FR2858772A1 - Antibacterial composition containing cationic peptide and antibiotic, useful particularly for treating infections by Gram-negative species, also new conjugates of peptides and antibiotics - Google Patents

Abstract

Antibacterial composition (A), particularly directed against Gram-negative bacteria, comprises at least one peptide (P) of 10-25 amino acids (aa); and at least one antibiotic (AB). Antibacterial composition (A), particularly directed against Gram-negative bacteria, comprises at least one peptide (P) of 10-25 amino acids (aa); and at least one antibiotic (AB). (P) comprises: (i) two domains, positively charged at neutral pH, of 3-9 aa, at least 2/3 of which are cationic aa; (ii) between these two domains, a group of 2 or 3 non-cationic aa; and (iii) at the C or N terminus of (P), a group of 0-10 (preferably 0-5) non-hydrophobic and/or positively charged aa, provided that positively charged aa are not directly adjacent to a domain of (i). An independent claim is also included for a compound of formula (A)m-(X)p-(P)n (I) A : residue of an antibacterial compound; P : residue of a peptide defined above; X : covalent bond between A and P or a spacer arm; m, n : 1-3; p : zero or an integer no greater than n+m. ACTIVITY : Antibacterial. No details of tests for antibacterial activity are given. MECHANISM OF ACTION : None given.

Description

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ANTI-BACTERIAL COMPOSITION, PARTICULARLY AGAINST GRAM NEGATIVE BACTERIA, COMPRISING A PEPTIDE AND AN ADVANTAGEALLY HYDROPHOBIC ANTI-BACTERIAL AGENT
The present invention relates to the field of anti-bacterial treatments and more particularly methods and compositions for treating infections by gram-negative bacteria in humans, animals or plants.

 Peptides capable of destroying bacteria have been described in the prior art (Park CB, Kim HS, Kim SC.

Biochem Biophys Res Commun. 1998 Mar 6; 244 (1): 253-7). PCT International Patent Application No. WO 01/64738 has also reported peptides capable of reacting with aminoglycans and transporting molecules of interest in eukaryotic or prokaryotic cells.

 The amount of antibiotic molecules entering the bacterium depends on its structure and mechanisms involved in the transport of substrates. Gram-negative bacteria are structurally distinguishable from gram-positive bacteria by the presence of two membranes that make up the bacterial envelope. If all bacteria have an inner membrane, Gram-negative bacteria have an extra single outer membrane. This hydrophobic outer membrane is a semi-permeable barrier that prevents the penetration of antibiotics but porins, proteins forming channels, allow the entry of small hydrophilic solutes such as nutrients and antibiotics of the family of penicillins and tetracyclines but exclude the penetration of large hydrophilic molecules and antibiotics of the macrolide / ketolide family.

 An important cause of therapeutic failures against Gram-negative bacteria is the emergence of strains

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 resistant. Some resistances are linked to a reduction in the permeability of bacterial membranes (quantitative / qualitative modifications of porins).

Other resistances are the presence of a membrane protein which causes the rejection of the antibiotic by an active efflux mechanism. The development of new types of anti-bacterial molecules or the use of non-active commercial antibiotics on Gram-negative bacteria requires their entry and delivery through selective bacterial membranes.

 It is an object of the present invention to provide novel methods and compositions for effectively treating infections with Gram-negative bacteria even when they have developed antibiotic resistance.

 This goal is achieved through the use of peptides able to cross the outer membrane of gram-negative bacteria and, through this translocation of the membrane, to deliver molecules of interest that can not otherwise penetrate inside bacteria, because of their physicochemical properties.

 The work carried out in the context of the present invention concerned Bodipy and tetramethylrhodamine which are hydrophobic fluorescent molecules excluded by the outer membrane of Gram-negative bacteria. These fluorescent tracers were chosen in order to evaluate the internalization properties of the peptides of the invention chemically linked to hydrophobic molecules. The translocation of fluorescent tracers in the Gram-negative bacterium was qualitatively evaluated on Escherichia coli and Pseudomonas aeruginosa.

 The present invention therefore firstly relates to an anti-bacterial composition, more particularly

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 directed against Gram-negative bacteria, comprising the combination of: a) at least one peptide of 10 to 25 amino acid residues comprising: i) two positively charged domains at neutral pH each consisting of 3 to 9 acid residues at least two-thirds of which are cationic amino acids, ii) between said positively charged domains, a group of two to three non-cationic amino acid residues, iii) at one or both of the N or C terminus of the peptide, a group of 0 to 10 and preferably 0 to 5 amino acid residues selected from the group consisting of non-hydrophobic amino acids and positively charged amino acids, but in the case of a residue of Positively charged amino acid is not directly adjacent to the positively charged domains. b) at least one anti-bacterial compound.

 Advantageously, in the peptides of the invention above, the cationic amino acids of the two positively charged domains are chosen from the group comprising arginine and lysine.

 It is preferred in the peptides of the above invention that the non-cationic amino acids of the group between said positively charged domains are non-hydrophobic amino acids, selected for example from the group consisting of: glutamic acid, serine, glycine , leucine, glutamine.

 The orientation of the amino acid sequences according to the invention is typically N-terminal to C-terminal. However, according to another embodiment, the orientation may be reversed, i.e. the amino acid sequences are oriented from C-terminal to N-terminal.

