INFECTIONS CAUSED BY GRAM NEGATIVE PATHOGENS

The present invention is within the field of biomedicine and pharmacy, and refers specifically to peptides against anti-outer membrane protein A (OmpA) to treat the infections caused by Gram negative pathogens.

STATE OF THE ART

Acinetobacter baumannii is a Gram negative coccobacillus non-fermenting with high clinical significance due to the substantial increase in the number of severe nosocomial infections that causes, mainly in intensive care units (ICUs), and to its ability to develop resistance to most of the antimicrobial agents used in clinical practice [Mortensen and Skaar. Cell. Microbiol. 2012; 14: 1336-44]. The most common infections caused by A. baumannii are, in order of frequency, pneumonia, bacteremia, urinary tract infections, surgical site infections and meningitis [McConnell MJ, Actis L, Pachon J. FEMS Microbiol. Rev. 2013; 37: 130-55]. In a prospective study performed at the University Hospital Virgen del Rocio in Seville in 1993, it was found that 18% of patients infected or colonized with A. baumannii developed bacteremia, forming the 27% of bacteremia by Gram-negative bacilli, with an associated mortality rate of 34% [Cisneros et al. Clin. Infect. Dis. 1996; 22: 1026-32]. In another study performed in the same center in 2003, A. baumannii caused approximately 9% of the ventilator-associated pneumonia (VAP), with a crude mortality rate of 40% [Garnacho et al. Crit. Care Med. 2003; 31 : 2478-82]. Moreover, A. baumannii is responsible for other infections such as meningitis and ventriculitis in patients undergoing neurosurgery or head trauma with a mortality rate of 25% [Jimenez-Mejias et al. Clin. Infect. Dis. 1997; 24: 932-5]. In a project performed by the inventors (FIS

PS09/01427), the crude mortality of patients with VAP by A. baumannii was 26%; 16% of them developed secondary bacteremia with an associated mortality rate of 62.5%. Thus, A. baumannii has grown from a microorganism with questionable pathogenicity to a bacterial agent with clinical importance associated with high mortality [Munoz-Price and Weinstein. N. Engl. J. Med. 2008; 358: 1271 -81].

On the other hand, infections with A. baumannii have become very difficult to treat due to the high rate of strains resistant to all or almost all antimicrobial agents used in clinical practice [McConnell MJ, Actis L, Pachon J. FEMS Microbiol. Rev. 2013; 37: 130-55]. These multidrug resistant (MDR) strains, including resistance to carbapenems, are sometimes susceptible to polymyxins (colistin and polymyxin B) [Asensio et al. Enferm. Infecc. Microbiol. Clin. 2008; 26: 199-204]. Despite the demonstration by the inventors that A. baumannii lost virulence after acquisition of resistance to colistin [Lopez-Rojas et al. J. Infect. Dis. 201 1 ; 203: 545-8; Lopez-Rojas et al. J. Infect. Dis. 201 1 ; 204: 1 147-8], ciprofloxacin [Smani et al. Clin. Microbiol. Infect. 2012; 18: E1 -4], and imipenem

[Fernandez-Cuenca et al. Int. J. Antimicrob. Agents 201 1 ; 38: 548-9], the attributable mortality and the difficulty to eradicate A. baumannii have classified this bacterium as highly successful human pathogen [Perez et al. Expert Rev. Anti Infect. Ther. 2008; 6: 269-71].

