Anti-bacterial composition, especially for controlling gram-negative bacteria, comprising a peptide and an advantageously hydrophobic anti-bacterial agent

Translation of description of equivalent WO 2005018650 A2

COMPO SITI ON ANT I - I BACTER UNION, P LUS

PARTY ESPECIALLY AGAINST BACTERIA GRAM NEGATIVE

PEPTIDE HAVING AN AGENT AND ANTI -BACTERIEN

ADVANTAGEOUSLY HYDROPHOBICALLY

The present invention concerns the field of anti-bacterial treatments and more particularly to methods and compositions to treat infections by gram negative bacteria in humans, animals or plants. It has been described in the prior art peptides capable of destroying bacteria (CB Park, Kim HS, Biochem Biophys Ki SC Res Commun 1998 Mar 6; 244 (1):.. 253-7). It has also been reported in International Patent Application PCT No. WO 01/64738 peptides capable of reacting with the aminoglycans and transport in eukaryotic or prokaryotic cells of molecules of interest. The amount of antibiotic molecules penetrates the bacteria depends on its structure and mechanisms involved in the transport of substrates. Gram-negative bacteria are distinguished structurally from Gram positive bacteria by the presence of two membranes constituting the bacterial envelope. If all the bacteria have an inner membrane, Gram negative bacteria have an additional single outer membrane. This hydrophobic outer membrane is a semi-permeable barrier that opposes the penetration of antibiotics but porins, proteins forming channels, allow entry of small hydrophilic solutes such as nutrients and antibiotics penicillin and tetracycline but exclude penetration big hydrophilic molecules and antibiotics of macrolide / ketolide. A major cause of treatment failure against Gram-negative bacteria is the emergence of resistant strains. Some resistances are related to a reduction of the permeability of the bacterial membranes (quantitative changes / qualitative porins). Other strengths include the presence of a membrane protein that causes the release of the antibiotic by an active efflux mechanism. The development of new types of anti-bacterial molecules or use of non-active commercial antibiotics against Gram-negative bacteria requires their entry and delivery across selective bacterial membranes. The present invention is specifically to provide new methods and compositions to effectively treat infections by gram negative bacteria even when they have developed resistance to antibiotics. This object is achieved thanks to the use of peptides capable of crossing the outer membrane of Gram-negative bacteria and, via this translocation of the membrane, to deliver molecules of interest that can not otherwise penetrate into bacteria, because of their physicochemical properties. Means penetration inside the bacteria, that the peptides of the invention facilitate or allow the penetration of molecules of interest in bacteria. The terms penetration and internalization are used synonymously below. The work in the context of the present invention have concerned Bodipy and the tetramethylrhodamine which are hydrophobic fluorescent molecules excluded by the outer membrane of Gram negative bacteria. These fluorescent labels were chosen to assess the properties of internalization of the peptides of the invention are chemically linked with hydrophobic molecules. Translocation of fluorescent tracers in the Gram-negative bacterium was evaluated qualitatively on Escherichia coli and Pseudomonas aeruginosa. The present invention therefore firstly relates to an antibacterial composition, more particularly directed against the Gram negative bacteria, comprising combining: a) at least one peptide of 10 to 25 amino acid residues comprising: i) two positively charged domains at a neutral pH constituted each have 3 to 9 amino acid residues in at least two thirds are cationic amino acids, ii) between said positively charged domains, a group of two to three acid residues noncationic amino, iii) has one and either N- or C-terminal of the peptide, a group of 0 to 10 and preferably from 0 to 5 amino acid residues selected from the group consisting of amino acids non-hydrophobic and positively charged amino acids, but in the case of an amino acid positively charged residue thereof is not directly adjacent to the positively charged domains. b) at least one anti-bacterial compound. Thus, the peptides of the invention are especially useful for the preparation of a pharmaceutical composition for the treatment of infection, particularly with gram negative bacteria, composition wherein said peptide through the membrane of bacteria so as to deliver to the interior thereof an antibacterial compound which it is associated in said composition. Advantageously, in the peptides of the invention above, the cationic amino acids of both positively charged domains are selected from the group consisting of arginine and lysine. It is preferred in the peptides of the invention above that the non-cationic amino acids of the group between said positively charged domains are amino acids: - non-hydrophobic, for example selected from the group comprising glutamic acid, serine, glycine, and glutamine, or - leucine (hydrophobic amino acid).

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 can be reversed, that is to say that amino acid sequences are oriented C-terminal to N-terminal. Preferred peptides for the compositions according to the invention are selected from the group comprising the following sequences (N-terminal to C- terminal direction): - DPV3: Lys Arg Lys Arg Arg Glu Arg Ser Arg Lys Arg Lys Arg Arg Glu Ser ( SEQ ID No.l) - DPV3.10: Lys Arg Lys Arg Arg Glu Arg Ser Ala Arg Arg Arg Arg Ser Leu Pro Arg His (SEQ ID NO.2) - DPV6: Arg Gly Pro Arg Gly Ser Glu Lys Lys Arg Lys Arg Lys Arg Leu Lys Pro (SEQ ID NO.3) - DPV7: Lys Arg Gly Lys Gly Lys Leu Lys Lys

Gly Lys Arg Lys Asp Pro (SEQ ID NO.4) - DPV7b: Gly Lys Arg Lys Lys Lys Gly Lys Gly Lys Leu Lys Arg Pro Ser Arg Arg (SEQ ID NO.5) - DPV15: Leu Arg Arg Glu Arg Ser Gin Arg Leu Arg Arg Glu Arg Gln Arg Ser (SEQ ID NO.6) - DPV15b: Ala Tyr Gly Asp Arg Leu Arg Arg Glu Arg Gln Arg Ser Leu Arg Arg Arg Glu Arg Gln Arg Ser (SEQ ID NO.7) - DPV10 7: Val Leu Gly Lys Arg Leu Lys Arg His Val Arg Pro Val Thr Arg Arg Met Asp Val (SEQ ID

