a peptide variant of osteocrine,wherein the lebetin derivative or lebetin fragment is lebetin or a lebetin fragment in which one or more amino acid residues have been chemically modified, or a peptide variant of lebetin or a lebetin fragment, and wherein said ANP peptide derivative is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full-length ANP form, a form having a structure that is an antiparallel dimer of ANP, a polymer consisting of ANP monomers, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted, or added, an ANP peptide in which the Phe amino acid of the loop has been substituted at position 8, an ANP peptide in which the Phe amino acid has been substituted at position 26 or the Ser amino acid at position 25, urodilatin, fsANP or a linearized version of ANP with disulfide bridge cleavage.

[0017] In a particular embodiment, said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, is used in combination with an antibiotic.

[0018] Another object of the invention relates to a pharmaceutical composition comprising (A) a natriuretic peptide selected from the group consisting of a lebetin, a lebetin fragment and a lebetin derivative or a lebetin fragment, and (B) an antibiotic, wherein the lebetin derivative or lebetin fragment is a lebetin or a lebetin fragment in which / of which one or more amino acid residues have been chemically modified or a peptide variant.

[0019] The application also describes a pharmaceutical composition comprising (A) a natriuretic peptide selected from the group consisting of (i) osteocrine, an osteocrine propeptide or an osteocrine derivative, (ii) lebetin, a lebetin fragment or a lebetin derivative or a lebetin fragment, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative, and (B) an antibiotic, for use in a method of therapeutic treatment of a bacterial infection, in particular a chronic bacterial infection, associated with a bacterial biofilm in a subject.

[0020] Another object of the invention relates to an antibacterial pharmaceutical combination comprising (A) a natriuretic peptide selected from the group consisting of (i) osteocrine, an osteocrine propeptide or an osteocrine derivative, (ii) lebetin, a lebetin fragment or a lebetin derivative or a lebetin fragment, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative, and (B) an antibiotic for simultaneous, separate or sequential use in the therapeutic treatment of a bacterial infection, in particular a chronic bacterial infection, associated with a bacterial biofilm in a subject, in which said natriuretic peptide disperses said bacterial biofilm, wherein the osteocrine derivative is an osteocrine in which one or more amino acid residues have been chemically modified or a peptide variant of osteocrine, wherein the lebetin derivative or lebetin fragment is a lebetin or a lebetin fragment in which one or more amino acid residues have been chemically modified or a peptide variant of lebetin or a lebetin fragment, and wherein said ANP peptide derivative is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full-length ANP form, a form having a structure that is an antiparallel dimer of ANP, a polymer consisting of ANP monomers, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted, or added, an ANP peptide in which the Phe amino acid of the loop at position 8 has been substituted, an ANP peptide in which the amino acid Phe has been substituted at position 26 or the amino acid Ser at position 25,Urodilatine, fsANP, or a linearized version of ANP with disulfide bridge cleavage.

[0021] The present invention also relates to the use in vitro Or ex vivoof a natriuretic peptide selected from the group consisting of (i) osteocrine, an osteocrine propeptide or an osteocrine derivative, (ii) lebetin, a lebetin fragment or a lebetin derivative or a lebetin fragment, and (iii) an ANP peptide, an ANP propeptide or an ANP peptide derivative, for dispersing a bacterial biofilm, wherein the osteocrine derivative is an osteocrine in which one or more amino acid residues have been chemically modified or a peptide variant of osteocrine, wherein the lebetin derivative or a lebetin fragment is a lebetin or a lebetin fragment in which / of which one or more amino acid residues have been chemically modified or a peptide variant of lebetin or a lebetin fragment, and wherein said ANP peptide derivative is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the complete form of ANP, a form having a structure that is an antiparallel dimer of ANP,a polymer consisting of ANP monomers, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted or added, an ANP peptide in which the Phe amino acid of the loop has been substituted at position 8, an ANP peptide in which the Phe amino acid has been substituted at position 26 or the Ser amino acid at position 25, urodilatine, fsANP or a linearized version of ANP with disulfide bridge cleavage. Detailed description of the invention Natriuretic peptides

[0022] By "natriuretic peptide", we mean here a member of the family of eukaryotic hormones and neurohormones composed of 3 main members: atrial natriuretic peptide (" Atrial Natriuretic Peptide " or ANP, the B-type natriuretic peptide (" Brain Natriuretic Peptide " or BNP and C-type natriuretic peptide (" C-type Natriuretic Peptide" or CNP. These peptides are primarily synthesized and released by cardiomyocytes and endothelial cells. Besides these three main members, the natriuretic peptide family also includes osteocrine and "natriuretic-like" peptides such as lebetin.

[0023] These various natriuretic peptides exhibit a certain similarity in terms of sequence. All, except osteocrine and lebetin 1β, possess a 17-amino-acid loop formed by a disulfide bridge. Of the 17 amino acids that make up this loop, 11 are common to ANP and lebetin 2α. Osteocrine also has a conserved sequence signature in this region, with 7 amino acids common to ANP. This conserved region of the natriuretic peptide sequence allows their binding to human natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C). Furthermore, ANP, osteocrine, and lebetin 2α have been shown to bind with high affinity to the AmiC sensor of P. aeruginosa (Rosay et al. (2015) mBio 25:e01033-15). In contrast, many differences in the amino acid sequence of their C and N terminal ends are observed, explaining their different physiological activities.It is interesting to note that, in addition to the active end products presented below, the synthesis, maturation, and partial degradation of natriuretic peptides can lead to the production of numerous variants possessing physiological activity. Many members of this family exist in humans and animals: urodilatin, described below, uroguanylin, guanylin, VNP, DNP, and KNP.

[0024] Ventricular natriuretic peptide (VNP) was initially identified in eel as a peptide with physiological functions similar to those of ANP and has since been identified in many fish species (Kawakoshi et al. (2004) J. Mol. Endocrinol. 32:547-555).

[0025] Dendroapsis natriuretic peptide (DNP) has been identified in the venom of the green mamba snake (Schweitz et al. (1992) J. Biol. Chem. 267:13928-13932) and Krait natriuretic peptide (KNP) has been identified in the venom of the Bungarus flaviceps snake (Sridharan et al. (2015) Biochem. J. 469:255-266).

[0026] Numerous other compounds similar to natriuretic peptides are routinely identified in viper venoms, such as lebetins isolated from the venom of Macrovipera lebetina, large reptiles such as Komodo dragons (Natriuretic peptide toxin Var2) (Fry et al. (2009) Proc. Natl. Acad. Sci. USA 106:8969-8974) and, similarly, in scorpion venom (Alves et al. (2013) Toxicon 74:19-26). They are generally called peptides with natriuretic-like activity. Among these, the preferred natriuretic peptides are the L2α (Gly1-Gly38) and L1β (Asp2-Gly13) lebetins. Finally, it is described in bacteria Escherichia colithe presence of an enterotoxin called ST (“heat-stable enterotoxin”) which has a peptide structure similar to human guanylin and uroguanylin and which exhibits natriuretic-like activity.

[0027] The natriuretic peptide used in the context of the invention is selected from the group consisting of (i) osteocrine, an osteocrine propeptide or an osteocrine derivative, (ii) lebetin, a lebetin fragment or a lebetin derivative or a lebetin fragment, and (iii) an ANP peptide, an ANP propeptide or an ANP peptide derivative.

[0028] By "ANP", we mean here the peptide corresponding to the main mature form resulting from the cleavage of the N-terminal fragment of the pro-hormone proANP.

[0029] Typically, human ANP is a 28-amino-acid peptide derived from the cleavage of the N-terminal fragment of the 126-amino-acid pro-hormone proANP. Human ANP is composed of amino acids 99 to 126 of the pro-hormone proANP.

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

[0031] By "ANP propeptide" we mean here any peptide resulting from the expression of the gene encoding preproANP, from the maturation and possibly successive cleavages of preproANP, and comprising the peptide sequence of ANP.

[0032] The term "ANP propeptide" thus includes the prohormone proANP (typically 126 amino acids in humans), which carries ANP in its C-terminal region, and the preprohormone preproANP (typically 151 amino acids in humans), which corresponds to the protein produced in humans by the NPPA gene. PreproANP carries a signal peptide that is cleaved to form proANP.