Pseudomonas aeruginosa, other Gram negative aerobic bacillus, causes nosocomial infections in patients undergoing invasive procedures in ICUs with cystic fibrosis, or immunosuppressed [Schechner et al. Clin. Infect. Dis. 2009; 48: 580-6; Gaynes and Edwards. Clin. Infect. Dis. 2005; 41 : 848-54; Lyczak et al. Clin. Microbiol. Rev. 2002; 15: 194-222]. This pathogen produces a wide range of infections such as pneumonia, skin and soft tissue infections, urinary tract infection, ocular infection, bacteremia, septicemia and endocarditis. Data provided in 201 1 by the Spanish Society of Preventive Medicine, Public Health and Hygiene in the Study of Nosocomial Infections in Spain (EPINE) showed that P. aeruginosa was the second most common etiologic agent isolated in nosocomial infections (10.2%) after Escherichia coli (17.6%). This study showed also a remarkable increase in the prevalence of respiratory infections and bacteremia in the period 1990-201 1 . Furthermore, It was reported that the rate of mortality in VAP and bacteremia caused by P. aeruginosa varied between 34% and 68%, and between 18% and 61 %, respectively [El Solh et al. Am. J. Respir. Crit. Care Med. 2008; 178: 513-9, Parkins et al. Infection 2010; 38: 25-32].

In a multicenter national study performed in Spain in 2008, the mortality rate of patients with bacteremia caused by P. aeruginosa was 29%. 8% of them were caused by strains resistant to carbapenems [Pena et al. Antimicrob. Agents Chemother. 2012; 56: 1265-72]. The incidence of pneumonia by P. aeruginosa acquired in the community is much less common than in the hospital, and is associated with mortality rate ranging between 50% and 100% [Fujitani et al. Chest 201 1 ; 139: 909-19].

In recent years, the treatment of infections caused by P. aeruginosa has become more difficult due to the emergence of MDR strains. The high frequency of infections caused by MDR strains has increased the use of colistin [Molina et al. Expert Opin. Pharmacother. 2009; 10: 281 1 -28]. The report from European Network for Antimicrobial Resistance Surveillance (EARS-Net) in 2012 highlights the high percentage of P. aeruginosa clinical isolates resistant to aminoglycosides, ceftazidime, fluoroquinolones, piperacillin/ tazobactam and carbapenems, especially in the South and East Europe. 15% of these isolates were resistant to at least three classes of antibiotics (MDR strains), and 5% were resistant to five classes of antimicrobials (pandrug-resistant (PDR) strains). In the United States, the rates of resistance to cefepime, ceftazidime, imipenem, piperacillin/

tazobactam, aztreonam, and colistin/polymexin B were 8.5%, 9.6%, 13.1 %, 1 1 .3%, 12.2%, and 3.2% respectively [Master et al. Ann. N. Y. Acad. Sci. 2013; 1277:1 -7]. £ coli, another Gram negative pathogen with high clinical importance, causes community and nosocomial infections including urinary and gastrointestinal tract infections, intraabdominal infection, and bacteremia. In recent decades, £. coli is increasingly resistant to antimicrobial agents such as beta-lactams, fluoroquinolones and aminoglycosides. £ coli resistance to broad-spectrum beta-lactams due to the production of extended spectrum beta-lactamase (ESBL) has increased from 18% to 28% between 2008 and 201 1.

Recently, resistance to carbapenems has increased due to the production of

carbapenemases [Nordmann et al. Emerg. Infect. Dis. 201 1 ; 17: 1791 -8; Walsh Int. J. Antimicrob. Agents. 2010; 36: S8-S14]. In the United States, 140,000 nosocomial infections by Enterobacteriaceae occur each year; approximately 26,000 and 9,300 of them are caused by ESBL-producing Enterobacteriaceae (EPE) and carbapenems- resistant Enterobacteriaceae (CRE), respectively. The 57% and 50% of all bacteremia by EPE and CRE, respectively, cause the mortality of patients [Antibiotic resistance threat in United States 2013. U.S. Department of Health and human Services, Centers for Disease Control and Prevention]. In Spain, the analysis of 4758 episodes of bacteremia by £ coli collected through a blood culture surveillance program between 1991 and 2007 showed that bacteremia was associated with 9% of patients mortality [Ortega et al. J. Antimicrob. Chemother. 2009; 63: 568-74].