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Preferred peptides for the compositions according to the invention are chosen from the group comprising the following sequences (N-terminal to C-terminal orientation) - DPV3: Arg Lys Arg Arg Arg Arg Ser Arg Arg Lys Arg Arg Arg Glu Ser (SEQ ID N0.1) - DPV3.10: Arg Arg Lys Arg Arg Arg Glu Ser Arg Arg Arg Ala Arg Arg Pro Arg Arg Leu (SEQ ID N0.2) - DPV6: Gly Arg Arg Arg Ser Gly Lys Arg Lys Arg Arg Lys Arg Leu Lys Pro (SEQ ID NO: 3) - DPV7: Gly Lys Arg Lys Lys Lys Gly Lys Lys Gly Lys Lys Arg Asp Pro (SEQ ID N0.4) - DPV7b: Gly Lys Arg Lys Lys Lys Gly Lys Leu Gly Arg Arg lys Arg Arg lys (SEQ ID N0.5) - DPV15: Arg Arg Glu Arg Gln Ser Arg Arg Arg Arg Glu Arg Gln Ser Arg (SEQ ID N0.6) - DPV15b: Gly Ala Asp Asp Arg Arg Arg Arg Glu Arg Gln Ser Arg Arg Arg Arg Glu Arg Arg Gln Ser Arg (SEQ ID N0.7) - DPV1047: Val Lys Arg Gly Leu Lys Leu Arg His Val Arg Pro Arg Val Arg Arg Met Asp Val (SEQ ID N0.8 ) - DPV11: Ala Lys Thr Arg Argly Gly Lys Arg Ser Gly (SEQ ID N0.9) - DPV1121: Val Lys Arg Gly Leu Lys Leu Arg Gln Lys Tyr Asn Lys Arg Ala Met Asp Tyr (SEQ ID NO.11)
Among these, the invention especially relates to the following peptides: DPV3, DPV3. 10, DPV6, DPV7, DPV7b, DPV15 and DPV15b.

 The alignment of the above sequences shows positively charged domains of the following sequences: Arg Arg Arg Lys Arg Arg (SEQ ID N0.13) Arg Arg Arg (SEQ ID No. 14) Arg Ly Lys Lys Lys Gly Lys (SEQ ID NO: 15) Arg Arg Glu Arg (SEQ ID No. 16) Arg Arg Arg Glu Arg (SEQ ID No. 17)

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Arg Arg Ala Arg Arg Ser Pro Arg (SEQ ID N0.18) - Arg Lys Arg Lys Arg Lys Arg Leu Lys (SEQ ID N0.19) - Arg Lys Arg (SEQ ID N0.20) - Arg Arg Arg Lys Lys Ser Arg (SEQ ID N0.21) - Arg Arg Arg Arg Arg (SEQ ID N0.22) - Arg Arg Arg Arg Arg Arg (SEQ ID N0.23)
Preferably, the sequence domains SEQ ID NO. 13 to 17 are on the N-terminus side of the peptide, whereas the SEQ ID NO. 18 to 23 are on the C-terminal side of the peptide.

Alignment of the above sequences also revealed groups of two to three non-cationic amino acid residues between the positively charged domains of the following sequences:
Glu Ser, Ser Gly Glu, Leu Gly, Gln Ser
The anti-bacterial compounds present in the compositions according to the invention are preferably chosen from those having physicochemical properties rendering them incapable of crossing the membrane of Gram-negative bacteria. Most preferably, it is hydrophobic anti-bacterial compounds. Examples of such compounds include antibiotics of the macrolide family, ketolides such as erythromycin, clarithromycin, azithromycin, telithromycin.

 The anti-bacterial compounds may also be antisense oligonucleotides.

 The evaluation of the DPV peptides above to show their ability to cross the membranes of Gram-negative E. coli or P. aeruginosa bacteria and to deliver Bodipy into the bacterium, whereas this is a hydrophobic molecule normally excluded by the outer membrane of Gram-negative bacteria which represents a semi-

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 permeable. In addition, it appears that these peptides have the ability to cross the outer membrane of both strains of Gram-negative bacteria and accumulate in the bacterial cytoplasm by a non-energy-dependent and nontoxic mechanism for the bacterium.

 The work carried out in the context of the invention has revealed some internalization differences between the two strains of P. aeruginosa and E. coli bacteria.

Thus it seems that the peptide DPV7b is more internalizing in P. aeruginosa than in E. coli. This difference could be explained by differences in the structure of the outer membrane between the two strains of bacteria.

 These peptides are therefore useful for the preparation of anti-bacterial pharmaceutical compositions, more particularly against gram-negative bacteria, where they are associated with one or more anti-bacterial agents.

 The compositions of the invention are useful both preventive and curative.

 The compositions according to the invention also advantageously comprise one or more pharmaceutically acceptable carriers, diluents or excipients generally used with this type of agent.

 The peptides of the invention can be prepared by chemical synthesis or by genetic engineering in a prokaryotic cell such as a bacterium, in a eukaryotic cell such as a yeast, a CHO cell or an NSO cell, in a transgenic animal, for example in milk. rabbit, goat, sheep, cow, etc. transgenic or in a transgenic plant as, for example, in tobacco plants, etc.

 The invention also relates to functional equivalents of the peptides defined above, such as peptides comprising modifications resulting from processes

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 post-translational agents such as glycosylation or chemical modifications such as coupling with lipids, sugars, nucleotide sequences, provided that these modifications do not modify the anti-bacterial and / or antifungal activity of said peptides according to the tests given in the part experimental below. Functional equivalents also include peptides of which one or more amino acids are D-conforming amino acids. The invention also covers retropeptides and retro-inverso-peptides.

 The combination of the compositions according to the invention may consist of one or more peptides described above and one or more anti-bacterial compounds, also, unless otherwise indicated, the singular used for the definition of the active agents (peptide and antibacterial compound) of the composition covers the plural.

 The composition according to the invention may be produced by the combination of peptide (s) and antibacterial compound (s) in a mixture or a product in which one or more identical or different peptides are covalently bound to a or several identical or different compounds, possibly via a spacer arm. Such products are especially products of formula (I) which will be described below.

 In the case of the administration of peptide and anti-bacterial compound in a mixture, these two active agents of the anti-bacterial composition of the invention can be presented separately, each in a suitable pharmaceutical form, and united in a same packaging.

However, to facilitate the simultaneous administration of the active agents, it is generally preferred to prepare the drug in a single pharmaceutical form containing the active agents.

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 two active ingredients in mixture, and possibly a suitable pharmaceutical excipient.

 Of course, a product consisting of a peptide directly or indirectly bound to an anti-bacterial compound must be considered as constituting on its own an association according to the invention and which can be used as the sole active ingredient.

 For example, a peptide and an anti-bacterial compound can be combined by establishing a chemical bond therebetween. In particular, it is possible to amidify an amine function of the peptide, or to esterify one or more alcohol functions of the peptide, with an acid group present at the level of an anti-bacterial compound or derivative thereof. An amidation product is thus obtained which constitutes the active product of the composition of the invention. It is also possible to add to one and / or the other N and / or C terminus of the peptide an amino acid residue whose side chain allows coupling with an antibacterial compound, such as for example a cysteine residue which the SH group is reactive. Examples of such products are those of formula (I) which are described below.