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

[0034] The 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] The proANP typically consists of the amino acid sequence NPMYNAVSNADLMDFKNLLDHLEEKMPLEDEVVPPQVLSEPNEEAGAALSPLPEVPPWT GEVSPAQRDGGALGRGPWDSSDRSALLKSKLRALLTAPRSLRRSSCFGGRMDRIGAQS GLGCNSFRY (SEQ ID NO: 3).

[0036] PreproANP typically consists of the amino acid sequence

[0037] By "ANP derivative" we mean natural or synthetic derivatives of ANP.

[0038] By "ANP derivative" we mean here an ANP peptide as defined above in which one or more amino acid residues have been chemically modified, for example by alkylation, acylation, amidation, methoxylation, ester formation, amide formation, insertion of a lipophilic substituent, addition of polyethylene glycol (PEG), addition of a carbohydrate chain.

[0039] By "ANP derivative" we also mean here truncated forms of ANP corresponding to amino acids 4 or 5 to 28 of the full form of ANP, forms having a structure that is an antiparallel dimer of ANP, in particular the β-ANP homodimer, a polymer made up of ANP monomers.

[0040] By "ANP derivative" we also mean peptide variants of ANP, i.e. intermediate forms obtained during the synthesis, maturation and partial degradation of ANP as defined above, as well as ANP peptides in which 1 to 12, in particular 1 to 10, in particular 1 to 5, in particular 1 to 4, in particular 1 to 3, more particularly 1 or 2 amino acids have been deleted, substituted or added and retaining the activity of wild ANP.

[0041] In a particular embodiment, the ANP derivative is an ANP peptide in which the amino acid Phenylalanine (Phe) of the loop has been substituted at position 8 (and replaced by an amino acid Alanine for example).

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

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

[0044] In another particular embodiment, the ANP derivative is an elongated form of the 12-amino-acid ANP peptide, such as fsANP, which is an elongated form of ANP with 12 amino acids on the C-terminal side and is present in individuals carrying 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 linearized version of ANP, with cleavage of the disulfide bridge (initially located between amino acids 7 and 23). Typically, the ANP derivatives that can be used in the context of the present invention are preferably modified compared to ANP so as to have a longer half-life than ANP and / or to have a lower affinity than ANP for endogenous human ANP receptors.

[0046] By "osteocrine" we mean here a hormone acting as a regulator of dendritic growth in the developing cerebral cortex in response to a sensory experience.

[0047] Typically, human osteocrine is a 50-amino-acid peptide derived from the cleavage of the N-terminal fragment of the 106-amino-acid pro-osteocrine prohormone. Human osteocrine is composed of amino acids 83 to 132 of the pro-osteocrine prohormone.

[0048] The term "osteocrine propeptide" refers to the 106-amino-acid pro-osteocrine prohormone, which carries osteocrine in its C-terminal region, and / or the 132-amino-acid prepro-osteocrine pre-hormone, which corresponds to the protein produced in humans by the OSTN gene. Prepro-osteocrine carries a signal peptide that is cleaved to form the 106-amino-acid pro-osteocrine.

[0049] Osteocrine typically consists of the amino acid sequence SFSGFGSPLDRLSAGSVDHKGKQRKVVDHPKRRFGIPMDRIGRNRLSNSR (SEQ ID NO: 5).

[0050] The pro-osteocrine typically consists of the amino acid sequence VDVTTTEAFDSGVIDVQSTPTVREEKSATDLTAKLLLLDELVSLENDVIETKKKRSFSGFG SPLDRLSAGSVDHKGKQRKVVDHPKRRFGIPMDRIGRNRLSNSRG (SEQ ID NO: 6).

[0051] The prepro-osteocrine typically consists of the amino acid sequence MLDWRLASAHFILAVTLTLWSSGKVLSVDVTTTEAFDSGVIDVQSTPTVREEKSATDLTA KLLLLDELVSLENDVIETKKKRSFSGFGSPLDRLSAGSVDHKGKQRKVVDHPKRRFGIPM DRIGRNRLSNSRG (SEQ ID NO: 7).

[0052] In the context of the present invention, the term "osteocrine propeptide" also includes any peptide resulting from the maturation and cleavage of prepro-osteocrine and pro-osteocrine.

[0053] By "osteocrine derivative" we mean here natural or synthetic derivatives of osteocrine.

[0054] By "osteocrine derivative" we mean here an osteocrine as defined above in which one or more amino acid residues have been chemically modified, for example by alkylation, acylation, amidation, methoxylation, ester formation, amide formation, insertion of a lipophilic substituent, addition of polyethylene glycol (PEG), addition of a carbohydrate chain.

[0055] By "osteocrine derivative", we also mean osteocrine peptide variants, that is, intermediate forms obtained during the synthesis, maturation and partial degradation of osteocrine as defined above.

[0056] By "lebetin," we mean here a peptide derived from viper venom. Macrovipera lebetina.

[0057] Lebetin as defined above is preferably lebetin 2α or lebetin 1β.

[0058] "Lebetin 2α" refers here to a peptide produced by the viper. Macrovipera lebetinadescribed in Barbouche et al. (1996) FEBS Lett. 392:6-10.

[0059] Typically, lebetin 2α is a peptide of 38 amino acids.

[0060] Lebetin 2α typically consists of the amino acid sequence GDNKPPKKGPPNGCFGHKIDRIGHSHSGLGCNKVDDNKG (SEQ ID NO: 8) with a disulfide bridge located between amino acids 14 and 30.

[0061] "Lebetin 1β" refers here to a peptide produced by the viper. Macrovipera lebetina described in Barbouche et al. (1998) Toxicon 36(12):1939-47.

[0062] Typically, lebetin 1β is a peptide of 12 amino acids.

[0063] Lebetin 1β typically consists of the amino acid sequence DNKPPKKGPPNG (SEQ ID NO: 9).

[0064] By "lebetin fragment" we mean here a fragment of at least 10 amino acids of said lebetin, preferably of lebetin 2α as defined above or of lebetin 1β as defined above.

[0065] The said lebetine fragment preferably comprises 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 at most 30 amino acids, preferably at most 25 amino acids, preferably at most 20 amino acids.

[0066] A lebetin 2α fragment includes, for example, amino acids 4 to 10 of the sequence SEQ ID NO: 8, preferably amino acids 2 to 13 of the sequence SEQ ID NO: 8.

[0067] A fragment of lebetin 2α consists, for example, of amino acids 1 to 13 of the sequence SEQ ID NO: 8.

[0068] Lebetin 1β of sequence SEQ ID NO: 9 is also a fragment of lebetin 2α consisting of amino acids 2 to 13 of sequence SEQ ID NO: 8.

[0069] By "lebetine derivative or lebetine fragment", we mean here natural or synthetic derivatives of lebetine or a lebetine fragment.

[0070] By "lebetine derivative or lebetine fragment" is meant herein a lebetine as defined above or a lebetine fragment as defined in which / which one or more amino acid residues have been chemically modified, for example by alkylation, acylation, amidation, methoxylation, ester formation, amide formation, insertion of a lipophilic substituent, addition of polyethylene glycol (PEG), addition of a carbohydrate chain.

[0071] By "lebetin derivative or lebetin fragment", we also mean peptide variants of lebetin or a lebetin fragment, i.e. intermediate forms obtained during the synthesis, maturation and partial degradation of lebetin or a lebetin fragment as defined above.

[0072] By "other natriuretic peptide" we mean here a natriuretic peptide that is not ANP, an ANP propeptide or an ANP derivative (such as urodilatin). Bacterial biofilm and bacterial infection

[0073] By “bacterial infection” we mean here an infection due to one or more species of bacteria, in particular Gram-negative and / or Gram-positive bacteria.

[0074] In one particular embodiment, the bacterial infection is a chronic bacterial infection.

[0075] In one particular embodiment, bacterial infection is an infection with a Gram-negative bacterium.