The most studied virulence factor in A. baumannii is the outer membrane protein A

(OmpA). OmpA is a porin with beta barrel conformation, highly conserved among bacterial species, especially in Gram negative bacteria such as P. aeruginosa and £ coli [Smith et al. FEMS Microbiol. Lett. 2007; 273: 1 -1 1]. The role of OmpA in A. baumannii is very diverse and has a variety of interesting biological properties in vitro and in vivo. Studies from our group and others showed that OmpA is involved in the adherence of A.

baumannii to human epithelial cells [Gaddy et al. Infect Immun. 2009; 77: 15 3150-60; Smani et al. PLoS One 2012; 7: e33073] and macrophages [Choi et al. Cell. Microbiol. 2005; 7: 1 127-38], by binding to fibronectin, an extracellular matrix protein [Smani et al. PLoS One 2012; 7: e33073]. Furthermore, it was reported that OmpA is involved in the induction of host cell death [Choi et al. Cell. Microbiol. 2005; 7: 1 127-38], in the formation of biofilm by A baumannii [Choi et al. Cell. Microbiol. 2005; 7: 1 127-38], and in the binding of A. baumannii to factor H that allow A baumannii to be resistent to serum [Kim et al. FEMS Microbiol. Lett. 2009; 301 : 224-31 ]. On the other hand, the inventors have demonstrated that recombinant OmpA was able to induce host cell death [McConnell and Pachon. Prot. Expr. Purif. 201 1 ; 77: 98-103] by translocating to nucleus of human lung epithelial cells [Choi et al. Cell. Microbiol. 2008; 10: 309-19.]. Moreover, it has been observed that OmpA-containing membrane vesicles secreted by A baumannii induced host cell death through the /V-terminal residue of OmpA [Jin et al. PLoS One 201 1 ; 6: e17027].

In P. aeruginosa, OprF is the homologous of OmpA. Generally, OprF is involved in the non specific diffusion of ionic particles and nutrients. It was reported that OprF plays an important role in the interaction of P. aeruginosa with host cells. OprF is involved in the adherence of P. aeruginosa to lung epithelial cells, glial cells, and caco-2 cells, and in their death [Azghani et al. Microb. Pathog. 2002; 33: 109-14; Krishnan and Prasadarao FEBS J. 2012: 279: 919-31 ; Sugawara et al. FEBS J. 2012; 279: 910-8; Wu et al. Science 2005; 309: 774-7]. Furthermore, OprF contributes in the pathogenecity of P. aeruginosa in experimental model of worms such as Caenorhabditis elegans, in the formation of biofilm, and in the modulation of quorum sensing [Fito-Boncompte et al. Infect Immun 201 1 ; 79: 1 176-86].

In £ coli, the importance of OmpA in its pathogenicity was established in various in vitro and in vivo experimental models. It is known that OmpA is a virulence factor of £ coli strains causing meningitis. OmpA is involved in the adherence and invasion of these strains into endothelium and astrocytes of nervous system [Meier et al. Infect. Immun. 1996; 64: 2391 -9; Prasadarao et al. Infect. Immun. 1996; 64: 146-53; Wu et al. J.

Neuropathol. Exp. Neurol. 2009; 68: 677-90]. Weiser and Gotschlich have demonstrated that £ coli OmpA-deficient was less virulent in rat experimental model [Weiser and

Gotschlich. Infect. Immun. 1991 ; 59: 2252-8]. Moreover, it was reported that OmpA is involved in the adherence of enteropathogenic £. coli to epithelial cells in the mucosal surfaces, to leucocytes and macrophages [Krishnan and Prasadarao. FEBS J. 2012; 279: 919-31 ]. On the other hand, OmpA is essential to promote the persistence of infection in the urogenital epithelium. Nicholson et al. have demonstrated that during the urinary tract infection, OmpA was overexpressed 20 to 30-fold [Nicholson et al. Infect. Immun. 2009; 77: 5245-51]. Moreover, it is known that OmpA can stimulate the murine dendritic cells to release pro-inflammatory cytokines [Torres et al. Infect. Immun. 2006; 74: 2676-85].