NO.8) - DPV11: Ala Lys Thr Gly Lys Arg Lys Arg Ser Gly (SEQ ID NO.9) - DPV1121 Val Arg Gly Lys Leu Lys Leu Lys Arg Gln Lys Arg Tyr Asn Ala Met Asp Tyr (SEQ ID NO. 11) Among them, the invention relates especially to the following peptides: DPV3, DPV3.10, DPV6, DPV7, DPV7b, DPV15 and DPV15b. The alignment of the above sequences reveals positively charged domains of the following sequences: - Arg Lys Lys Arg Arg Arg (SEQ ID NO.13) - Pro Arg Arg (SEQ ID NO.14) - Lys Arg 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) - Arg Arg Arg Arg Ala Pro Ser Arg (SEQ ID NO.18) - Lys Lys Arg Lys Arg Lys Arg Leu Lys (SEQ ID NO.19) - Lys Lys Arg (SEQ ID NO.20) - Lys Arg Lys Arg Pro Ser Arg (SEQ ID NO.21) - Arg Leu Arg Arg Glu Arg (SEQ ID NO.22) - Arg Leu Arg Arg Arg Glu Arg (SEQ ID NO.23) preferably, the domains of the sequences SEQ

ID NO. 13 to 17 are on the side of the N-terminus of the peptide, while the areas of sequences SEQ ID NO. 18-23 are on the side of the C-terminus of the peptide. Sequence alignment above also revealed groups of two to three residues non-cationic amino acid between the positively charged domains of the following sequences: Glu Ser Glu Ser Gly Leu Gly Gin Ser

Anti -bactériens present compounds in the compositions according to the invention are preferably chosen from those having the physicochemical properties rendering them incapable of crossing the membrane of gram negative bacteria. Most preferably, this is hydrophobic antibacterial compounds. Examples of such compounds include antibiotics of the macrolide family, ketolides 1 as erythromycin, clarithromycin, azithromycin, telithromycin. Antibacterial compounds may also be antisense oligonucleotides.

Evaluation of DPV above peptides to show their ability to pass through membranes of gram-negative E. coli or P. aeruginosa bacteria and deliver Bodipy in the bacteria, whereas the latter is a hydrophobic molecule normally excluded by the outer membrane of Gram negative bacteria which is a semi-permeable barrier. 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 mechanism and non-toxic for the bacteria. The work done in the context of the invention showed some differences in internalization between the two strains of P. aeruginosa bacteria and E. coli. Thus it appears that the peptide is DPV7b more internalizing in P. aeruginosa than in E. coli. This difference could be explained by differences in structures of the outer membrane between the two strains of bacteria. These peptides are therefore useful for the preparation of pharmaceutical compositions antibacterial, particularly against gram-negative bacteria, where they are associated with one or more anti-bacterial agents. The compositions of the invention are useful as a preventive than curative. The compositions according to the invention also advantageously comprise one or more carriers, diluents or excipients generally used with this type of agents. The peptides of the invention may be prepared by chemical synthesis or by genetic engineering in a prokaryotic cell such as a bacterium, in a eukaryotic cell such as yeast, CHO (Chinese Hamster Ovary), a NSO cell (Mouse myeloma cells), in a transgenic animal, such as rabbit, goat, sheep, cow, etc .. transgenic or in a transgenic plant such as, for example, in tobacco plants, etc .... The invention also concerns functional equivalents of the peptides defined above, such as peptides comprising of changes from post-translational processes such as glycosylation or chemical modifications such as coupling with lipids, sugars, nucleotide sequences as long as these changes do not alter the anti-bacterial activity and / or antifungal said peptides in accordance with the tests given in the experimental section below after. Functional equivalents also include peptides in which one or more amino acids are amino acids conformation D. The invention also covers the retro-peptides and retro-inverso-peptides.

The combination 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 active agents (and peptide anti-bacterial compound) of the composition covers the plural. The composition according to the invention can be achieved by the peptide combination (s) and compound (s) Anti-bacterial (s) in admixture or a product wherein one or more identical or different peptides are covalently bonded one or more identical or different compounds, optionally via a spacer arm. Such products are in particular the products of formula (I) which will be described below. In the case of the peptide administration and anti-bacterial compound in the mixture, these two active agents of the antibacterial composition of the invention may be presented separately, each in a suitable pharmaceutical form, and combined in a same package. However, to facilitate the simultaneous administration of active agents are generally preferred to prepare the drug in a single dosage form containing both active ingredients in the mixture and possibly a suitable pharmaceutical excipient. Of course, a product consisting of a peptide directly or indirectly linked to an anti-bacterial compound is to be regarded as constituting in itself a combination according to the invention and which can be used as the sole active ingredient. For example, a peptide and an anti-bacterial compound may be combined in establishing a chemical bond therebetween. Mention may in particular amidify an amino function of the peptide, or esterifying one or more alcohol functions of the peptide with an acid group present in a compound of anti-bacterial or derivative thereof. Thus, a product of amidation which is the active ingredient of the composition of the invention. May also be added to one and / or the other of the N and / or C terminal peptide of an amino acid residue whose side chain allows coupling with an anti-bacterial compound, such as a residue of cysteine ​​SH group which is reactive. Examples of such products are those of formula (I) which are described below. Indeed, the invention also relates to new products wherein the peptide and the anti-compound blight are bonded to each other by covalent bonding, optionally via at least one spacer arm. Such products include those which have the formula (I): (A-)_m(X)_p(-P)_not (I) wherein: A is the radical of an antibacterial compound, P is the residue of a peptide as defined above, and X represents either a covalent bond between A and P, or a spacer arm linking at least one residue with at least one P residue, 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 greatest of numbers m and n. It may indeed be graft one or more A and / or P on one spacer arm, or graft one or more AX groups on a rest P (and then m ≈ p and n = 1) or graft one or more XP groups on an a residue (and then n = p and m = 1).