 Indeed, the invention also relates to new products where the peptide and the anti-bacterial compound are covalently bonded to one another, optionally via at least one spacer arm.

 Such products are in particular those which correspond to the following formula (I): (A-) m (X) p (-P) n (I) in which: A is the residue of an antibacterial compound, P is the remainder of a peptide, as defined above, and X represents either a covalent bond between A and P, or a spacer arm connecting at least one residue A to at least one residue P, m is an integer ranging from 1 to 3 , n is an integer that can go

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 from 1 to 3, and p represents zero or an integer at most equal to the largest of the numbers m and n.

 It is possible either to graft one or more A and / or P residues on a single spacer arm, or to graft one or more groups AX on a remainder P (and then m = p and n = 1), or to graft one or more XP groups on a remainder A (and then n = p and m = 1). When p = zero, either one or more remains A are directly related to a remainder P (and n = 1), or one or more remains P are directly related to a remainder A (and m = 1).

 The products of formula (I) can be used in the form of salts, in particular in the form of alkali metal salts such as sodium or potassium salts; these salts are, for example, those of the phosphate groups, if they are present, phenolic groups (in the case of salicylic acid), etc. The products of formula (III), where appropriate, may also be used in the form of addition salts (for example in the hydrochloride form) when these products contain an amine group.

 The bonds between the spacer arm and the A and P residues or directly between A and P are covalent bonds.

These covalent bonds can be formed, as indicated above, between carboxylic ester, carboxylic amide, thiocarboxylic ester or thiocarboxylic amide groups.

 The residues of anti-bacterial compound (A) and peptide (P) are derivatives of anti-bacterial compound or peptide, of which one or more chemical groups have been either deleted or modified so as to allow the formation of covalent bonds directly. between A and P or indirectly via a spacer arm.

 It may be acyl functions of antibacterial compounds having a carboxylic group capable of forming a bond with the spacer arm or the peptide,

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last having a primary amine or a hydroxyl group capable of forming a covalent bond with the spacer arm or the corticoid
The spacer arms may in particular be bivalent residues of bi-functional aliphatic compounds, such as compounds having reactive functional groups at each of their ends, each of which makes it possible to form covalent bonds with A and with P. These compounds may be, for example, compounds which have both an amino group and a carboxylic (or thiocarboxylic) group, or compounds which have both an amino group and a hydroxyl group.

 In the formula (I), the group X (leaving aside its end functional groups) represents in particular a divalent aliphatic group optionally interrupted by one or more heteroatoms O - or - S - or by one or more heteroatomic groups - NH - or - CO - NH -.

 Compounds capable of giving, after reaction with the peptide and the anti-bacterial compound or their derivatives, products of formula (I) in which A and P are linked by spacer arms, are, for example, alpha-, beta- or gamma-amino alkanecarboxylic acids, in particular natural alpha-amino acids such as glycine, alanine, valine or leucine, or peptides, especially dipeptides or tripeptides. As indicated in the examples, it may advantageously be a cysteine residue.

 The spacer agents may also be hydroxy-carboxylic acids such as lactic acid, glycolic acid, aldonic acids (gluconic, mannonic, galactonic, ribonic, arabinonic, xylonic and erythronic acid) and the corresponding lactones or dilactones

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 (eg lactide, glycolide, delta-glucolonactone, delta-valeronactone), or even aldaric acids.

 The functional groups optionally present on the spacer arm and not involved in the binding with A or P may be used to graft other A and / or P residues so as to obtain compounds of formula (III) for which m and / or n are greater than 1. This is the case, for example, with hydroxyl groups of the hydroxy acids, the second carboxylic group of the diacid carboxylic amino acids, the second amino group of the diamino amino acids, the hydroxyl group of the hydroxylated amino acids, and the like.

The spacer arm may advantageously consist of a binding molecule capable of allowing the delayed release of one and / or the other of the A or P residues, in particular by protecting them from degradation after administration. The spacer arm may also consist of a targeting molecule for targeting a particular organ or tissue for the delivery of purine or corticoid residues,
To prepare the compounds of formula (III), conventional methods of organic synthesis are used.

For example, to prepare amides or esters, a carboxylic compound can be reacted in the form of a carboxylic acid halide (or thiocarboxylic acid), or in the form of a mixed anhydride, or in the form of an activated ester, for example a p-nitrophenyl ester. The acid can also be activated with a coupling agent such as dicyclohexylcarbodiimide.

 Since the compounds of formula (I) include peptide residues, they can be prepared using, in particular, methods known in peptide chemistry.

 Of course, when the compounds of which A, P or X derive from the formula (I), comprise several functions

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 may be reacted, it is necessary to operate either by using the reagents in stoichiometric proportions (according to the number of precursors of A and / or P that we want to react), or by temporarily protecting the reactive functions of which we do not do not want them to react. For this purpose the methods of temporary protection of said reactive functions are used. These methods of temporary protection are well known, especially those that have been developed during research on peptide synthesis. For example, the -NH 2 groups may be protected by carbobenzoxy, phthaloyl, t-butoxycarbonyl, trifluoroacetyl or toluenesulfonyl groups; the carboxylic groups can be protected in the form of benzyl esters, tetrahydropyranyl esters or t-butyl esters; The alcohols may be protected as esters (eg acetates), as tetrahydropyranyl ethers, benzyl ethers or trityl ethers, or as acetals (including in the form of acetonides in the case of vicinal glycols). The protection and possible deprotection reactions of various chemical groups are known and described in the literature.

 Phosphatation or dephosphatation reactions of the primary alcohol of nucleotides or nucleosides can be carried out using natural enzymes (eg phosphatases, phosphokinases).

 The anti-bacterial compositions of the invention, and in particular those comprising a compound of formula (I), may be administered by any of the accepted modes of administration for the therapeutic agents and of course orally, sublingually, nasally pulmonary, rectal or parenteral (eg intravascular, intramuscular, transcutaneous, intra

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 articular). Mention may also be made of systemic, topical or central administration, for example by intracranial surgery or intraocular administration. We can also mention subcutaneous implantation of biodegradable implants.