[0076] In one particular embodiment, bacterial infection is an infection with a bacterium of the genus Pseudomonas, more specifically of the species Pseudomonas aeruginosa.

[0077] In another particular embodiment, bacterial infection is an infection with a Gram-positive bacterium.

[0078] In one particular embodiment, bacterial infection is an infection with a bacterium of the genus Staphylococcusmore specifically of the species Staphylococcus aureus.

[0079] The term "bacterial biofilm" refers to a community of bacteria adhering to each other and to a surface. Biofilm production is characterized by the secretion of a matrix that can adhere to many types of surfaces, providing cohesion and protection for the bacteria that make up this structure.

[0080] In the context of the invention, the bacterial biofilm comprises at least the bacterial species responsible for the infection to be treated.

[0081] Within the framework of the invention, said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, disperses the bacterial biofilm.

[0082] By “dispersion of bacterial biofilm” or “destruction of bacterial biofilm,” we mean that the bacteria forming the community constituting the bacterial biofilm separate from each other and from the surface, so that the biofilm diminishes or even disappears, typically making the bacteria accessible to antibiotics again.

[0083] In a particular embodiment, said bacterial biofilm is dispersed at least 50%, in particular at least 55%, in particular at least 60%, in particular at least 65%, in particular at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%.

[0084] Advantageously, said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, disperses the bacterial biofilm without killing the bacteria constituting this biofilm.

[0085] It should be noted, however, that when said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, is used in combination with an antibiotic, the bacteria constituting the biofilm may be killed by said antibiotic, particularly when they are released following the dispersive effect of the natriuretic peptide, in particular ANP, an ANP propeptide or an ANP derivative. Therapeutic application

[0086] The present invention relates to a natriuretic peptide selected from the group consisting of (i) osteocrine, an osteocrine propeptide or osteocrine derivative, (ii) lebetin (preferably lebetin 2α or lebetin 1β), a lebetin fragment or a lebetin derivative or a lebetin fragment, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative, wherein the osteocrine derivative is an osteocrine in which one or more amino acid residues have been chemically modified or a peptide variant of osteocrine, wherein the lebetin derivative or a lebetin fragment is a lebetin or a lebetin fragment in which / of which one or more amino acid residues have been chemically modified or a peptide variant of lebetin or a lebetin fragment, and wherein said derivative of ANP peptide is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full-length ANP form.a form having a structure that is an antiparallel dimer of ANP, a polymer consisting of ANP monomers, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted or added, an ANP peptide in which the Phe amino acid of the loop has been substituted at position 8, an ANP peptide in which the Phe amino acid has been substituted at position 26 or the Ser amino acid at position 25, urodilatine, fsANP or a linearized version of ANP with disulfide bridge cleavage for its use in a therapeutic treatment method for a bacterial infection associated with a bacterial biofilm, as defined in section , "Bacterial biofilm and bacterial infection" above in a subject, in which said natriuretic peptide disperses said bacterial biofilm.

[0087] By "subject" we mean here a human or an animal such as a non-human mammal, a bird or a fish.

[0088] In one particular embodiment, the subject suffers from a chronic respiratory condition, such as cystic fibrosis, chronic obstructive pulmonary disease, or bronchiectasis. Preferably, the subject suffers from cystic fibrosis.

[0089] In another particular embodiment, the subject suffers from severe burns (major burn) or a surface infection.

[0090] In another embodiment, the subject wears an implant and / or a prosthesis.

[0091] In a particular embodiment, said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, is used in combination with an antibiotic.

[0092] By "antibiotic" we mean here a natural or synthetic substance that destroys or blocks the growth of bacteria.

[0093] Antibiotics include: Antibiotics that inhibit bacterial envelope synthesis, including: ∘ beta-lactams, in particular: ▪ penicillins, including group A penicillins such as amoxicillin; group G and V penicillins such as benzathine benzylpenicillin, benzathine penicillin, benzathine phenoxymethylpenicillin, penicillin G and penicillin V; group M penicillins such as cloxacillin and oxacillin; carboxypenicillins such as ticarcillin; ureidopenicillins such as piperacillin; aminidopenicillins such as pivmecillinam; and temocillin; ▪ carbapenems such as ertapenem, imipenem and meropenem; ▪ Monobactams such as aztreonam; ▪ Cephalosporins, including first-generation cephalosporins such as cefaclor, cefadroxil, cefalexin, cefalotin, cefazolin and cefradine;Second-generation cephalosporins such as cefamandole, cefoxitin, cefuroxime sodium, and cefuroxime axetil; and third-generation cephalosporins such as cefixime, cefpodoxime proxetil, cefodiam hexetil, cefepime, cefotaxime, cefpirome, ceftazidime, and ceftriaxone; fosfomycins such as fosfomycin and fosfomycin trometamol; glycopeptides such as teicoplanin and vancomycin; lipopeptides such as daptomycin; and polymyxins such as polymyxin B and polymyxin E or colistin; protein synthesis inhibitor antibiotics, including: ∘ aminoglycosides such as amikacin sulfate, gentamicin, neomycin, netilmycin, spectinomycin, streptomycin and tobramycin;• Macrolides and related drugs, in particular: • True macrolides such as amphotericin B, azithromycin, clarithromycin, erythromycin, josamycin, midecamycin, and roxithromycin; • Lincosamides such as clindamycin and lincomycin; • Ketolides such as telithromycin; and • Synergistins such as pristinamycin; • Phenicoles such as thiamphenicol; • Tetracyclines such as chlortetracycline, doxycycline, lymecycline, methylenecycline, minocycline, and tigecycline; • Fusidic acids such as fusidic acid; and ∘ oxazolidinones such as linezolid and tedizolid; nucleic acid synthesis inhibitor antibiotics, including: ∘ quinolones such as pipemidic acid, flumequine, enoxacin, lomefloxacin, norfloxacin, ciprofloxacin, ofloxacin, pefloxacin, levofloxacin and moxifloxacin;• Quinoline antibiotics such as hydroxyquinoline; • Mupirocin; and • Rifamycin; antibiotics that inhibit folic acid synthesis, including sulfonamides such as sulfadiazine, sulfamethizole, sulfafueazole, and sulfamethoxazole; and antibiotics with complex or poorly understood mechanisms, including: • Nitrates, in particular: • Nitrofurans such as nitrofurantoin and nifuroxazide; and • Nitroimidazoles such as metronidazole, omidazole, and tinidazole; and • Ethambutol, isoniazid, pyrazinamide, rifabutin, and rifampicin.

[0094] In a particular embodiment, said 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 envelope 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 envelope, in particular a polymyxin such as polymyxin B or colistin.

[0095] In a particular embodiment, the antibiotic is an aminoglycoside antibiotic, a quinolone, a carbapenem, or a polymyxin. More specifically, the antibiotic may be an aminoglycoside antibiotic or a quinolone.

[0096] In another particular embodiment, said antibiotic is tobramycin, ciprofloxacin, imipenem and / or colistin.

[0097] More specifically, the antibiotic in question may be tobramycin and / or ciprofloxacin.

[0098] The inventors have shown that the combined use of the ANP peptide with these antibiotics has a synergistic effect on biofilm destruction. While not bound by the theory, it is hypothesized that the ANP peptide, by dispersing the bacteria constituting the biofilm, makes them more accessible to antibiotics, which are thus more effective.

[0099] In another particular embodiment, said natriuretic peptide is used in combination with at least one other natriuretic peptide as defined in section "Natriuretic peptides".

[0100] Thus, in a particular embodiment, when said natriuretic peptide is ANP, an ANP propeptide or ANP peptide derivative, it can be used in combination with osteocrine, osteocrine propeptide or osteocrine derivative, and / or with lebetin (preferably lebetin 2α or lebetin 1β), a lebetin fragment or a lebetin derivative or a lebetin fragment.

[0101] Alternatively, in another particular embodiment, when said natriuretic peptide is osteocrine, an osteocrine propeptide or osteocrine derivative, it may be used in combination with ANP, ANP propeptide or ANP peptide derivative and / or with lebetin (preferably lebetin 2α or lebetin 1β), a lebetin fragment or a lebetin derivative or a lebetin fragment.