When p = zero or one or more radicals A are directly related to a rest P (and n = 1), one or more radicals P are directly linked to an A residue (and m = 1). The products of formula (I) may be used in the form of salts, particularly as alkali metal salts such as sodium or potassium salts; these salts are for instance those of the phosphate groups, if present, phenolic groups (as in salicylic acid), etc. One can also use the products of formula (I), where appropriate in the form of addition salts (e.g. as hydrochloride) when these products contain an amino group. The bonds between the spacer arm and the radicals A and P or directly between A and P are covalent bonds. Such covalent bonds can be formed, as indicated above, between, carboxylic ester, carboxylic amide, thiocarboxylic ester or thiocarboxylic amide. antibacterial compound of the radicals (A) and peptide (P) are antibacterial compound derivatives or peptide, one or more chemical groups have been either deleted or modified to permit covalent bond formation directly between A and P or indirectly via a spacer arm. It may be functions acyl anti -bactériens compounds having a carboxyl group capable of forming a bond with the spacer arm or peptide, the latter having a primary amino or a hydroxyl group capable of forming a covalent bond with the spacer arm or the anti-bacterial compound. The spacer arm may include bivalent radicals of aliphatic bifunctional compounds, such as compounds having at each of their ends reactive functional groups each capable of forming covalent bonds with A and P. These compounds may be for example compounds who possess both an amino group and a carboxyl group (or thiocarboxylic) or compounds that possess both an amino group and a hydroxyl group. In formula (I), the group X (disregarding its end functional groups) especially represents 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 derivatives thereof, of formula (I) wherein A and P are connected by spacer arms, are for example alpha-, beta- or gamma-amino alkane, particularly alpha acids - natural amino acids such as glycine, alanine, valine or leucine, or peptides including dipeptides or tripeptides. As indicated in the examples, it can advantageously be a cysteine ​​residue. The spacers agents may also be hydroxy-carboxylic acids such as lactic acid, glycolic acid, aldonic acids (gluconic, mannonic, galactonic, ribonic, arabinonic, xylonic and erythronic) and the corresponding lactones or dilactones (eg, lactide, glycolide, glucolonactone delta, delta-valéronactone) or the aldaric acids. The functional groups optionally present on the spacer arm and not involved in the bond with A or P may be used to graft other A and / or P in order to obtain compounds of formula (I) wherein m and / or n are greater than 1. This is the case for example, hydroxyl groups hydroxy acids, the second carboxylic group of dicarboxylic amino acids, the amino group of the second dia ines amino acids, the hydroxyl group of the hydroxyl amino acids, etc. The spacer arm is advantageously constituted a binding molecule capable of enabling the delayed release of one and / or the other of the radicals A or P, in particular by protecting from degradation after administration. The spacer arm can also constituted a vectorization molecule to target a particular organ or tissue for the issue remains of anti -bactérien compound. To prepare compounds of formula (I), conventional methods of organic synthesis are used. For example, to prepare amides or esters, can be reacted a carboxylic compound as a carboxylic acid halide (or thiocarboxylic), or in the form of a mixed anhydride, or in the form of an activated ester, for example an ester of p-nitrophenyl. Can also activate the acid using a coupling agent such as dicyclohexylcarbodiimide. Since the compounds of formula (I) include peptide residues, can be prepared in particular using the methods known in peptide chemistry. Of course, where the compounds which derive A, P or X in formula (I) include several functions which can react, it is necessary to operate either using the reagents in stoichiometric amounts (depending on the number of products precursors A and / P or one wants to react), or temporarily protecting the reactive functions of which one does not wish they react. This is done using the methods of temporary protection said reactive functions. These methods of temporary protection are well known, including those that were developed during research on the synthesis of peptides. For example, -NH₂ may be protected by carbobenzoxy, phthaloyl, tert-butoxycarbonyl, trifluoroacetyl, toluenesulfonyl; carboxylic groups can be protected as benzyl esters, esters of tetrahydropyranyl or t-butyl esters; alcohols can be protected as esters (e.g. acetates), in the form of tetrahydropyranyl ethers, ethers of benzyl or trityl ethers, as well as acetals (including in the form of acetonides in the case of vicinal glycols). Protection reactions and deprotection of various chemical groups are known and described in the literature. Reactions phosphating or phosphate removal of the primary alcohol of nucleotides or nucleosides can be implemented using the natural enzymes (e.g. phosphatase, phosphokinase).

The antibacterial compositions of the invention, in particular those comprising a compound of formula (I) may be administered by any of the accepted modes of administration for therapeutic agents and of course by oral, sublingual, nasal , pulmonary, rectally or parenterally (for example intravascular, intramuscular, transcutaneous, intra-articular). Mention may also be systemic administration, topical or central, for example by surgically intracranial or intraocular administration. We can also mention the subcutaneous implantation of biodegradable implants. For this purpose, they can be presented in any form allowing administration by: - Orally (in particular in the form of gel capsules, drinkable solutions or emulsions, powders, gels, granules, lozenges or tablets), tablets, capsules, soft capsules, including formulations with delayed release or prolonged, pills, powders, granules, elixirs, tinctures, suspensions, syrups, emulsions. This form of presentation is particularly suited for the passage of the intestinal barrier and the most common use of anti-bacterial and / or fungal compounds. parenterally, usually by intramuscular or intravenous injection by infusion. 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 or prolonged release formulations such as the inclusion of the peptides in micro or biodegradable nano particles of lipid formulation or dextran formulation or PLGA or equivalent. This form of presentation is particularly suitable for passage of the blood-brain barrier and hospital use of anti-bacterial compounds and / or antifungal. One possibility for parenteral administration employs the implantation of a slow-release or sustained-release system that maintains a constant dose level. Another possibility consists in fixing by adsorption or other peptides of the invention on a support, such as a catheter, a prosthesis or biological glue. - Nasally (for example solutions to be administered as drops or sprays), - pulmonary route (solutions in a pressurized bottle for aerosols) - rectally (suppositories), - dermal (e.g. creams, ointments or patches , also known as patch or patches), - transmucosally such as sublingually (solutions in a pressurized bottle, or tablets disintegrating oral). These pharmaceutical forms are prepared in the usual way and may contain excipients and appropriate conventional vehicles.