 For this purpose, they may be presented in any form allowing administration by: - orally (in particular in the form of capsules, oral solutions or emulsions, powders, gels, granules, tablets or tablets) , tablets, capsules, soft capsules, including delayed release or extended release formulations, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. This form of presentation is more particularly adapted to the passage of the intestinal barrier and the most common use of anti-bacterial and / or antifungal compounds.

 parenteral route, usually by intramuscular or intravenous injection. The injectable compositions may be prepared in conventional forms, either in suspension or liquid solution or in solid form suitable for extemporaneous dissolution in a suitable liquid, including delayed-release or extended-release formulations such as the inclusion of peptides in micro or biodegradable nanoparticles of lipid formulation or formulation of dextran or PLGA or the like. This form of presentation is more particularly adapted to the passage of the blood-brain barrier and the hospital use of anti-bacterial and / or antifungal compounds.

 One possibility for parenteral administration is the implantation of a slow-release system or

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 sustained release that maintains a constant level of dose.

 Another possibility is to adsorb or otherwise fix the peptides of the invention on a support, such as a catheter, a prosthesis or a biological glue.

 - Nasal (eg solutions to be administered in the form of drops or sprays), - Pulmonary route (solutions in pressurized bottle for aerosols), - Rectal route (suppositories), - Dermal route (eg creams, ointments or transdermal patches , also called patches or patches), transmucosal route such as sublingually (solutions in pressurized bottle, or oral disintegrating tablets).

 These dosage forms are customarily prepared and may contain suitable conventional excipients and carriers.

 Other usual topical preparations include creams, ointments, lotions, gels and aerosol sprays. The latter are more particularly adapted for the treatment of bacterial and / or fungal bronchopulmonary infections.

 The compositions of the invention can also be used in the cosmetic field, as a primarily preventive, and then consisting of creams, nail polish, hygiene products of the genital organs, toothpaste pastes, oral hygiene solutions or to include in slow diffusion micro-particles, in the aqueous phase, included for example in layers, cotton swabs, dressings, cotton cottons,

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 removing make-up, sanitary napkins or animal bedding.

 Depending on the mode of administration, the compounds may be in solid, semi-solid or liquid form.

 For solid compositions, such as tablets, pills, powders or granules in the free state or included in capsules, the combination may be combined with: - diluents, for example lactose, dextrose, sucrose, mannitol sorbitol, cellulose and / or glycine; lubricants, for example silica, talc, stearic acid, its magnesium or calcium salt and / or polyethylene glycol; binders, for example magnesium aluminum silicate, starch paste, gelatin, gum tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; where appropriate, disintegrants, for example starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and / or - absorbents, dyes, flavoring agents and sweeteners. The excipients may be, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and pharmaceutical grade analogs.

 For semi-solid compositions, such as suppositories, the excipient may, for example, be a fat emulsion or suspension, or based on polyalkylene glycol, such as polypropylene glycol.

 Liquid compositions, in particular injectable or to be included in a soft capsule, can be prepared for example by dissolution, dispersion, etc. active ingredient in a pharmaceutically pure solvent such as, for example,

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 for example, water, physiological saline, aqueous dextrose, glycerol, ethanol, an oil and the like.

 The compositions according to the invention may also be administered in the form of liposome-like delivery systems, such as in the form of small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, including cholesterol, stearylamine or phosphatidylcholines. In one embodiment, a liquid component film may be hydrated with an aqueous solution of the drug to form a lipid layer encapsulating the drug.

 The compositions according to the invention can be sterilized and / or contain adjuvants and non-toxic auxiliary substances such as preservatives, stabilizers, wetting agents or emulsifiers, dissolving agents, salts for adjusting the osmotic pressure. and / or buffers. In addition, they may also contain other substances of therapeutic interest. The compositions are prepared, respectively, by conventional methods of mixing, granulating or coating and contain from about 0.1 to 75%, preferably from about 1 to 50%, of active ingredient.

 The peptides and anti-bacterial agents of the combination of the composition according to the invention can also be coupled with soluble polymers such as targetable drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxy-ethyl-aspanamide-phenol or polyethylene oxide-polylysine substituted with palmitoyl residues, dextran. In addition, the compounds according to

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 The invention may be coupled to a class of biodegradable polymers useful for achieving a controlled release of a drug, for example, poly (lactic acid), poly (epsilon-caprolactone), poly (hydroxybutyric acid), polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipatic block hydrogel copolymers.

 The dosage for administration of the compositions according to the invention is chosen according to many factors including the type, species, age, weight, sex and medical condition of the subject, the severity of the state to be treated, the route of administration; the state of renal and hepatic function of the subject and the nature of the particular compound, or salt, used. A normally experienced physician or veterinarian will readily determine, and prescribe, the effective amount to prevent, antagonize or halt the progress of the medical condition to be treated.

 A composition according to the invention may contain from 0.1 to 99%, preferably from 1 to 70% of active principle.

 By way of example, the oral dosages of the compositions according to the invention will be between approximately 0.5 and 1 mg / day orally and, preferably, provided in the form of tablets containing 0.5, 1, 2.5 , 5, 10, 15, 25, 50, 100, 250, 500 and 1,000 mg of active ingredient. Effective plasma concentrations will be obtained from a dosage ranging from 0.002 mg to 50 mg per kg of body weight per day.

 The compositions of the invention may be administered in the form of single daily doses, or in two, three or four doses per day.

 Other advantages and characteristics of the invention will become apparent from the examples which follow, given by way of

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illustrative, and in which reference will be made to the attached drawings where:
Figure 1 shows the formula of Bodipy FL N- (2-aminoethyl) maleimide.

 Figure 2 shows the formula of Tetramethylrhodamine-6-maleimide.

 Figure 3 shows the internalization of DPV-Bodipy conjugates in E. coli.

 FIG. 4 represents the immunolabeling of the outer membrane after internalization of the DPV3-bodipy conjugate.

 Figure 5 shows the internalisation of DPV-Bodipy conjugates in P. aeruginosa.

 Figure 6 gives confocal microscopy images of P. aeruginosa.

 Figure 7 shows the internalisation of the DPV3-TMR conjugate in E. coli.