[0102] Alternatively, in another particular embodiment, when said natriuretic peptide is lebetin (preferably lebetin 2α or lebetin 1β), a lebetin fragment or a derivative of lebetin or a lebetin fragment, it may be used in combination with ANP, ANP propeptide or ANP peptide derivative and / or with osteocrine, osteocrine propeptide or osteocrine derivative.

[0103] The combined use of several natriuretic peptides has the advantage of allowing the use of lower doses of each compound, thus limiting potential side effects even more effectively.

[0104] In this particular embodiment, said natriuretic peptide may further be used in combination with an antibiotic as described above, possibly in a sequential manner (said natriuretic peptide first, then the antibiotic).

[0105] In a particular embodiment, said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, is used in combination with any other suitable substance for the treatment of the subject suffering from bacterial infection as described above, possibly sequentially (said natriuretic peptide first, then the suitable substance).

[0106] In particular, when the subject suffers from a chronic respiratory disease, said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, is preferably used in combination with a substance suitable for the treatment of this chronic respiratory disease, possibly sequentially (said natriuretic peptide first, then the suitable substance).

[0107] Thus, in the embodiment in which said subject suffers from cystic fibrosis, said natriuretic peptide, in particular said ANP peptide, ANP propeptide or ANP peptide derivative, is preferably used in combination with a substance for mucociliary clearance, such as Dnase (for example Pulmozyme ®< ), and / or bronchodilator, possibly in a sequential manner (a substance for mucociliary clearance first, then said natriuretic peptide).

[0108] In these particular embodiments, said natriuretic peptide may further be used in combination with an antibiotic as described above and / or with another natriuretic peptide as described above, preferably with an antibiotic as described above.

[0109] In the context of the invention, the term "treatment" or "treat" means to reverse, relieve, or inhibit the progression of the disease to which this term applies, or one or more symptoms of that disease.

[0110] By "therapeutically effective quantity," we mean a sufficient amount of the compound of interest to disperse the bacterial biofilm in the treatment of bacterial infection, at a reasonable risk / benefit ratio applicable to any medical treatment. It should be clearly understood, however, that the total daily use of the compounds used in the context of this invention will be determined by the treating physician based on their medical judgment.The therapeutically effective dose level specific to a particular individual will depend on a number of factors, including the disease being treated and its severity, the activity of the specific compounds used, age, weight, general health, sex, and diet, the timing of administration, the route of administration and the rate of excretion of the specific compounds used, the duration of treatment, medications used in combination or concurrently with the specific compounds used, and similar factors well-known in the medical field. For example, it is well-known in the field to start compound doses at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is obtained.

[0111] The compounds used in the context of the invention can be administered in the form of a pharmaceutical composition comprising pharmaceutically acceptable excipients, and optionally delayed-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0112] The terms "pharmaceutical" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce adverse reactions, allergic reactions, or other adverse effects when administered to a mammal, particularly a human. A pharmaceutically acceptable vehicle or excipient is defined as a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating agent, or formulation aid of any kind.

[0113] The form of pharmaceutical compositions comprising the compounds used in the context of the invention and the route of administration will naturally depend on the disease to be treated, the severity of the disease, the age, weight and gender of the patient, etc.

[0114] The compounds used in the context of the invention can be formulated for administration by air or inhalation (in particular by nebulization), oral, parenteral, intranasal, intravenous, intramuscular, topical, subcutaneous or intraocular route, for example.

[0115] In a particular embodiment, the compounds used in the context of the invention are administered by airborne or inhalation route (in particular by nebulization).

[0116] In a particular embodiment, the natriuretic peptide, in particular the ANP peptide (or propeptide or derivative), used in the context of the invention is administered at a daily dose of 0.3 ng / ml to 3,000 ng / ml.

[0117] In another particular embodiment, the antibiotic used in combination is administered at a maximum daily dose of 600 mg (two times 300 mg per day). Pharmaceutical composition and pharmaceutical combination

[0118] The present invention also relates to a pharmaceutical composition comprising (A) a natriuretic peptide selected from the group consisting of a lebetin (preferably lebetin 2α or lebetin 1β), a lebetin fragment or a derivative of lebetin or a lebetin fragment and (B) an antibiotic as defined in section "Therapeutic application"above, wherein the lebetin derivative or lebetin fragment is a lebetin or lebetin fragment in which / which one or more amino acid residues have been chemically modified or a peptide variant.

[0119] In one particular embodiment, the composition according to the invention further comprises a pharmaceutically acceptable vehicle or excipient as defined above.

[0120] In another particular embodiment, the composition according to the invention further comprises another natriuretic peptide as defined in section " Natriuretic peptides " above.

[0121] The present invention also relates to an antibacterial pharmaceutical combination comprising (A) a natriuretic peptide selected from the group consisting of (i) osteocrine, osteocrine propeptide or osteocrine derivative, (ii) lebetin (preferably lebetin 2α or lebetin 1β), a lebetin fragment or a lebetin derivative or a lebetin fragment, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative as defined in section " Natriuretic peptides above and (B) an antibiotic as defined in section "Therapeutic application" above for simultaneous, separate or sequential use in the therapeutic treatment in a subject as defined above of a bacterial infection associated with a bacterial biofilm as defined in section "Bacterial biofilm and bacterial infection" above, in which said natriuretic peptide disperses said bacterial biofilm, wherein the osteocrine derivative is an osteocrine in which one or more amino acid residues have been chemically modified or a peptide variant of osteocrine, wherein the lebetin derivative or lebetin fragment is a lebetin or a lebetin fragment in which one or more amino acid residues have been chemically modified or a peptide variant of lebetin or a lebetin fragment, and wherein said ANP peptide derivative is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full-length ANP form, a form having a structure that is an antiparallel dimer of ANP, a polymer consisting of ANP monomers, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted, or added, an ANP peptide in which the Phe amino acid of the loop at position 8 has been substituted, an ANP peptide in which the amino acid Phe has been substituted at position 26 or the amino acid Ser at position 25,Urodilatine, fsANP, or a linearized version of ANP with disulfide bridge cleavage.

[0122] In the context of the invention, the term "combination" or "pharmaceutical combination" defines either a fixed combination in a single dosage unit form, or a kit for combined administration in which the natriuretic peptide and the antibiotic can be administered independently at the same time or separately at time intervals that allow the partners in the combination to show a synergistic effect.

[0123] The compounds of the combination of the invention can therefore be formulated in one or two separate pharmaceutical combinations, each composition being for the same route of administration or for different routes of administration.

[0124] In a particular embodiment, said pharmaceutical combination according to the invention further comprises (C) another natriuretic peptide as defined in section " "Natriuretic peptides" and / or (D) another substance suitable for the treatment of the subject suffering from the bacterial infection as defined in section "Therapeutic application" above.

[0125] In this particular embodiment, the combination may be a fixed combination in a single dosage unit form, or a kit for combined administration in which the natriuretic peptide, the antibiotic, the other natriuretic peptide and / or the other substance suitable for the treatment of the subject, may be administered independently at the same time or separately at time intervals which allow the partners of the combination to show a synergistic effect.

[0126] In this embodiment, the compounds of the combination can therefore be formulated in one, two, three or four separate pharmaceutical combinations, each composition being for the same route of administration or for different routes of administration.

[0127] To prepare the pharmaceutical compounds used in the context of the invention, an effective amount of the compounds used in the context of the invention can be dissolved or dispersed in a pharmaceutically acceptable vehicle or aqueous medium.

[0128] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions, for example, in micronized form. In all cases, the form must be sterile and free-flowing to facilitate administration by syringe. It must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria, viruses, or fungi.

[0129] The vehicle can be a solvent or a dispersion medium comprising, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, or other), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining a required particle size in the case of a dispersion, and by using surfactants, stabilizing agents, cryoprotectants, or antioxidants. Prevention of microbial action can be achieved by antibacterial and antifungal agents. In many cases, it will be preferable to include isotonic agents, such as sugars or sodium chloride.