Other conventional topical preparations include creams, ointments, lotions, gels and aerosol sprays. They are more particularly suited to the treatment of bacterial bronchopulmonary infections and / or fungal infections. The compositions of the invention can also be used in cosmetics, essentially preventive, and then consisting of creams, nail polish, hygiene products genitals, toothpastes, mouth hygiene solutions or include in micro-particles slow release, in aqueous phase, included for example in diapers, cotton swabs, bandages, cotton cleansing, sanitary napkins or animal bedding. Depending on the mode of administration, the compounds can be in solid, semi-solid or liquid. For solid compositions such as tablets, pills, powders or granules in the free state or included in capsules, the association can 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 polyethyleneglycol; - Binders, for example magnesium aluminum silicate, starch paste, gelatin, 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, colorants, flavors and sweeteners. The excipients may be for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and pharmaceutically like. For semi-solid preparations such as suppositories, the excipient can be, for example, be an emulsion or oily suspension, or based on polyalkylene glycol, such as polypropylene glycol. The liquid compositions, in particular injectable or included in a soft capsule can be prepared for example by dissolving, etc. the active ingredient in a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, oils and the like. The compositions according to the invention may also be administered in the form of the liposome type delivery systems, such as in the form of small vesicles Unila ellaires, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines. In one embodiment, a film of liquid components may be hydrated with an aqueous solution of 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, stabilizing, wetting or emulsifying, agents promoting dissolution, salts for regulating the osmotic pressure and / or buffers. In addition, they may also contain other substances of therapeutic interest. The compositions are prepared, respectively, by conventional mixing, granulating or coating and contain about 0.1 to 75%, preferably from about 1 to 50%, active ingredient. The peptides and anti-bacterial agents of the combination of the composition according to the invention may also be coupled with soluble polymers such as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxy-ethyl-aspana ide-phenol or poly (ethylene oxide) - polylysine substituted with palmitoyl residues, dextran. In addition, the compounds according to the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, poly (lactic acid), poly (epsilon-caprolactone), poly ( hydroxybutyric acid), polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and hydrogel copolymers crosslinked or amphipathic sequences. The dose for administration of the compositions according to the invention is chosen based on many factors including type, species, age, weight, sex and medical condition of the subject, the severity of the condition being treated, the route of administration; the condition of the renal and hepatic function of the subject and the particular compound or salt employed. A doctor or normally experienced veterinarian will readily determine and prescribe the effective amount to prevent, frustrate or stop the progress of the medical condition being treated. A composition according to the invention may contain 0.1 to 99%, preferably 1 to 70% active ingredient. As examples, the oral dosages of the compositions according to the invention will be between about 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 1000 mg of active ingredient. Effective plasma concentrations will be obtained from a dosage ranging from 0.002 mg to 50 mg per kg body weight per day. The compositions of the invention may be administered in the form of a single daily dose or in two, three or four doses per day. Other advantages and features of the invention emerge from the examples which follow, given by way of illustration, and in which reference will be made to the accompanying drawings where: Figure 1 represents the formula Bodipy^® FL N- (2-aminoethyl) maleimide. 2 shows the formula of tetramethylrhodamine-6-maleimide. Figure 3 shows the internalization of DPV ~ Bodipy in E. coli. Figure 4 shows immuno-marking outer membrane after internalization DPV3-bodipy. 5 represents the internalization of DPV Bodipy in ~ P. aeruginosa. Figure 6 shows confocal microscopy images of P. aeruginosa. Figure 7 shows the internalization of DPV3-TMR conjugate in E. coli. I - Materials and Methods. 1.1) Fluorescent tracers. - The Bodipy^® FLN- (2-aminoethyl) maleimide (Bodipy) (Molecular Probes Cat # B-10250), which has the molecular formula C₂₀H₂₁BF₂NOT₄O₃ , The molecular weight of 414.22 Da, absorbance of 504nm and emission of 510nm (green fluorescence), and the structural formula is shown in Figure 1. tetramethylrhodamine-6-maleimide (TMR) (Molecular Probes Cat # T-6028), the formula molecular is C₂₈H₂₃NOT₃0₅, The molecular weight of 481.51 Da, absorbance of 541nm and 567nm emission (red fluorescence) and the structural formula is shown in Figure 2. These two fluorescent molecules contain a maleimide reactive group for chemical coupling of the thiol group of cysteine ​​of the peptide. 1.2) The peptide vectors (DPVs). The peptide sequences below were used: - DPV3: Lys Arg Lys Arg Arg Glu Arg Ser Arg Lys Arg Lys Arg Arg Glu Ser (SEQ ID No.l) with Cys (cysteine) to its C-terminal, - DPV3.10: Lys Arg Lys Arg Arg Glu Arg Ser

Arg Arg Arg Arg Ala Ser Leu Pro Arg His (SEQ ID NO.2) with a Cys residue at its C terminus - DPV6: Arg Gly Pro Arg Gly Ser Glu Lys Lys Arg Lys Arg Lys Arg Leu Lys Pro (SEQ ID NO.3) with a Cys residue at its C terminus - DPV7: Gly Lys Arg Lys Lys Lys Gly Lys Leu Lys Arg Gly Lys Asp Pro (SEQ ID NO.4) with a Cys residue at its C terminus - DPV7b: Gly Lys Arg Lys Lys Lys Gly Lys Gly Lys Leu Lys Arg Pro Ser Arg Arg (SEQ ID NO.5) with a Cys residue at its C terminus - DPV15: Leu Arg Arg Glu Arg Gln Arg Ser Leu Arg Arg Glu Arg Gln Arg Ser (SEQ ID NO.6) with a Cys residue at its C terminus - DPV15b: Ala Tyr Gly Asp Arg Leu Arg Arg

Glu Arg Ser Gin Arg Leu Arg Arg Arg Glu Arg Ser Gln Arg (SEQ ID NO.7) with a Cys residue at its N terminus, - DPV1047 Val Leu Gly Lys Arg Leu Lys Arg His Val Arg Pro Val Thr Arg Arg Met Asp Val (SEQ ID NO.8) with a Cys residue at its N terminus - DPV11: Ala Lys Thr Gly Lys Arg Lys Arg Gly Ser (SEQ ID NO.9) with a Cys residue at its

Terminal C - DPV12: Gly Lys Gln Lys Arg Ser Glu Lys Arg Lys Asp Val Phe (SEQ ID NO.10) with a Cys residue at its C terminus - DPV1121 Val Lys Arg Leu Lys Leu Gly Arg