 I - Materials and Methods.

 I.1) Fluorescent tracers.

 - Bodipy @ FLN- (2-aminoethyl) maleimide (Bodipy) (Molecular Probes Cat # B-10250), whose molecular formula is C20H21BF2N4O3, The molecular weight is 414.22, the absorbance of 504nm and the emission of 510nm (green fluorescence), and the structural formula is given in Figure 1.

 - Tetramethylrhodamine-6-maleimide (TMR) (Molecular Probes Cat # T-6028), whose molecular formula is C28H23N3O5, the molecular weight is 481.51, the absorbance is 541nm and the emission is 567nm (red fluorescence) and the structural formula is given in Figure 2.

 These two fluorescent molecules contain a maleimide reactive group allowing the chemical coupling on the thiol function of the cysteine of the peptide.

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 1.2) Peptide vectors (DPVs).

 The peptides of sequences below were used: - DPV3: Arg Lys Arg Arg Lys Arg Glu Ser Arg Lys Lys Arg Arg Arg Glu Ser (SEQ ID NO.1) with a Cys residue at its C terminus, - DPV3. 10: Arg Lys Arg Arg Arg Glu Ser Arg Arg Ala Arg Arg Arg Pro Arg His Leu (SEQ ID N0.2) with a Cys residue at its C terminus, - DPV6: Gly Arg Arg Arg Ser Gly Lys Arg Arg Arg Lys Arg Lys Arg lys (SEQ ID N0.3) with a Cys residue at its C terminus, - DPV7: Gly Lys Arg Lys Lys Gly Lys Lys Leu Gly Lys Arg Asp Pro Lys (SEQ ID N0.4) with a Cys residue at its C terminus, - DPV7b: Gly Lys Arg Lys Lys Lys Gly Lys Leu Gly Lys Lys Arg Pro Arg Ser Arg (SEQ ID N0.5) with a Cys residue at its C terminus, - DPV15: Leu Arg Arg Glu Arg Gln Ser Arg Arg Arg Arg Glu Arg Gln Ser Arg (SEQ ID N0.6) with a Cys residue at its C terminus, - DPVl5b: Gly Ala Asp Asp Arg Arg Arg Arg Arg Glu Arg Gln Ser Arg Leu Arg Arg Arg Glu Arg Gl Gl n Ser Arg (SEQ ID N0.7) with a Cys residue at its N-terminus, - DPV1047: Arg Lys Arg Gly Leu Lys Leu Arg His Val Arg Pro Arg Val Arg Arg Met Asp Val (SEQ ID N0.8) with a Cys residue at its N terminus, - DPV11: Ala Lys Thr Gly Lys Arg Lys Arg Ser Gly (SEQ ID N0.9) with a Cys residue at its C terminus, - DPV12: Gln Gly Lys Ser Lys Arg Glu Lys Asp Arg Val Phe lys (SEQ ID NO: 10) with a Cys residue at its C terminus,

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 - DPV1121: Lys Arg Gly Leu Lys Leu Arg Gln Lys Tyr Asn Lys Arg Ala Met Asp Tyr (SEQ ID N0.11) with a Cys residue at its N terminus, - DPV19: Asn Pro Gly Val Ser Thr Val Val Leu Gly Ala Tyr Asp Arg Arg Arg Arg Glu Arg Gln Ser Arg (SEQ ID N0.12) with a Cys residue at its N terminus.

 The peptide syntheses were carried out according to the techniques known to those skilled in the art. The peptides are soluble in water.

 The peptides have a cysteine residue at the N- or C-terminal position to allow conjugation to the fluorescent tracer.

 1.3) The products controls.

 Bodipy and TMR are chemically coupled to a cysteine residue and serve as a negative internalization control.

 1.4) Method of chemical coupling.

 Bodipy or TMR solutions are prepared at a final concentration of 50mM dimethylformamide (DMF). DPV solutions are prepared at a final concentration of 10 mM DMF. 200 l of the Bodipy solution or TMR are mixed with 700 l of the DPV solution. After incubation for 2 hours at room temperature, in the dark, 2 ml of H 2 O and 8 ml of dichloromethane (DCM) are added. The solution is vortexed and centrifuged for 2 minutes at 3000g. The aqueous phase is removed and stored.

Four successive DCM extractions are carried out. The aqueous phases are collected in a glass flask and placed for 1 hour at -80 ° C. before being lyophilized for at least 18 hours. The powder obtained is stored under argon at -20 ° C., protected from light.

 1.5) Conservation of the conjugates in solution.

 The DPV-Bodipy and DPV-TMR conjugates are stored diluted to 3mM in H2O at -20 ° C, protected from light.

 1.6) HPLC analysis of the conjugates.

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- For Bodipy conjugates:
Luna column 100 3 C18 100x4.6 mm
Solvent A: 0.1% TFA in H20 Solvent B: 0.1% TFA in CAN
Gradient: 5% B - 60% in 10 min, 60 - 90% in 1 min, 90% B in 3 min Flow: 1. 2 ml / min; volume injected: 10 l; the concentration of the sample injected is 1 mg / ml in 0. 1% TFA
Detector: DAD: 214 nm, 300 nm.

- For TMR conjugates:
Column Luna 100 3p C18 100x4.6 mm
Solvent A: 0.1% TFA in H20
Solvent B: 0. 1% TFA in CAN
Gradient: 5% B 60% in 10 min, 60 90% in 1 min, 90% B in 3 min Flow: 1. 2 ml / min; volume injected: 20 l; the concentration of the sample injected is 1 mg / ml in 0. 1% TFA
Detector: DAD: 220 nm.

 1.7) Bacterial strains.

- Escherichia coli ATCC 25922 - Pseudomonas aeruginosa ATCC 27853
1.8) Internalisation protocol.

 I.8.a) Evaluation of the penetration of the conjugates in the bacterium at 37 C.