[0130] Sterile injectable solutions can be prepared by incorporating the active compounds into a required quantity of the appropriate solvent along with several other ingredients, followed by sterilization through filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other required ingredients. For sterile powders used in the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization, which produce a powder of the active ingredient plus any desired additional ingredients from a solution of these ingredients that has been previously sterilized by filtration.

[0131] For parenteral administration in an aqueous solution, for example, the solution should preferably be appropriately buffered if necessary, and the liquid diluent made isotonic with sufficient saline or glucose solution. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0132] The formulations of pharmaceutical compositions for administration by inhalation are well known to those skilled in the art. In general, the active ingredients are delivered as an aerosol spray from a metered-dose pressurized inhaler using a suitable propellant gas, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide, as a powder administered using a dry powder inhaler, or as an aqueous liquid aerosol using a nebulizer.Nebulizers for delivering a liquid aerosol can be categorized into jet nebulizers operated by a pressurized airflow using a portable compressor or a central air supplier in the hospital, ultrasonic nebulizers incorporating a piezo-crystal to provide the energy to generate the aerosol out of an ultrasonic fountain, and electronic nebulizers based on the principle of a perforated vibrating membrane. Non-therapeutic uses

[0133] The present invention also relates to the use in vitro Or ex vivoof a natriuretic peptide selected from the group consisting of (i) osteocrine, an osteocrine propeptide or osteocrine derivative, (ii) lebetin (preferably lebetin 2α or lebetin 1β), a lebetin fragment or a lebetin derivative or a lebetin fragment and (iii) an ANP peptide, an ANP propeptide or an ANP peptide derivative, for dispersing a bacterial biofilm, wherein the osteocrine derivative is an osteocrine in which one or more amino acid residues have been chemically modified or a peptide variant of osteocrine, wherein the lebetin derivative or lebetin fragment is lebetin or a lebetin fragment in which one or more amino acid residues have been chemically modified, or a peptide variant of lebetin or a lebetin fragment, and wherein said ANP peptide derivative is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full-length ANP form, a form having a structure that is an antiparallel dimer of ANP, a polymer consisting of ANP monomers, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted, or added, an ANP peptide in which the Phe amino acid of the loop has been substituted at position 8, an ANP peptide in which the Phe amino acid has been substituted at position 26 or the Ser amino acid at position 25, urodilatin, fsANP or a linearized version of ANP with disulfide bridge cleavage.

[0134] Natriuretic peptide, in particular ANP, ANP propeptide or ANP peptide derivative, can thus typically be used in antifouling applications (“ antifouling " , for example on boats or in pipelines.

[0135] This disclosure also describes a method, not covered by the claims, in vitro Or ex vivo of dispersing a bacterial biofilm on a surface, comprising the application to said surface of a composition comprising a natriuretic peptide selected from the group consisting of (i) osteocin, osteocrine propeptide or osteocrine derivative, (ii) lebetin (preferably lebetin 2α or lebetin 1β), a lebetin fragment or a lebetin derivative or a lebetin fragment, and (iii) an ANP peptide, an ANP propeptide or an ANP peptide derivative, as defined in section "Natriuretic peptides" above.

[0136] This surface can typically be the surface of a pipeline, the surface of a boat, or the surface of an implantable device prior to implantation.

[0137] The application of said composition to said surface can be carried out by any appropriate technique, depending on the formulation of the composition.

[0138] The present invention will be described in more detail by the examples and figures below. Brief description of the sequences

[0139] SEQ ID NO Description Sequence 1 ANP amino acid sequence 2 Amino acid sequence of urodilatin 3 proANP amino acid sequence 4 Amino acid sequence of preproANP 5 Osteocrine amino acid sequence 6 Pro-osteocrine amino acid sequence 7 Prepro-osteocrine amino acid sequence 8 Amino acid sequence of lebetin 2α 9 Amino acid sequence of lebetin 1β DNKPPKKGPPNG Brief description of the figures

[0140] There Figure 1 shows graphs representing the growth of P. aeruginosa in the presence of different concentrations of ANP. Examples Example 1 : Effect of ANP alone (0.1 µM) on forming biofilms, on preformed biofilms of P. aeruginosa and on the growth of P. aeruginosa.

[0141] This example shows the action of ANP on preformed biofilms of P. aeruginosa. Materials and methods Substances tested, bacterial strains and bacterial cultures

[0142] The strain of P. aeruginosa wild PA14 used was from Harvard Medical School (Noston, MA) (Liberati et al. (2006) Proc. Natl. Acad. Sci. USA 103:2833-2838).

[0143] The bacterial strains were cultured at 37°C in Luria Bertani (LB) medium under agitation. Formation of P. aeruginosa biofilm under dynamic conditions

[0144] After 3 hours of pre-culture in LB medium at 37°C, P. aeruginosa was inoculated at an OD 600 = 0.08 in LB medium and subcultured for 2 h.

[0145] ANP (Calbiochem Merck) was added 2 hours after the start of the culture, a time corresponding to the middle of the exponential growth phase of the bacteria. The final bacterial density and the absence of contamination were checked by inoculation.

[0146] Biofilms were formed under dynamic conditions at 37°C in a 3-channel flow chamber as described in Bazire et al. (2010) J. Bacteriol. 192:3001-3010. Briefly, from an 18-hour pre-culture, a bacterial suspension with an OD600 of 0.08 prepared in sterile physiological saline was injected into each channel of the flow chamber. The bacteria were left for 2 hours at 37°C under static conditions (without flow) to adhere to the glass slide. Biofilm formation was then studied under a flow of LB medium (2.5 ml / h) for 24 hours at 37°C. Destruction of P. aeruginosa biofilm under dynamic conditions

[0147] To study the impact of ANP on preformed biofilms over 24 h, 300 µl of ANP (0.1 µM) or 300 µl of sterile ultrapure water (control condition) were injected into different channels of the flow chamber. The preformed biofilm was treated for 2 h at 37°C under static conditions. After 2 h, the flow of medium was reapplied for 15 min to remove any cells that had detached from the biofilm during the treatment.

[0148] Biofilms were observed using confocal laser scanning microscopy (Zeiss LSM710 (Zeiss)) after being labeled for 15 min with 5 µM Syto9 green fluorochrome (Invitrogen). Images were acquired from the different layers of the biofilm, allowing for three-dimensional reconstruction; at least three images were taken from different points within the same channel of the flow chamber. The files from each image were then analyzed using COMSTAT software (Heydorn et al. (2000) Microbiology 146:2395-2407). Three images from at least three independent experiments were analyzed, allowing the determination of the mean and maximum thicknesses of the biofilms as well as the bacterial biovolume. Cell culture

[0149] The human type II A549 lung epithelial cell line (ATCC-CCL185TM, ATCC Manassas, VA) 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 routine practice, cells were seeded in a 25 mL flask and used at 80% confluence.

[0150] For cytotoxicity assays, cells were seeded in 24-well plates at a final density of 3 × 10⁵ cells per well and cultured for 48 h before use. A minimum of 24 h prior to infection assays, cells were deprived of antibiotics and fetal bovine serum by the addition of fresh serum-free medium. Measurement of cytosolic lactate dehydrogenase (LDH) release by A549 cells

[0151] LDH is a stable cytosolic enzyme released into the culture 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, exposed or not to ANP (concentration from 1 µM to 10 nM), was determined using the Cytotox 96 enzyme assay (Promega, Charbonnières, France). A549 cells were incubated for 6 h with a control (untreated) or with P. aeruginosaPA14 (pre-treated with ANP) at an infection multiplicity of 10. A lysis buffer, consisting of a 9% Triton X-100 solution in water, was used to determine the maximum LDH potentially released by A549 cells under experimental conditions (100% LDH release). A background level was established using the culture medium alone, and defined as 0% LDH release, to eliminate the contribution of the culture medium. The percentage of LDH release in the cell population was then calculated using the equation: %LDH = DO é chantillon DO 100 % × 100

[0152] The test was sensitive enough to measure an LDH concentration equivalent to 1% lysis of the cell population. Results Effect of ANP on a preformed P. aeruginosa biofilm

[0153] The inventors studied the potential anti-biofilm activity of ANP on an established biofilm of P. aeruginosa.With this in mind, after 24 hours of biofilm formation of P. aeruginosa In a dynamic flow system, the inventors exposed bacteria to 0.1 µM ANP for 2 hours. Under these conditions, the inventors observed biofilm dispersion that was disrupted by the ANP, and the mushroom-shaped structures observed under control conditions and characteristic of a biofilm of P. aeruginosa, were absent after exposure to ANP. Bacterial biomass was reduced after 2 hours of exposure to 0.1 µM ANP by 81.4 ± 4.1% (P < 0.001) compared to the biomass present in the control condition. In parallel, the inventors observed a significant reduction in biofilm thickness after 2 h of ANP exposure.