From this literature, it is evident that OmpA or its homologous protein are main virulence factors in A. baumannii, P. aeruginosa and E. coli. Therefore, OmpA or its homologous protein are good candidates for development of molecules to stop the infections caused by these pathogens. In the last years, different studies reveal a substantial increase in antimicrobial resistance of A. baumannii, P. aeruginosa and E. coli. This increase creates a major problem due to the lack of other therapeutic alternatives, aggravated by the lack of development of new antimicrobial agents against Gram negative bacilli by the pharmaceutical industry. This last justifies the need to investigate other non-therapeutic antimicrobial alternatives. Among them, we find the inhibition of OmpA or its homologous protein in order to block the bacterial infection progression. To summarize, the frequency of severe nosocomial infections caused by MDR and PDR Gram negative pathogens has increased significantly over the recent decades. Due to this trend, it is necessary to develop new approaches for the treatment of nosocomial infections caused by these pathogens. Summary of the invention

The use of inhibitors of OmpA as directed therapy described in the present invention represents a new therapeutic approach to reduce the morbidity and mortality derived from the infections caused by Gram negative bacilli.

A first aspect of the invention refers to a cyclic peptide having 4-8 amino acids

independently selected from the group consisting of: an aromatic amino acid selected from the group consisting of Trp, 3Pal, 2Nal,1 Nal, Phe, Phe(4-CF3), Tyr, Tyr(P03H2), Tyr(Me), 5FTrp, 6FTrp and Bip; Pro or a proline derivative selected from the group consisting of Pro(4-NH ₂ ), Pro(3-NH ₂ ), Pro(N3), Acylated 4-NH ₂ -Pro, Acylated 3-NH ₂ -Pro,

Alkylated 4-NH ₂ -Pro, Alkylated 3-NH ₂ -Pro, Ala, Pip and /V-MeAla; a positively charged amino acid selected from the group consisting of Arg, hArg, Arg(Me), Orn, Lys, hLys, Dab Dap and His; Ser; and Cys, for use as medicament, wherein when the selected amino acid is Cys, there are two in the cyclic peptide and are forming a disulfide bridge. Peptide of the invention from now on, able to block OmpA. In a preferred embodiment of this aspect of the invention, the peptide is a cyclic hexapeptide. More preferably, the peptide is selected from the list including the peptide sequence SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 or any of their salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. Most preferably, the peptide is selected from the peptides of formula (I), formula (II), formula (III), formula (IV), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), formula (XI), formula (XII), formula (XIII), formula (XIV), formula (XV), formula (XVI), formula (XVII) and formula (XVIII). Even more preferably the peptide is selected from the peptides of SEQ ID NO: 1 formula (I), SEQ ID NO: 2 formula (II), SEQ ID NO: 3 formula (III), SEQ ID NO: 4 formula (IV), SEQ ID NO: 8 formula (VIII), SEQ ID NO: 10 formula (X), SEQ ID NO: 1 1 formula (XI),SEQ ID NO: 14 formula (XIV) ) and SEQ ID NO:18 formula (XVIII). And even more preferably the peptide is selected from the peptides of SEQ ID NO: 1 formula (I), SEQ ID NO: 2 formula (II), SEQ ID NO: 3 formula (III), SEQ ID NO: 4 formula (IV). Even more preferably the peptide is SEQ ID NO: 1 formula (I).

In other preferred embodiment, the amino acids are in D- or L-enantiomer form. It means aminoacids can all be D, all L or combinations of L and D.

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (I).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (II).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (III).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (IV).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (VI).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (VII).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (VIII). In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (IX).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (X).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (XI).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (XII).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (XIII).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (XIV).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (XV).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (XVI).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (XVII).