Lys Gln Lys Arg Tyr Asn Ala Met Asp Tyr (SEQ ID NO.11) with a Cys residue at its N terminus - DPV19: Asn Gly Val Pro Ser Thr Val Leu Gly Val Tyr Ala Asp Arg Leu Arg Arg Glu Arg Gln Ser Arg (SEQ ID NO.12) with a Cys residue at its N terminus. The peptide syntheses were performed according to known techniques of the art. The peptides are soluble in water. The peptides have a cysteine ​​residue at the N- or C-terminal position to permit conjugation to fluorescent tracer. 1.3) Controls products. Bodipy and TMR are chemically coupled to a cysteine ​​residue and used as a negative control for internalization. 1.4) Chemical coupling method. Solutions or Bodipy TMR were prepared at a final concentration of 50 mM in dimethylformamide (DMF). DPVs solutions were prepared at a final concentration of lOmM in DMF. 200 .mu.l of the solution Bodipy TMR or mixed with 700 .mu.l of the CAE solution. After incubation for 2 hours at room temperature in the dark, 2 ml of H20 and 8ml 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 extractions are performed with DCM. The aqueous phases are combined in a glass vial and placed for 1 hour at -80 ° C before being lyophilized at least 18 hours. The resulting powder was stored under argon at -20 ° C protected from light. 1.5) Conservation conjugates in solution. The DPV ~ Bodipy and DPV-TMR are preserved diluted to 3 mM in H20 to -20 ° C, protected from light. 1.6) HPLC analysis of the conjugates. - For the combined Bodipy: Column Luna 3μ C18 100A 100x4.6 mm Solvent A: 0.1% TFA in the H₂0 Solvent B: 0.1% TFA in 1 acetonitrile (CAN) Gradient: 5% B to 60% in 10 min, 60% to 90% B in 1 min, 90% B for 3 min, 5% B for 2 min flow: 1.2 ml / min; Volume injected: 10 .mu.l; the concentration of the injected sample was 1 mg / ml in 0.1% TFA Detector: DAD: 214nm, 300 nm. - For TMR conjugates: Column Luna 3μ C18 100A 100x4.6 mm Solvent A: 0.1% TFA in the H₂0 Solvent B: 0.1% TFA in 1 acetonitrile (CAN) Gradient: 5% B to 60% in 10 min, 60% to 90% B in 1 min, 90% B for 3 min, 5% B for 2 min flow: 1.2 ml / min; Volume injected: 20 .mu.l; the concentration of the injected sample was 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) Protocol internalization. 1.8. a) Evaluation of Penetration of the conjugates in the bacterium at 37 ° C. Bacteria in culture exponential phase were centrifuged and washed 3 times with lOmM sodium phosphate buffer, pH7. (NAPB buffer). The bacterial concentration was adjusted to 1x10^e cfu / ml (colony forming units) in the NAPB buffer. 50 .mu.l of the bacterial suspension are deposited on blade poly-L-Lysine. After incubation for 30 minutes at 37 ° C in a humid chamber, immobilized on the blade bacteria are rinsed 3 times with the NAPB buffer. 50 .mu.l of conjugate solution CAE ~ Bodipy or DPV-TMR or product controls are placed on the bacteria. After a 30 minute incubation at 37 ° C in a humid chamber, protected from light, the slides are rinsed 3 times with the buffer NAPB. Bacteria can be fixed on the slide by a 20 minute incubation at 37 ° C protected from light. A drop of PBS / Glycerol 50% is deposited on the slide and covered with a glass coverslip. After sealing the glass on the slide, the fluorescence of the bacteria was observed under optical microscope Leica epifluorescence (objective 40X or 63X immersion). The images are taken with the Nikon Coolpix digital camera at maximum zoom and with adapter 0.63 X. A more detailed analysis is performed confocal microscope Bio-Rad MRC 600 (BIO-RAD Microscience Ltd., Hemel Hempstead, England) equipped an optical inverted microscope and an objective to X100 immersion. Bacteria were visualized by their fluorescence after excitation by a Krypton / Argon laser. Various cuts of bacteria 0.1 - 0.2μm are made. I.8.b) Evaluation of Penetration of the conjugates in the bacterium at + 4 ° C. The method described above (1.8. A) has been amended as follows. Bacteria immobilized on a poly-L-lysine and rinsed 3 times with the NAPB buffer were incubated for 24 hours at + 4 ° C before the addition of fluorescent conjugates DPV preincubated at 4 ° C. All subsequent steps are performed at 4 ° C with cold solutions. 1.9) Immuno-labeling of the outer membrane of bacteria: indirect immunofluorescence. Bacteria immobilized on a poly-L-lysine are rinsed 3 times with NAPB buffer and incubated for 30 minutes at laboratory temperature with a NAPB / BSA solution 0.05% (bovine serum albumin). The bacteria were incubated 30 min at laboratory temperature with mouse monoclonal anti-endotoxin (Biovalley Cat # C55157; batch # 212529) diluted in NAPB / BSA 0.05%, washed several times with NAPB / 0.05% BSA and then incubated 30 minutes at laboratory temperature, protected from light, with a second antibody: rabbit anti-mouse polyclonal antibody conjugated to tetramethyl rhodamine (TRITC) (Jackson ImmunoResearch # 315-026-003 Cat, batch # 47511 ) or fluorescein (FITC) ((Jackson ImmunoResearch Cat # 715- 095-150, batch # 51038). After several washes with NAPB buffer, a drop of PBS / Glycerol 50% is deposited on the slide and covered with a glass slide. After sealing the cover glass on the slide, the fluorescence of the bacteria is observed in a confocal microscope as described previously (§ 1.8. a). 1.10) Evaluation of the antibacterial activity of the conjugates. The minimum inhibitory concentrations (MIC) are determined by the microdilution method in liquid medium (NCCLS M7A5) for all bacterial species in 96-well polystyrene plate. A single colony of E. coli ATCC 25922 or P. aeruginosa ATCC 27853 is suspended in 3 to 5 ml of Mueller-Hinton culture medium (MH) and incubated at 37 ° C overnight with stirring. From the overnight culture, a culture in the exponential phase of growth of the strain is carried out; the MH medium was seeded at l / 50th with overnight culture and incubated for 2 hours at 37 ° C with stirring. The bacterial concentration was adjusted to lxl0⁶ cfu / ml (colony forming units) in the MH medium. 50 .mu.l of bacterial inoculum was distributed by wells containing an equal volume of the diluted conjugate solution of half half in adequate culture medium (0-1 microM). The cultures were incubated at

37 ° C in ambient air for 16 to 20 hours. The MIC expressed in .mu.M is the first concentration not exhibiting bacterial growth.