[0154] Similar results were obtained when the pre-formed biofilm was exposed to ANP for 30 minutes. Under these conditions, the inventors observed that the biofilm was strongly dispersed by the ANP, and the mushroom-shaped structures observed under control conditions were characteristic of a biofilm of P. aeruginosa, were absent after exposure to ANP. The remaining bacterial biomass was reduced after exposure to ANP (30 minutes) by 80.2 ± 2.0% (P < 0.05) compared to the biomass present under control conditions. Effect of ANP on the growth of P. aeruginosa

[0155] In order to determine a potential direct effect of ANP on the growth of P. aeruginosa, The inventors studied the impact of ANP at 1 µM and 0.1 µM on the growth of P. aeruginosa P14 in a liquid medium. None of the tested concentrations of ANP affected bacterial growth, as shown in Figure 1.

[0156] Thus, the effect of ANP on preformed biofilms cannot be attributed to an action on bacterial growth. Effect of ANP on bacterial cytotoxicity towards cultured human lung cells

[0157] The cytotoxic activity of P. aeruginosa exposure to different concentrations of ANP (1 µM to 10 nM) was studied using A549 lung cells. The inventors observed that ANP, at any concentration, had no impact on the cytotoxic activity of P. aeruginosa compared to lung cells, as shown in the table below. Experimental conditions % of LDH (dead cells) PA14 untreated 56,4 ± 5,5 PA14 + ANP (10⁻⁶ < M) 58,9 ± 5,8 PA14 + ANP (10⁻⁷ < M) 53,5 ± 5,3 PA14 + ANP (10⁻⁸ < M) 54,5 ± 5,5 Conclusion

[0158] The inventors have thus shown in this example that ANP, used at a concentration of 0.1 µM, completely prevents the formation of biofilm. P. aeruginosa and strongly destroys a pre-formed biofilm of P. aeruginosa.

[0159] For a long time, studies aimed at preventing biofilm formation focused on the initial stages of biofilm formation, including biofilm adhesion and maturation. The drug concentrations needed to inhibit biofilm formation are generally much lower than those required to destroy or disrupt a pre-formed biofilm.

[0160] The main advantage of ANP, as highlighted by the inventors, is that its use at a concentration of 0.1 µM for 2 hours or even 30 minutes is sufficient to disperse approximately 80% of the biofilm structure. This is particularly relevant, especially for use in synergy with antibiotics.

[0161] This effect of ANP on the biofilm of P. aeruginosais more important than the previously observed inhibitory effect on formation with CNP and is of additional interest because CNP slightly increased bacterial virulence and cytotoxicity by activating bacterial quorum-sensing systems.

[0162] Here, the inventors demonstrated that ANP, despite its strong anti-biofilm action, did not kill bacteria and did not increase cytotoxicity. P. aeruginosa compared to cultured human lung cells. The fact that the bacteria are not killed is particularly interesting because it prevents the emergence of resistant strains. Example 2 : Dose-dependent effect of ANP on preformed P. biofilms. aeruginosa

[0163] This example shows the action of ANP alone on preformed biofilms of P. aeruginosa depends on the dose used and is visible from 0.3 ng / ml. Materials and methods

[0164] The materials and methods used in this example are identical to those described above in Example 1. (Destruction of P. aeruginosa biofilm under dynamic conditions). Results

[0165] The inventors studied the potential anti-biofilm activity of ANP on an established biofilm of P. aeruginosa to different concentrations of ANP: 0.3 ng / ml, 3 ng / ml and 30 ng / ml and compared with that presented in example 1 ([ANP] = 300 ng / ml or 0.1 µM), after an exposure of 2 h or 30 min.

[0166] The inventors observed that the biofilm was strongly dispersed by ANP even at the lowest concentration of 0.3 ng / ml and after an exposure of 30 min.

[0167] The results obtained are described in the following table. Experimental conditions Biovolume (2h) (µm 3< / µm 2< ) % inhibition (2h) Biovolume (30 min) (µm 3< / µm 2< ) % inhibition (30 min) PA14 untreated 21,1 ¶ 22,19 ANP (0.3 ng / ml) 10,6 45,1 (**) 9,5 57,5 (*) ANP (3 ng / ml) 6,3 65,9 (***) 8,8 59 ,1 (*) ANP (30 ng / ml) 4,4 72,5 (***) 6,7 69,0 (*) ANP (300 ng / ml) 4,9 81,4 (***) 4,4 80,2 (*) ¶ : Average of all controls. * : p < 0.05 vs untreated biofilms; ** : p < 0.01 vs untreated biofilms; *** : p < 0.001 vs untreated biofilms.

[0168] This example therefore confirms the value of ANP, which is active at very low concentrations, and from 30 minutes of exposure. Example 3 : Effect of osteocrine on preformed biofilms of P. aeruginosa

[0169] This example shows the action of osteocrine alone on preformed biofilms of P. aeruginosa at a dose of 10⁻⁸< M. Materials and methods

[0170] The materials and methods used in this example are identical to those described above in Example 1. (Destruction of P. aeruginosa biofilm under dynamic conditions). Results

[0171] The inventors studied the potential anti-biofilm activity of osteocrine on an established biofilm of P. aeruginosa to the concentration of 10 -8< M and after an exposure of 2 h.

[0172] The inventors observed that the biofilm was strongly and significantly dispersed by osteocrine. Experimental conditions Biovolume (2h) (µm 3< / µm 2< ) % inhibition (2h) PA14 untreated (control) 29,8 Osteocrine (10⁻⁸< M) 12,1 59,4 (***) Example 4 : Effect of the Lebetin 2α peptide on preformed biofilms P. aeruginosa

[0173] This example shows the action of the lebetin L2 alpha peptide alone on preformed biofilms of P. aeruginosa to the dose of 10 -8< M. Materials and methods

[0174] The materials and methods used in this example are identical to those described above in Example 1, ( Destruction of P. aeruginosa biofilm under dynamic conditions ) . Results

[0175] The inventors studied the potential anti-biofilm activity of the lebetin L2 alpha peptide on an established biofilm of P. aeruginosa to the concentration of 10 -8< M and after an exposure of 2 h.

[0176] The inventors observed that the biofilm was strongly and significantly dispersed by the lebetin L2 alpha peptide. Experimental conditions Biovolume (2h) (µm 3< / µm 2< ) % inhibition (2h) PA14 untreated (control) 29,7 Lebetin L2alpha (10⁻⁸ < M) 8,9 70,2 (***) Example 5 : Effect of the Lebetin 1β peptide on preformed biofilms P. aeruginosa

[0177] This example shows the action of the lebetin L1 beta peptide alone on preformed biofilms of P. aeruginosa to the dose of 10 -8< M. Materials and methods

[0178] The materials and methods used in this example are identical to those described above in Example 1, ( Destruction of P. aeruginosa biofilm under dynamic conditions ) . Results

[0179] The inventors studied the potential anti-biofilm activity of the lebetin L1 Beta peptide on an established biofilm of P. aeruginosa to the concentration of 10 -8< M and after an exposure of 2 h.