 The bacteria in the exponential culture phase are centrifuged and washed 3 times with 10mM sodium phosphate buffer, pH7. 4 (NAPB buffer). The bacterial concentration is adjusted to 1x106 cfu / ml (Colony Forming Units) in NAPB buffer. 50 l of the bacterial suspension are deposited on a poly-L-Lysine slide. After incubation for 30 minutes at 37 ° C. in a humid chamber, the bacteria immobilized on the slide are rinsed 3 times with

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 the NAPB buffer. 50 l of DPV-Bodipy conjugate solution or DPV-TMR or control product are deposited on the bacteria. After incubation for 30 minutes at 37 ° C. in a humid chamber and protected from light, the slides are rinsed 3 times with the NAPB buffer. Bacteria can be fixed on the slide by incubating for 20 minutes at 37 ° C, protected from light. A drop of PBS / Glycerol 50% is deposited on the slide and covered with a glass slide. After sealing the coverslip on the slide, the fluorescence of the bacteria is observed under a Leica epifluorescence optical microscope (40X or 63X immersion objective). The images are taken with the Nikon digital camera coolpix at maximum zoom and with the adapter 0. 63 X. A more detailed analysis is carried out with a confocal microscope Bio-rad MRC 600 (BIO-RAD Microscience Ltd., Hemel Hempstead, England) , equipped with an inverted light microscope and an X100 immersion lens. The bacteria are visualized by their fluorescence after excitation by a Krypton / Argon laser. Several sections of bacteria of 0.1 - 0.2pm are made.

 I.8.b) Evaluation of the conjugate penetration in the bacterium at +4 C.

 The method described previously (I.8.a) has been modified as follows. The bacteria immobilized on a poly-Lysine slide and rinsed 3 times with the NAPB buffer are incubated for 24 hours at +4 ° C. before the addition of the pre-incubated fluorescent DPV conjugates at +4 ° C. All the following steps are performed at + 4 C with cold solutions.

 1.9) Immuno-labeling of the outer membrane of bacteria: indirect immunofluorescence.

 The bacteria immobilized on a poly-L-Lysine slide are rinsed 3 times with NAPB buffer and incubated for 30 minutes at laboratory temperature with a 0.05% NAPB / BSA solution (bovine serum albumin). The bacteria are incubated for 30 minutes at laboratory temperature with

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 the anti-endotoxin mouse monoclonal antibody (Biovalley Cat # C55157; 212529) diluted in 0.5% NAPB / BSA, washed several times with 0.05% NAPB / BSA and then incubated for 30 minutes at laboratory temperature; protected from light, with a second antibody: polyclonal anti-mouse rabbit antibody conjugated with tetramethyl rhodamine (TRITC) (Jackson ImmunoResearch Cat # 315-026-003, batch # 47511) or fluorescein (FITC) (( Jackson ImmunoResearch Cat # 715-095-150, batch # 51038) After several washes with the NAPB buffer, a drop of PBS / Glycerol 50% is deposited on the slide and covered with a glass coverslip.

After sealing the coverslip on the slide, the fluorescence of the bacteria is observed under a confocal microscope as described previously (# III.8.a).

 I.10) Evaluation of the anti-bacterial activity of the conjugates.

 Minimum inhibitory concentrations (MIC) are determined by the liquid microdilution method (NCCLS M7A5) for all bacterial species in 96-well polystyrene plates.

 An isolated colony of E. coli ATCC 25922 or P. Aeruginosa ATCC 27853 is suspended in 3-5 ml of Mueller-Hinton culture medium (MH) and incubated at 37 ° C overnight with shaking. From this night culture, an exponential phase growth culture of the strain is carried out; MH medium is inoculated at 1 / 50th with the overnight culture and incubated for 2 hours at 37 ° C. with stirring. The bacterial concentration is adjusted to 1x106 cfu / ml (Colony Forming Units) in the MH medium.

 50 μl of bacterial inoculum are distributed per well containing an equal volume of the conjugate solution diluted half to half in the appropriate culture medium (0 to 1

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  M). The cultures are incubated at 37 ° C. in ambient air for 16 to 20 hours.

 The MIC in M is the first concentration that does not show bacterial growth.

 II - Results.

 II.1) Internalization of DPV-Bodipy Conjugates in Gram-Negative Bacteria

 II.l.a) Qualitative evaluation of the internalization of DPV-Bodipy conjugates.

 - Qualitative evaluation in E. coli bacteria.

 Figure 3 shows the internalization of DPV-Bodipy conjugates in E. coli.

 The bacteria immobilized on a poly-L-Lysine slide are incubated with 1 M of DPV-Bodipy conjugate for 30 minutes at 37 ° C. The microscopy images (epifluorescence optical microscope, immersion objective X 63) show the penetration of the DPV conjugate. -Bodipy in living bacteria. A: DPV3, B: DPV3.10; C: DPV6; D: DPV7; E: DPV7b; F: DPV15; G: DPV15b; H: DPV1047; I: DPV11; J: DPV12; K: DPV1121; L: DPV19.

 The E. coli bacteria are incubated for 30 minutes at 37 ° C. with 1 M of DPV-Bodipy conjugate as described in paragraph I.8.a. The internalization of fluorescent DPV-Bodipy conjugates in unfixed bacteria is visualized under an epifluorescence microscope. No fluorescence is detected with the Cys-Bodipy control conjugate. As shown in Figure 3, several DPV-Bodipy conjugates cross bacterial membranes of E. coli bacteria to accumulate in the bacterial cytoplasm. DPV3 peptides. (Fig. 3B), DPV3 (Fig. 3A), DPV6 (Fig. 3C) and DPV15 (Fig. 3F) are highly penetrating and internalizing hydrophobic molecules. With peptides DPV11 and DPV12 (Fig. 31 and 3J), we obtain fluorescence levels

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 heterogeneous in the same bacterial population. The internalizing properties of these peptides are lower. The DPV19 peptide does not enter the bacteria (Fig. 3L).

 An identical pattern of internalization of DPV-Bodipy conjugates is observed after fixation of the bacteria.

 FIG. 4 represents the immunolabeling of the outer membrane after internalization of the DPV3-bodipy conjugate.

 E. coli bacteria immobilized on a poly-L-lysine slide are incubated with 3M of the DPV3-Bodipy conjugate at 37 ° C. for 30 minutes. After internalization, the outer membrane of living bacteria is detected by immunostaining using an anti-endotoxin mouse monoclonal antibody and a rabbit anti-mouse IgG polyclonal antibody coupled to TRITC. The localization of the DPV3-Bodipy conjugate (green fluorescence) and the immunolabeling of the outer membrane (red fluorescence) are observed under a confocal microscope. A: Original size of the image; B and D: Two bacterial enlargements of image A.