II - Results. 1 1. 1) Internal i sat ion s conjugate CAE ~ s Bodipy in Gram-negative bacteria. II. 1. a) Eva lua ti at on qua bed ive

1 'internal ization of DPV conjugates ~ Bodipy. - Evaluation qual itative in the bacterium E. coli. Figure 3 shows the internalization of DPV ~ Bodipy in E. coli. Bacteria immobilized on poly-L-Lysine blade are incubated with conjugate IμM DPV Bodipy ~ for 30 minutes at 37 ° C. The images of microscope (optical microscope epi-fluorescence, X 63 immersion lens) show penetration combined DPV ~ Bodipy in 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; The DPV19. The bacteria E. coli were incubated 30 minutes at 37 ° C with conjugate lμM DPV ~ Bodipy as described in paragraph 1.8. at. The internalization of DPV ~ fluorescent Bodipy in unfixed bacteria was visualized under epifluorescence microscope. No fluorescence is detected with the control conjugate Cys ~ Bodipy. As shown in Figure 3, several DPV ~ Bodipy through bacterial membranes of E. coli to accumulate in the bacterial cytoplasm. The DPV3.10 peptides (Fig. 3B), DPV3 (Fig. 3A), DPV6 (Fig. 3C) and DPV15 (Fig. 3F) are highly penetrating and internalizing of hydrophobic molecules. With DPV11 DPV12 and peptides (Fig. 3J and 31), we obtain heterogeneous fluorescence levels in the same bacterial population. The properties of internalization of these peptides are lower. DPV19 the peptide does not penetrate into the bacteria. (Fig. 3L). An identical profile internalization of DPV ~ Bodipy observed after fixing bacteria. Figure 4 shows immuno-marking outer membrane after internalization DPV3-bodipy. The E. coli bacteria immobilized on poly-L-Lysine slide were incubated with 3 microns of DPV3 ~ Bodipy at 37 ° C for 30 minutes. After internalization, the outer membrane of the living bacteria was detected by immuno-labeling using a monoclonal anti-endotoxin mouse and mouse anti-IgG rabbit polyclonal antibody coupled to TRITC. The location of DPV3 ~ Bodipy (green fluorescence) and immuno-marking outer membrane (red fluorescence) are observed in the confocal microscope. A: Original size of the image; B and D: Two magnifications of the image of bacteria A. To confirm the location of DPV-Bodipy conjugates in the bacterial cytoplasm, bacteria E. coli were incubated with 3 microns of DPV3-Bodipy as described in paragraph 1.8 and the outer membrane of the living bacteria was visualized by specific immunolabeling as described in paragraph I 9. Endotoxin is a specific constituent of the outer membrane of Gram-negative bacteria. The fluorescence of the bacteria was visualized with a confocal microscope (Figure 4). Internalization Bodipy is displayed by green fluorescence and the outer membrane is identified by a red fluorescence. The analysis of these images clearly shows that DPV3 peptide passes through the outer and inner membranes of the gram-negative bacterium E. coli and allows the accumulation Bodipy in the bacterial cytoplasm. - Qualitative evaluation in bacteria P. aeruginosa. 5 represents the internalization of DPV-Bodipy conjugates P. aeruginosa. Bacteria immobilized on slide were incubated with conjugate lμM DPV-Bodipy for 30 minutes at 37 ° C. The images of microscope (optical microscope epi-fluorescence, X 63 immersion lens) show penetration of DPV-Bodipy conjugate in the 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. Figure 6 shows confocal microscopy images of P. aeruginosa. The bacteria are immobilized on a poly-L-lysine and incubated with conjugate 3 microns DPV3-Bodipy (A) or conjugate DPV7-Bodipy (B) at 37 ° C for 30 min and then fixed on the slide. The bacteria are observed in confocal microscope. An enlargement of the original image of bacteria is presented. A same qualitative assessment is performed on P aeruginosa. 5 shows one internalization of conjugates into bacteria after 30 minutes of incubation. The properties of internalization of DPV are identical to those observed for E. coli except for DPV7b DPV6 and peptides that appear to be more internalizing in P. aeruginosa. Observation with a confocal microscope (Figure 6) of the bacteria incubated with DPV3 or DPV7b peptides shows that these peptides have the ability to cross the outer membrane and allow the accumulation Bodipy in the bacterial cytoplasm. Il.lb) Classification DPVs. As shown in Figures 3 and 5, the level of accumulation of DPV-Bodipy in the bacterial cytoplasm varies according to the DPV and according to the bacteria strain. Generally, 1 internalization of DPV is almost identical for the two bacterial strains studied. The DPV peptides can be classified into three major groups: - DPV3, DPV3.10: high internalization - DPV6, DPV7, DPV7b, DPV15: average internalization - DPV15b, DPV1047, DPV1121: low DPV3.10 the internalization peptides, DPV3, DPV6, and DPV7

DPV7b have previously been described as peptides cytoplasmic localization in eukaryotic cells (International Patent Application PCT published under

No. WO 01/64738) when they are chemically coupled to peroxidase protein or IgG. In contrast, the peptides DPV15, DPV15b, DPV1047 DPV1121 and have been described as nuclear localization peptides. It is important to note that the level of internalization of DPV "nuclear" is lower than that of DPV "cytoplasmic". The peptides having a cytoplasmic tropism are more internalizing in the bacterium. As shown in Table 1 below, the DPV19 peptides, and DPV11 DPV12 have no ownership of internalisation in the eukaryotic cell. The same property is observed with the prokaryotic cells, such as Gram-negative bacteria. Table 1: Qualitative evaluation 1 interna-zations of DPV-Bodipy conjugates in Gram-negative bacteria. Table 1 E. coli P. aeruginosa DPV3-Bodipy +++ +++ <img id="imgf000032_0001" he="90" wi="123" file="imgf000032_0001.tif" img-format="tif" img-content="table" orientation="portrait" inline="no" />