[0180] The inventors observed that the biofilm was strongly dispersed by the lebetin L1 Beta peptide. Experimental conditions Biovolume (2h) (µm 3< / µm 2< ) % inhibition (2h) PA14 untreated (control) 21,0 Lébetin L1 Beta (10 -8< M) 6,9 67,0 Example 6: Effect of a combination of ANP and tobramycin on preformed biofilms of P. aeruginosa

[0181] This example demonstrates the synergistic action of the ANP + tobramycin combination on preformed biofilms of P. aeruginosa. Materials and methods

[0182] The equipment and methods used in this example are identical to those described above in Example 1, with the following clarifications: To study the impact of ANP in combination with tobramycin on 24-h preformed biofilms (as described in Example 1), 300 µl of a mixture of ANP (1 nM) and tobramycin (50 µg / ml) or 300 µl of tobramycin alone (control condition) were injected into different channels of the flow chamber. The preformed biofilm was treated for 2 h at 37°C under static conditions. After 2 h, the flow of medium was reapplied for 15 min to remove any cells that had detached from the biofilm during the treatment.

[0183] Biofilms were observed by confocal laser scanning microscopy as described in Example 1. To test the membrane integrity of bacteria, a mixture of 5 µM SYTO 9 green and 0.3 µM propidium iodide (IP) was used (Live / Dead BacLight Bacterial Viability Kit, Invitrogen). Results

[0184] The inventors studied the anti-biofilm activity potential of the combination ANP (at 10⁻⁹ < M; 3 ng / ml) + tobramycin (at 50 µg / ml or 10 µg / ml) on an established biofilm of P. aeruginosa. The biovolume of the biofilm was determined and the results are described in the tables below. Experimental conditions Biovolume (µm 3< / µm 2< ) % inhibition PA14 untreated 13,5 Tobramycin (50 µg / ml) 3,26 75,9 ANP (10 -9< M) 4,53 66,4 Tobramycin (50 µg / ml) + ANP (10 -9< M) 0,5 96,3 Experimental conditions Biovolume (µm 3< / µm 2< ) % inhibition PA14 untreated 13,5 Tobramycin (10 µg / ml) 2,9 78,5 ANP (10 -9< M) 4,53 66,4 Tobramycin (10 µg / ml) + ANP (10 -9< M) 0,7 94,8

[0185] Under these conditions, the inventors observed that the biofilm was strongly dispersed by the combination of ANP (1 nM) + tobramycin (50 µg / ml), which acted synergistically to achieve 96.3% biofilm destruction. Similarly, the combination of ANP (1 nM) + tobramycin (10 µg / ml) acted synergistically to achieve 94.8% biofilm destruction. Example 7 : Effect of a combination of ANP and ciprofloxacin on preformed biofilms of P. aeruginosa

[0186] This example demonstrates the synergistic action of the ANP + ciprofloxacin combination on preformed biofilms of P. aeruginosa. Materials and methods

[0187] To study the impact of ANP in combination with ciprofloxacin on 24-hour preformed 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) were injected into different channels of the flow chamber. The preformed biofilm was treated for 2 h at 37°C under static conditions. After 2 h, the flow of medium was reapplied for 15 min to remove any cells that had detached from the biofilm during the treatment.

[0188] As described in Example 1, biofilms were observed by confocal laser scanning microscopy, after being labeled for 15 min with 5 µM of Syto9 green fluorochrome (Invitrogen). Results

[0189] The inventors studied the anti-biofilm activity potential of the combination of ANP (at 10⁻⁸ < M; 30 ng / ml) + ciprofloxacin (at 0.04 µg / ml or 0.01 µg / ml) on an established biofilm of P . aeruginosa.

[0190] The biovolume of the biofilm was determined and the results are described in the tables below. Experimental conditions Biovolume (µm 3< / µm 2< ) % inhibition PA14 untreated 12,6 Ciprofloxacin (0.04 µg / ml) 0,7 99,3 Ciprofloxacin (0.04 µg / ml) + ANP (10 -8< M) 0,5 99,5 Experimental conditions Biovolume (µm 3< / µm 2< ) % inhibition PA14 untreated 15,5 Ciprofloxacin (0.01 µg / ml) 1,55 90,0 Ciprofloxacin (0.01 µg / ml) + ANP (10 -8< M) 0,47 97,0

[0191] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by the ANP + ciprofloxacin combination, compared to treatment with the antibiotic ciprofloxacin alone.

[0192] Thus, the combination with ANP allows for 97% destruction of the biofilm in combination with a concentration of 0.01 µg / ml of ciprofloxacin. Example 8: Effect of a combination of ANP and colistin on preformed biofilms of P. aeruginosa

[0193] This example demonstrates the synergistic action of the ANP + colistin combination on preformed biofilms of P. aeruginosa. Materials and methods

[0194] To study the impact of ANP in combination with colistin on 24-hour preformed 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) were injected into different channels of the flow chamber. The preformed biofilm was treated for 2 h at 37°C under static conditions. After 2 h, the flow of medium was reapplied for 15 min to remove any cells that had detached from the biofilm during the treatment.

[0195] As described in Example 1, biofilms were observed by confocal laser scanning microscopy, after being labeled for 15 min with 5 µM of Syto9 green fluorochrome (Invitrogen). Results

[0196] The inventors studied the anti-biofilm activity potential of the combination ANP (at 10⁻⁸ < M; 30 ng / ml) + colistin (at 1 µg / ml) on an established biofilm of P. aeruginosa.

[0197] The biovolume of the biofilm was determined and the results are described in the tables below. Experimental conditions Biovolume (µm 3< / µm 2< ) % inhibition PA14 untreated 18,5 ANP (10⁻⁸< M) 4,9 73,5 Colistin (1 µg / ml) 2,1 88,6 Colistin (1 µg / ml) + ANP (10 -8< M) 0,5 97,3

[0198] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by the combination of ANP + colistin (1 µg / ml), compared to treatment with the antibiotic colistin alone or treatment with ANP alone.

[0199] Thus, the combination with ANP allows for 97.3% destruction of the biofilm in combination with a concentration of 1 µg / ml of colistin. Example 9 Effect of a combination of ANP and Imipenem on preformed biofilms of P. aeruginosa

[0200] This example demonstrates the synergistic action of the ANP + Imipenem combination on preformed biofilms of P. aeruginosa. Materials and methods

[0201] To study the impact of ANP in combination with Imipenem on 24-hour preformed 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) were injected into different channels of the flow chamber. The preformed biofilm was treated for 2 h at 37°C under static conditions. After 2 h, the medium flow was reapplied for 15 min to remove any cells that had detached from the biofilm during the treatment.

[0202] As described in Example 1, biofilms were observed by confocal laser scanning microscopy, after being labeled for 15 min with 5 µM of Syto9 green fluorochrome (Invitrogen). Results

[0203] The inventors studied the anti-biofilm activity potential of the combination ANP (at 10⁻⁸ < M; 30 ng / ml) + Imipenem (at 0.5 µg / ml) on an established biofilm of P. aeruginosa.

[0204] The biovolume of the biofilm was determined and the results are described in the tables below. Experimental conditions Biovolume (µm 3< / µm 2< ) % inhibition PA14 untreated 28,0 ANP (10⁻⁸< M) 6,9 75,4 Imipenem (0.5 µg / ml) 7,0 75,0 Imipenem (0.5 µg / ml) + ANP (10 -8< M) 0,6 97,9

[0205] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by the combination of ANP + Imipenem (0.5 µg / ml), compared to treatment with the antibiotic Imipenem alone or treatment with ANP alone.

[0206] Thus, the combination with ANP allows for 97.9% destruction of the biofilm in combination with a concentration of 0.5 µg / ml of Imipenem. Example 10 : Effect of a combination of ANP and polymyxin B on preformed biofilms of P. aeruginosa

[0207] This example demonstrates the synergistic action of the ANP + Polymyxin B combination on preformed biofilms of P. aeruginosa. Materials and methods

[0208] To study the impact of ANP in combination with polymyxin B on 24-hour preformed biofilms (as described in Example 1), 300 µl of a mixture of ANP (10 nM) and polymyxin B (4 µg / ml) or 300 µl of polymyxin B alone (control condition) were injected into different channels of the flow chamber. The preformed biofilm was treated for 2 h at 37°C under static conditions. After 2 h, the flow of medium was reapplied for 15 min to remove any cells that had detached from the biofilm during the treatment.