 In order to confirm the location of the DPV-Bodipy conjugates in the bacterial cytoplasm, the E. coli bacteria are incubated with 3M of the DPV3-Bodipy conjugate as described in Section III.8 and the outer membrane of the live bacteria is visualized by specific immuno-labeling as described in paragraph III 9.

Endotoxin is a specific constituent of the outer membrane of Gram-negative bacteria. The fluorescence of the bacteria is visualized under a confocal microscope (Figure 4).

The internalization of the Bodipy is visualized by a green fluorescence and the outer membrane is identified by a red fluorescence. The analysis of these images clearly shows that the DPV3 peptide crosses the membranes

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 external and internal gram-negative bacterium E. coli and allows the accumulation of Bodipy in the bacterial cytoplasm.

 - Qualitative evaluation in the bacterium P. aeruginosa.

 Figure 5 shows the internalisation of DPV-Bodipy conjugates in P. aeruginosa.

 The bacteria immobilized on a slide are incubated with 1 M of DPV-Bodipy conjugate for 30 minutes at 37 ° C. The microscopy images (epifluorescence optical microscope, immersion objective X 63) show the penetration of the DPV-Bodipy conjugate into the live bacteria. . A: DPV3, B: DPV3.10; C: DPV6; D: DPV7; E: DPV7b; F: DPV15; G: DPV15b; H: DPV1047; I: DPV11; J: DPV12; K: DPV1121; L: DPV19.

 Figure 6 gives confocal microscopy images of P. aeruginosa.

 The bacteria are immobilized on a poly-Lysine slide and incubated with 3 M of DPV3-Bodipy conjugate (A) or of DPV7-Bodipy conjugate (B) at 37 ° C. for 30 min and then fixed on the slide. Bacteria are observed under a confocal microscope. An enlargement of the original image of the bacteria is presented.

 The same qualitative assessment is carried out on P aeruginosa. Figure 5 shows the internalization of the conjugates in the bacteria after 30 minutes of incubation.

The internalization properties of DPVs are identical to those observed for E. coli except for the DPV7b and DPV6 peptides which seem to be more internalizing in P. aeruginosa. Confocal microscopic observation (FIG. 6) of the bacteria incubated with the DPV3 or DPV7b peptides shows that these peptides have the capacity to cross the outer membrane and allow the accumulation of Bodipy in the bacterial cytoplasm.

 II.l.b) Classification of DPVs.

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As shown in FIGS. 3 and 5, the level of accumulation of DPV-Bodipy conjugates in the bacterial cytoplasm is variable according to the DPV and according to the strain of bacteria. In general, the internalization of DPV is almost identical for the two strains of bacteria studied. The DPV peptides can be classified into three major groups: - DPV3, DPV3.10: high internalization - DPV6, DPV7, DPV7b, DPV15: medium internalization - DPV15b, DPV1047, DPV1121: weak internalization
The peptides DPV3.10, DPV3, DPV6, DPV7 and DPV7b have previously been described as cytoplasmic localization peptides in eukaryotic cells (PCT International Patent Application Publication No. WO 01/64738) when chemically coupled to the peroxidase protein or IgG. In contrast, the peptides DPV15, DPV15b, DPV1047 and DPV1121 have been described as nuclear localization peptides. It is important to note that the level of internalization of nuclear DPV is lower than that of cytoplasmic DPV. Peptides with a cytoplasmic tropism are the most internalising in the bacterium.

 As indicated in Table 1 below, peptides DPV19, DPV11 and DPV12 do not have internalizing property in the eukaryotic cell. The same property is observed with prokaryotic cells, such as Gram-negative bacteria.

 Table 1: Qualitative Evaluation of the Internalization of DPV-Bodipy Conjugates in Gram-Negative Bacteria

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<Tb>
<tb> E. <SEP> coli <SEP> P. <SEP> aeruginosa
<tb> DPV3-Bodipy <SEP> +++ <SEP> +++ <SEP>
<tb> DPV3.10-Bodipy <SEP> ++++ <SEP> ++++ <SEP>
<tb> DPV6-Bodipy <SEP> ++ <SEP> +++
<tb> DPV7-Bodipy <SEP> ++ <SEP> ++
<tb> DPV7b-Bodipy <SEP> + <SEP> +++
<tb> DPV15-Bodipy <SEP> ++ <SEP> ++
<tb> DPVl5b-Bodipy <SEP> + <SEP> +
<tb> DPV1047-Bodipy <SEP> + <SEP> +
<tb> DPV1121 # Bodipy <SEP> + <SEP> +
<tb> DPV19-Bodipy
<tb> DPV11-Bodipy <SEP> +/- <SEP> + / DPV12-Bodipy
<Tb>

II.l.c) Study of the effect of poly-L-Lysine (support of bacteria) on internalization.

 In order to confirm the previous results and to evaluate a possible interference of the poly-L-Lysine with the internalization of the conjugates, the E. coli bacteria are incubated with the DPV-Bodipy conjugates for 30 minutes at 37 ° C. and then washed in a controlled manner. extensively with the NAPB buffer before being immobilized and fixed or not on a poly-L-Lysine slide. The localization of the conjugates is visualized using an epifluorescence or confocal optical microscope. The internalization properties of the various DPVs do not differ from the results obtained previously. Poly-L-Lysine has no effect on the ability of DPV to cross bacterial membranes and enter the bacterium.

 II.l.d) Influence of temperature on the level of internalisation.

 In order to explain the internalisation mechanism previously observed, the ability of DPVs to internalize

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 +4 C was analyzed. The E. coli bacteria exponential growth phase are immobilized on poly-L-Lysine slide and then incubated for 24 hours at +4 C to abolish the energetic metabolism of the bacteria. The bacteria are then incubated with 3M of DPV7-Bodipy or Cyst-Bodipy (control) for 30 minutes at +4 ° C as described in paragraph I.8.b) and washed extensively before being visualized under a microscope. optical epifluorescence and confocal. To compare internalisation levels at 37 C and +4 C, the same experiment is conducted at 37 C as described in III.8.a.