II.lc) Study of the effect of poly-L-Lysine (support bacteria) on internalization. To confirm previous results and evaluate the potential interference of the poly-L-Lysine with the internalization of the conjugates, the bacteria E. coli were incubated with DPV-Bodipy for 30 minutes at 37 ° C and then extensively washed with the NAPB buffer before being immobilized and fixed or not on poly-L-Lysine blade. The location of the conjugates was visualized by light microscopy or confocal epifluorescence. The properties of various internalisation of DPV do not differ from previous results. Poly-L-Lysine has no effect on CAE's ability to cross the bacterial membrane and penetrate the bacteria. Il.ld) Effect of temperature on the level of internalisation. To explain the mechanism of internalization previously observed, the ability of DPV to internalize at 4 ° C was analyzed. The bacteria E. coli in exponential growth phase were immobilized on poly-L-lysine slide and incubated for 24 hours at + 4 ° C in order to abolish the energy metabolism of the bacteria. The bacteria are then incubated with 3 microns of DPV7 or Cyst ~ Bodipy-Bodipy (control) for 30 minutes at + 4 ° C as described in paragraph I.8.b) and extensively washed before being visualized optical microscope and confocal epifluorescence. To compare the levels of internalization at 37 ° C and + 4 ° C, the same experiment was conducted at 37 ° C as described in paragraph I .8. at. The level of internalization of the bacterium in DPV7 is identical whatever the temperature of the experiment. So it seems that the internalization of the conjugate DPV7 ~ Bodipy in E. coli is not an energy-dependent mechanism. The phenomenon is probably a passive translocation across bacterial membranes. II.2) Internalization of DPV3-TMR conjugate in E. coli. We have shown that some DPV peptides may cross the outer membrane of Gram-negative bacteria and the bacteria penetrate to internalize a hydrophobic compound as Bodipy which is normally excluded by this outer membrane. To validate the previous results and exclude any influence of fluorescent tracer Bodipy on internalization, identical experiments were conducted with a second hydrophobic fluorescent tracer, the TMR. The TMR differs Bodipy by physico-chemical properties such as its structure or the presence of a positive charge (Fig. 2). Figure 7 shows the internalization of DPV3-TMR conjugate in Ξ. coli. E. coli bacteria are immobilized on a poly-L-lysine and incubated with lμM of DPV3-TMR conjugate at 37 ° C for 30 min. After internalization, the outer membrane of the living bacteria was detected by immuno-labeling using a monoclonal anti-endotoxin mouse and mouse anti-IgG rabbit polyclonal antibody coupled to FITC. The location of DPV3-TMR conjugate (red fluorescence) and 1 'immunolabeling of the outer membrane (green fluorescence) were observed under a confocal microscope. A: Original size of the image; B and D:

Two enlargements of the image of bacteria A. The internalization DPV3 TMR is evaluated on E bacteria. coli immobilized on a poly-L-lysine or in suspension. The bacteria were incubated with conjugate lμM DPV3-TMR or Cyst-TMR control for 30 minutes at 37 ° C and then fixed or not on blade before being viewed under the optical microscope epifluorescence. Whatever the protocol used internalization, no fluorescence is detected with the conjugate control while DPV3 - TMR is shown by the red fluorescence of the bacteria. To confirm 1 internalization DPV3-TMR conjugate, the bacteria are immobilized on a poly-L-lysine and incubated with conjugate lμM at 37 ° C for 30 minutes. The immunolabeling of the outer membrane is performed using the monoclonal anti-endotoxin mouse and a mouse anti-rabbit IgG polyclonal antibody coupled to FITC. The location of DPV3-TMR conjugate was observed under the confocal microscope (Figure 7). The DPV3-TMR conjugate through the outer membrane, the bacterium enters and accumulates in the cytoplasm. This result is identical to those obtained with the Bodipy fluorescent tracer. 11.3) anti -bactérienne Activity DPV. To determine that the internalization of DPV does not cause the death of bacteria, 12 DPV-Bodipy and DPV3-TMR conjugate are tested for their anti-bacterial activity as described in paragraph 1.10) None of conjugates tested showed antibacterial activity on E. coli concentrations used in the experiments internalization. This experiment shows that 1 internalization conjugates does not affect bacterial viability. The mechanism for internalization into the bacterium is not toxic.

III - Internalization of a conjugate CAE ~ Antibiotic (eg CAE ~ Erythromycin) III .1) S ynthè sed a con j ugué

CAE ~ Antibiotic III .1. a) Activation of^f Erythro ycine by a heterobifunctional cross linker A solution of maleimidocaproic acid (MIC) (2.8 equivalents) and dicyclohexylcarbodiimide (DCC) (2.8 equivalents) in dimethylformamide (DMF) is stirred overnight at 0 ° C under argon and away from light. The precipitate (dicyclohexylurea) was removed by filtration, washed with DMF and then filtered again. An antibiotic solution (1.0 equivalent) and pyridine (5.0 equivalents) in DMF is stirred until complete dissolution. We add on this solution, the filtrate obtained above; the mixture is stirred 1 hour at room temperature. The solution is taken in distilled water, washed 4 times with dichloromethane (DCM). The organic phases obtained are combined and washed successively with hydrochloric acid (HCl) 0.1 N, two times with disodium carbonate (Na₂C0₃), Three times with water (H₂0). After drying over magnesium sulfate (MgS0₄) And concentration, the crude reaction product is purified by flash chromatography on silica (eluent CH₂C1₂ / MeOH). III. lb) Coupling of Erythromycin activated with a penetrating peptide DPV A penetrating peptide (1.0 equivalent) in a sodium phosphate buffer solution (NaH₂P0₄/N / A₂HP0₄) To 10 mM and pH 7.1 was stirred five minutes at room temperature under argon, protected from light. Then, antibiotic activated (1.5 to 2.0 equiv), dissolved in the minimum of DMF is added. The solution is stirred until complete conversion of the peptide (HPLC monitoring). Distilled water was then added and the aqueous phase is extracted three times with the same volume of dichloromethane to remove excess activated antibiotic. The aqueous phase is then lyophilized and the obtained solid was purified by preparative HPLC. The antibiotic-penetrating peptide conjugate is thus isolated with yields of between 45 and 100% and 90% higher purities. 111.2) Evaluation of the antimicrobial activity of DPV-Erythromycin. The minimum inhibitory concentrations (MIC) of conjugates are determined by the microdilution method in liquid medium according NCCLS- standards M7A5 (National Committee for Clinical Laboratory Standards - Document M7A5) for all bacterial species in 96-well polystyrene plate. Protocol: A single colony of bacteria (eg, E. coli and P. aeruginosa) is suspended in 3 to 5 ml of Mueller-Hinton culture medium (MH) and incubated at 37 ° C overnight with stirring. From the overnight culture, a culture in the exponential phase of growth of the strain is carried out; the MH medium was seeded at l / 50th with overnight culture and incubated for 2 hours at 37 ° C with stirring. The bacterial concentration was adjusted to lxl0^ε cfu / ml (colony forming units) in the MH medium. 50 .mu.l of bacterial inoculum was distributed per well containing an equal volume of the diluted conjugate solution half in half in the adequate culture medium (512 to 0.5 mcg / trtx). The cultures are incubated at 37 ° C in ambient air for 16 to 20 hours. The MIC expressed in micrograms / ml (Units