[0209] As described in Example 1, biofilms were observed by confocal laser scanning microscopy, after being labeled for 15 min with 5 µM of Syto9 green fluorochrome (Invitrogen). Results

[0210] The inventors studied the anti-biofilm activity potential of the combination ANP (at 10⁻⁸ < M; 30 ng / ml) + Polymyxin B (at 4 µg / ml) on an established biofilm of P. aeruginosa.

[0211] The biovolume of the biofilm was determined and the results are described in the tables below. Experimental conditions Biovolume (µm 3< / µm 2< ) % inhibition PA14 untreated 18,2 ANP (10⁻⁸< M) 4,5 75,3 Polymyxin B (4 µg / ml) 7,4 59,3 Polymyxin B (4 µg / ml) + ANP (10 -8< M) 3,0 83,5

[0212] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by the combination of ANP + Polymyxin B (4 µg / ml), compared to treatment with the antibiotic Polymyxin B alone or treatment with ANP alone.

[0213] Thus, the combination with ANP allows for the destruction of 83.5% of the biofilm in combination with a concentration of 4 µg / ml of Polymyxin B. Example 11 : Effect of sequential treatment with ANP followed by tobramycin on preformed biofilms of P. aeruginosa.

[0214] This example demonstrates the sequential action of ANP treatment followed by tobramycin treatment on preformed biofilms of P. aeruginosa. Materials and methods

[0215] The equipment and methods used in this example are identical to those described above in Example 1, with the following clarifications: To study the impact of sequential exposure to ANP followed by tobramycin on 24-h preformed biofilms (as described in Example 1), 300 µl of ANP (at 1 nM) (control condition) were injected into two channels of the flow chamber. The preformed biofilm was treated for 2 h at 37°C under static conditions. After 2 h, the flow of medium was reapplied for 15 min to remove any cells that had detached from the biofilm during the treatment. Then, 300 µl of tobramycin (50 µg / ml) or 300 µl of sterile MilliQ water (control condition) were injected into the two channels of the flow chamber, which had been pre-exposed to ANP. The second treatment of the preformed biofilm was carried out again for 2 hours at 37°C under static conditions.After 2 hours, the flow of medium was reapplied for 15 minutes to eliminate any cells that may have detached from the biofilm under the action of the treatment.

[0216] As described in Example 1, biofilms were observed by confocal laser scanning microscopy, after being labeled for 15 min with 5 µM of Syto9 green fluorochrome (Invitrogen). Results

[0217] The inventors studied the anti-biofilm activity potential of sequential exposure of ANP (at 10⁻⁹ < M; 3 ng / ml) followed by tobramycin (at 50 µg / ml) on an established biofilm of P . aeruginosa.

[0218] The biovolume of the biofilm was determined and the results are described in the tables below. Experimental conditions Biovolume (µm 3< / µm 2< ) % inhibition PA14 untreated 13,1 ANP (10 -9< M) 4,1 68,7 ANP (10⁻⁹ < M) (2h) then Tobramycin (50 µg / ml) (2h) 0,7 94,7

[0219] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by a double sequential treatment of ANP (10 -9< M) (2h) then tobramycin (50 µg / ml) (2h) compared to treatment with ANP alone.

[0220] Thus, the sequential treatment of ANP (2h) then tobramycin (2h) allows for a destruction of 94.7% of the biofilm.

Claims

1. Natriuretic peptide selected from the group consisting of (i) osteocrin, an osteocrin propeptide or osteocrin derivative, (ii) a lebetin, a lebetin fragment or a derivative of lebetin or of a lebetin fragment, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative, for use thereof in a method for the therapeutic treatment of a bacterial infection associated with a bacterial biofilm in a subject, wherein said natriuretic peptide disperses said bacterial biofilm, wherein the osteocrin derivative is an osteocrin in which one or more amino acid residues have been chemically modified or an osteocrin peptide variant, wherein the derivative of lebetin or of a lebetin fragment is a lebetin or a lebetin fragment in which one or more amino acid residues have been chemically modified or a lebetin or lebetin fragment peptide variant, and wherein said ANP peptide derivative is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full form of ANP, a form having a structure which is an antiparallel dimer of ANP, a polymer consisting of monomers of ANP, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted or added, an ANP peptide in which the amino acid Phe of the loop at position 8 has been substituted, an ANP peptide in which the amino acid Phe at position 26 or the amino acid Ser at position 25 has been substituted, urodilatin, fsANP or a linearized version of ANP with cleavage of the disulfide bridge.

2. Natriuretic peptide for use thereof according to Claim 1, wherein said bacterial infection is a Pseudomonas aeruginosa infection.

3. Natriuretic peptide for use thereof according to Claim 1 or 2, wherein said subject is suffering from a chronic respiratory disease, in particular cystic fibrosis.

4. Natriuretic peptide for use thereof according to Claim 1 or 2, wherein said subject carries an implant or wears a prosthesis, or is suffering from significant burns (serious burn victim) or from a surface infection.

5. Natriuretic peptide for use thereof according to any one of Claims 1 to 4, wherein said natriuretic peptide is used in combination with an antibiotic.

6. Natriuretic peptide for use thereof according to Claim 5, wherein said antibiotic is an aminoglycoside antibiotic or a quinolone.

7. Natriuretic peptide for use thereof according to one of Claims 1 to 6, wherein said natriuretic peptide is used in combination with at least one other natriuretic peptide.

8. Pharmaceutical composition comprising (A) a natriuretic peptide selected from the group consisting of a lebetin, a lebetin fragment and a derivative of lebetin or of a lebetin fragment, and (B) an antibiotic, wherein the derivative of lebetin or of a lebetin fragment is a lebetin or a lebetin fragment in which one or more amino acid residues have been chemically modified or a peptide variant.

9. Antibacterial pharmaceutical combination comprising (A) a natriuretic peptide selected from the group consisting of (i) osteocrin, an osteocrin propeptide or osteocrin derivative, (ii) a lebetin, a lebetin fragment or a derivative of lebetin or of a lebetin fragment, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative, and (B) an antibiotic for simultaneous, separate or sequential use in the therapeutic treatment of a bacterial infection associated with a bacterial biofilm in a subject, wherein said natriuretic peptide disperses said bacterial biofilm, wherein the osteocrin derivative is an osteocrin in which one or more amino acid residues have been chemically modified or an osteocrin peptide variant, wherein the derivative of lebetin or of a lebetin fragment is a lebetin or a lebetin fragment in which one or more amino acid residues have been chemically modified or a lebetin or lebetin fragment peptide variant, and wherein said ANP peptide derivative is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full form of ANP, a form having a structure which is an antiparallel dimer of ANP, a polymer consisting of monomers of ANP, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted or added, an ANP peptide in which the amino acid Phe of the loop at position 8 has been substituted, an ANP peptide in which the amino acid Phe at position 26 or the amino acid Ser at position 25 has been substituted, urodilatin, fsANP or a linearized version of ANP with cleavage of the disulfide bridge.

10. In vitro or ex vivo use of a natriuretic peptide selected from the group consisting of (i) osteocrin, an osteocrin propeptide or osteocrin derivative, (ii) a lebetin, a lebetin fragment or a derivative of lebetin or of a lebetin fragment, and (iii) an ANP peptide, an ANP propeptide or an ANP peptide derivative, for dispersing a bacterial biofilm, wherein the osteocrin derivative is an osteocrin in which one or more amino acid residues have been chemically modified or an osteocrin peptide variant, wherein the derivative of lebetin or of a lebetin fragment is a lebetin or a lebetin fragment in which one or more amino acid residues have been chemically modified or a lebetin or lebetin fragment peptide variant, and wherein said ANP peptide derivative is a truncated form of ANP corresponding to amino acids 4 or 5 to 28 of the full form of ANP, a form having a structure which is an antiparallel dimer of ANP, a polymer consisting of monomers of ANP, an ANP peptide in which 1 to 4 amino acids have been deleted, substituted or added, an ANP peptide in which the amino acid Phe of the loop at position 8 has been substituted, an ANP peptide in which the amino acid Phe at position 26 or the amino acid Ser at position 25 has been substituted, urodilatin, fsANP or a linearized version of ANP with cleavage of the disulfide bridge.