In a prefered embodiment of the first aspect of the invention the peptide is a peptide of formula (XVIII).

A particular embodiment of the invention refers to a cyclic peptide from 4 and 8 amino acids selected from tryptophan, proline, and arginine, peptide of the invention from now on able to block OmpA, for use as a medicament. In a preferred embodiment of this aspect of the invention, the peptide of the invention is cyclic. In a preferred embodiment of this aspect of the invention, the peptide is a cyclic hexapeptide. More preferably, the peptide is selected from the list including the peptide sequence SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or any of their salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. Most preferably, the peptide is selected from the peptides of formula (I), formula (II), formula (III) and formula (IV). Even more preferably the peptide is SEQ ID NO: 1 formula (I).

In a prefered embodiment of this aspect of the invention the peptide is a peptide of formula (I).

In a prefered embodiment of this aspect of the invention the peptide is a peptide of formula (II). In a prefered embodiment of this aspect of the invention the peptide is a peptide of formula (III).

In a prefered embodiment of this aspect of the invention the peptide is a peptide of formula (IV).

Another aspect of the invention refers to a composition, particularly a pharmaceutical composition, comprising a peptide of the invention, composition of the invention from now on, for use as a medicament. A preferred embodiment of this aspect of the invention refers to a pharmaceutical composition comprising at least one peptide of the invention, composition of the invention from now on, for use as a medicament. Preferably, the composition comprises an acceptable pharmaceutical carrier, and most preferably, the composition comprises other active ingredient.

Anotheraspect of the invention refers to a combined preparation, combined preparation of the invention from now on, which comprises a peptide of the invention, or a composition of the invention, and additionally other active ingredient.

Another aspect of the invention refers to a pharmaceutical form that comprises a peptide of the invention, or a composition of the invention. Preferably, the pharmaceutical form of the invention is selected from the list including plaster, pomade, paste, cream, solution, suspension, emulsion, lotion, liniment, gel, hydrogel, hydrocolloid, foam, powder, spray, or any their combinations.

Another aspect of the invention refers to the peptide of the invention, the pharmaceutical composition of the invention, the combined preparation of the invention, or the the pharmaceutical form of the invention for use as a medicament.

Another aspect of the invention refers to the peptide of the invention, the pharmaceutical composition of the invention, the combined preparation of the invention, or the

pharmaceutical form of the invention for use as in the treatment, alleviation and/or prevention of infections caused by Gram negative pathogens, or alternatively, the peptide of the invention, the composition of the invention, or the pharmaceutical form of the invention for use in the treatment, alleviation and/or prevention of infections caused by Gram negative pathogens.

Another aspect of the invention relates to novel cyclic peptides which are selected from the group consisting of: a) a cyclic peptide having 4-8 amino acids independently selected from the group consisting of: tryptophan, arginine, proline and a proline derivative selected from the group consisting of , Pro(4-NH ₂ ), Pro(3-NH ₂ ), Pro(N3), Acylated 4-NH ₂ -Pro, Acylated 3-NH ₂ -Pro, Alkylated 4-NH ₂ -Pro, Alkylated 3-NH ₂ -Pro, Ala, Pip and /V-MeAla; b) a cyclic peptide having 4-8 amino acids independently selected from the group consisting of: proline, arginine and a tryptophan derivative selected from the group consisting of Trp, 3Pal, 2Nal,1 Nal, Phe, Phe(4-CF3), Tyr, Tyr(P03H2), Tyr(Me), 5FTrp, 6FTrp and Bip; c) a cyclic peptide having 4-8 amino acids independently selected from the group consisting of: proline, tryptophan, and a positively charged amino acid selected from the group consisting of Arg, hArg, Arg(Me), Orn, Lys, hLys, Dab Dap and His; and d) a cyclic peptide having 4-8 amino acids independently selected from the group consisting of: proline, tryptophan, arginine, serine, alanine, and cysteine wherein when the selected amino acid is cysteine, there are two in the cyclic peptide and are forming a disulfide bridge.