International) is the first concentration not exhibiting bacterial growth. The determination of the minimal bactericidal concentration (MBC) is performed after reading the MIC plates. The CMB is the lowest concentration of conjugate that inhibits bacterial growth on the subculture agar (&lt;0.1% of survivors).

IV - Evaluation of the antibacterial activity of the combination of a DPV peptide and 1 erythromycin A by the method of IV-1 board) Materials and Methods Peptides selected: DPV3 and DPV3.10 antibacterial compound: Erythromycin (Sigma E0774) Bacterial strains: E. coli ATCC 25922 and P. aeruginosa ATCC 27853. This method is carried out in polystyrene 96-well microplates in the Mueller-Hinton culture medium (MH) and involves exposing a suspension bacteria to different concentrations of DPV peptide and erythromycin, used alone or in combination. The selected final concentrations of erythromycin and peptide ranging from 256 respectively to 4μg / ml and from 256 to 2μg / ml. The dilution ranges are prepared according to a geometrical progression of ratio 2 25μl of MH medium in product solutions whose concentration is four times higher than the desired final concentration or the middle of 25μl MH (for rows 0) are distributed in the wells according to the diagram below (Table 2) to obtain a final volume of 50 .mu.l per well: Table 2: distribution of product solutions middle MH (X marked wells were not used) Table 2

<img id="imgf000038_0001" he="68" wi="156" file="imgf000038_0001.tif" img-format="tif" img-content="table" orientation="portrait" inline="no" /><img id="imgf000039_0001" he="18" wi="156" file="imgf000039_0001.tif" img-format="tif" img-content="table" orientation="portrait" inline="yes" />

A single colony of E. coli ATCC 25922 or P. aeruginosa ATCC 27853 is suspended in 3 to 5 ml of MH culture medium and incubated at 37 ° C overnight with stirring. From the overnight culture, a culture in the exponential phase of growth of the strain is carried out; the MH medium was seeded at l / 50th with overnight culture and incubated for 2 hours at 37 ° C with stirring. The bacterial concentration was adjusted to 5xl0⁵ - 10^s cfu / ml (colony forming units) in the MH medium. 50 .mu.l of bacterial inoculum was distributed per well containing an equal volume of the peptide solution and / or erythromycin. The MIC of peptide and 1 'éryhromycine is determined as the lowest concentration that causes the absence of bacterial growth (absence of turbidity) following 18 hours of culture in an incubator at 37 ° C. The MIC is expressed in g / ml (mg / 1). For each row of wells, the first wells containing the combination of DPV peptide and the erythromycin and showing no visible growth were noted to calculate the fraction for each index of the inhibitory concentration (FIC) using the formula next: FIC = (MIC of peptide 1 with erythromycin / MIC of peptide alone) + (MIC 1 erythromycin with peptide / MIC of erythromycin alone) This index quantifies the association. A lower index not exceeding 0.5 indicates a synergy, an index greater than 2 antagonism, an additive effect is shown by an FIC range between 0.5 and 1, and an indifference effect, by FIC with values ​​between 1 and 2. IV.2) Results gram-negative bacteria such as E. coli and P. aeruginosa are resistant to antibiotics of the macrolide as 1 erythromycin due to the non-penetration of the antibiotic through the outer membrane of the bacterium. To assess internalizing properties of DPVs peptides identified previously and their ability to facilitate the penetration of an antibiotic in the macrolide antibacterial activity of 1 erythromycin on E. coli and P. aeruginosa was evaluated in combination with peptide or DPV3 DPV3.10 that known to the board. The synergistic effect of the combination of peptides DPV3 and 3.10 with the erythromycin is shown in Tables 3 and 4. In the presence of DPV3, a synergistic effect with the erythromycin is observed only on E. coli showing that this peptide allows the entry of erythromycin in E coli. The association with DPV3.10 1 erythromycin is synergistic on both bacterial strains. DPV3.10 the peptide to the non-toxic concentration of 32μg / ml allows penetration (1 internalization) of erythromycin. Table 3: Determination of MICs and FICs 1 erythromycin in combination with DPV3 Table 3

<img id="imgf000040_0001" he="42" wi="157" file="imgf000040_0001.tif" img-format="tif" img-content="table" orientation="portrait" inline="yes" /><img id="imgf000041_0001" he="16" wi="157" file="imgf000041_0001.tif" img-format="tif" img-content="table" orientation="portrait" inline="yes" /> * Values ​​in parentheses are concentrations (mcg / ml) added DPV3 Table 4: Determination of MICs and FICs of erythromycin in the presence of peptide DPV3.10 Table 4

<img id="imgf000041_0002" he="48" wi="157" file="imgf000041_0002.tif" img-format="tif" img-content="table" orientation="portrait" inline="yes" /> * Values ​​in parenthesis are its s concentrations (mcg / ml) of DPV3. 10 aj outed