 The degree of internalization of DPV7 in the bacterium is identical regardless of the temperature of the experiment. It therefore seems that the internalisation of the DPV7-Bodipy conjugate in E. coli is not an energy-dependent mechanism. The phenomenon is probably a passive translocation through bacterial membranes.

 II.2) Internalization of the DPV3-TMR conjugate in E. coli.

 We have shown that some DPV peptides can cross the outer membrane of Gram-negative bacteria and enter the bacterium to internalize a hydrophobic compound such as the Bodipy that is normally excluded by this outer membrane.

 In order to validate the results obtained previously and exclude any influence of the Bodipy fluorescent tracer on internalization, identical experiments were carried out with a second hydrophobic fluorescent tracer, TMR. TMR differs from Bodipy by physicochemical properties such as its structure or the presence of a positive charge (Fig. 2).

 Figure 7 shows the internalisation of the DPV3-TMR conjugate in E. coli.

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 The E. coli bacteria are immobilized on a poly-L-Lysine slide and incubated with 1M of the DPV3-TMR conjugate at 37 ° C. for 30 min. After internalization, the outer membrane of living bacteria is detected by immunostaining using an anti-endotoxin mouse monoclonal antibody and a FITC-coupled mouse anti-mouse IgG rabbit polyclonal antibody. The localization of the DPV3-TMR conjugate (red fluorescence) and the immunolabeling of the outer membrane (green fluorescence) are observed under a confocal microscope. A: Original size of the image; B and D: Two bacterial enlargements of image A.

 The internalization of the DPV3 TMR conjugate is evaluated on E. coli bacteria immobilized on a poly-L-Lysine slide or in suspension. The bacteria are incubated with 1 M of DPV3-TMR conjugate or the Cyst-TMR control for 30 minutes at 37 ° C., and then fixed or not on a slide before being visualized under an epifluorescence optical microscope. Whatever the internalisation protocol used, no fluorescence is detected with the control conjugate while the DPV3TMR is visualized by the red fluorescence of the bacterium.

 In order to confirm the internalisation of the DPV3-TMR conjugate, the bacteria are immobilized on a polyL-Lysine slide and incubated with 1 M of conjugate at 37 ° C. for 30 minutes. Immunolabeling of the outer membrane is accomplished using mouse anti-endotoxin monoclonal antibody and rabbit anti-mouse IgG polyclonal antibody coupled to FITC. The localization of the DPV3-TMR conjugate is observed under a confocal microscope (FIG. 7). The DPV3-TMR conjugate crosses the outer membrane, enters the bacterium and accumulates in the cytoplasm. This result is identical to those obtained with the Bodipy fluorescent tracer.

 II.3) Anti-bacterial activity of DPV conjugates.

 To determine that the internalization of DPV conjugates does not induce the death of bacteria, the 12 conjugates

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 DPV-Bodipy and the DPV3-TMR conjugate are tested for anti-bacterial activity as described in Section I.10) None of the conjugates tested showed anti-bacterial activity on E. coli at the concentrations used in internalisation experiments. This experiment shows that the internalisation of the conjugates does not affect the bacterial viability. The internalization mechanism in the bacterium is not toxic.

Claims (11)

1) Anti-bacterial composition, more particularly directed against gram-negative bacteria, comprising the combination of: a) at least one peptide of 10 to 25 amino acid residues comprising: i) two positively charged domains at neutral pH consisting of from 3 to 9 amino acid residues of which at least two thirds are cationic amino acids, ii) between said positively charged domains, a group of two to three non-cationic amino acid residues, iii) to one and or the other of the N or C terminus of the peptide, a group of 0 to 10 and preferably 0 to 5 amino acid residues selected from the group consisting of non-hydrophobic amino acids and positively charged amino acids, but in the the case of a positively charged amino acid residue is not directly adjacent to the positively charged domains. b) at least one anti-bacterial compound.  2) A composition according to claim 1, characterized in that (i) the cationic amino acids of the two positively charged domains are selected from the group consisting of arginine and lysine, and that (ii) the non-cationic amino acids of group between said positively charged domains are non-hydrophobic amino acids, selected for example from the group consisting of: glutamic acid, serine, glycine, leucine, glutamine.  3) Composition according to one of claims 1 or 2, characterized in that the peptide is selected from the group consisting of the following sequences: SEQ ID NO.1, SEQ ID <Desc / Clms Page number 33>  NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.11.  4) Composition according to any one of claims 1 to 3, characterized in that the peptide is selected from the group consisting of the following sequences: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO5., SEQ ID NO. 6, SEQ ID NO.  5) Composition according to any one of claims 1 to 3, characterized in that the anti-bacterial compound is selected from those having physico-chemical properties making it unable to cross the membrane of gram negative bacteria.  6) Composition according to any one of the preceding claims, characterized in that the antibacterial compound is hydrophobic.  7) Composition according to any one of the preceding claims, characterized in that the anti-bacterial compound is chosen from the group comprising the following compounds: antibiotics of the macrolide family, ketolides such as erythromycin, clarithromycin, azithromycin, telithromycin.  8) Composition according to any one of the preceding claims, characterized in that it comprises the association of peptide (s) of anti-bacterial compound (s) either in the form of a mixture or of a product in which one or more identical or different peptides are covalently bound to one or more identical or different anti-bacterial compounds, optionally via a spacer arm. <Desc / Clms Page number 34>  9) Composition according to any one of the preceding claims, characterized in that it comprises a product of formula (I) below: (A-) m (X) p (-P) n (I) in which: A is the remainder of an antibacterial compound, P is the residue of a peptide, as defined in the preceding claims, and X represents either a covalent bond between A and P, or a spacer arm connecting at least one residue A to at least one a remainder P, m is an integer ranging from 1 to 3, n is an integer ranging from 1 to 3, and p represents zero or an integer at most equal to the largest of the numbers m and n.  10) A product of formula (I) below: (A-) m (X) p (-P). (I) as defined in claim 9.  11) Use of a peptide of formula (I) as defined in any one of claims 1 to 4 for the preparation of an antibacterial pharmaceutical composition, more particularly against gram-negative bacteria, wherein said peptide is associated with less an anti-bacterial compound as defined in one of claims 1 or 5 to 7.