It is part of the invention an implantable medical device comprising a cyclic peptide, or a combined preparation as defined above.

As used herein, the term "medical device" makes reference to an instrument,

apparatus, implant or related article that is used to diagnose, prevent, or treat a disease or another medical condition, and which does not achieve its purposes

through chemical action within or on the body. In use a medical device will be held into contact with some part of the body. The medical device may be a catheter of a prosthesis.

It is also part of the invention, the implantable medical device defined above comprising the cyclic peptide or the combined preparation as defined above for use as a medicament, and more particularly, for use in the treatment, relief and/or prevention of infections caused by Gram negative pathogens.

A further aspect of the present invention relates to the non-therapeutic use of a combined preparation as defined above, to inhibit growth of bacteria outside the human body. In a particular embodiment, the combined preparation are used to inhibith growth of bacteria over a medical device by contacting and/or depositing an amount onto the device outside the human body. In another particular embodiment, the peptide or the combined preparation are used to inhibit the formation of bacterial biofilms or to reduce the amount of already formed bacterial biofilms outside the human body. Description of the figures

Fig. 1. Effect of the inhibitors SEQ ID NO: 1 (A), SEQ ID NO: 2 (B), SEQ ID NO: 3 (C), SEQ ID NO: 4 (D), and SEQ ID NO: 5 (E) on the adherence of A baumannii to A549 cells. A549 cells were infected for 2 h by A baumannii ATCC 17978 strain (10 ⁸ cfu/ml) pretreated with these inhibitors (0.25, 0.5 and 1 mg/ml) for 30 min. Representative results of three independent experiments are shown. P<0.05: ^* between infected A549 cells, and infected A549 cells with inhibitors treatment. Fig. 2. Effect of SEQ ID NO: 1 on the adherence of A baumannii, P. aeruginosa and £ coli to A549 cells. A549 cells were infected for 2 h by A baumannii (10 ⁸ cfu/ml): ATCC 17978 strain (A), 77 strain (B) and 1 13-16 strain (C), by P. aeruginosa (10 ⁸ cfu/ml): PA01 strain (D), and by £ coli (10 ⁸ cfu/ml): ATCC 25922 strain (E), pretreated with SEQ ID NO: 1 (0.25 and 0.5 mg/ml) or with SEQ ID NO: 5 (0.5 mg/ml) for 30 min. Representative results of three independent experiments are shown. P<0.05: ^* between infected A549 cells and infected A549 cells with SEQ ID NO: 1 treatment.

Fig. 3. Effect of SEQ ID NO: 1 on the adherence/invasion of A baumannii, P. aeruginosa and £ coli by A549 cells. A549 cells grown on coverslip in 24-wells plates were infected for 2 h by A baumannii (10 ⁸ cfu/ml): ATCC 17978 strain (A), 77 strain (B) and 1 13-16 strain (C), by P. aeruginosa (10 ⁸ cfu/ml): PA01 strain (D), and by £. coli (10 ⁸ cfu/ml):

ATCC 25922 strain (E), pretreated with SEQ ID NO: 1 (0.25 and 0.5 mg/ml) or with SEQ ID NO: 5 (0.5 mg/ml) for 30 min. The bacterial adherence/invasion into A549 cells was determined by immunofluorescence using specific antibodies against OMPs of A

baumannii, P. aeruginosa and £ coli (stained in green), and against human fibronectin

(stained in red). A549 cells attached by one or more bacterial colonies were considered positive cells. Representative results of three independent experiments are shown.

Fig. 4. Effect of SEQ ID NO: 1 on the interaction of A baumannii, P. aeruginosa and £ coli with fibronectin. A baumannii (10 ⁸ cfu/ml): ATCC 17978 strain (A), 77 strain (B) and

1 13-16 strain (C), P. aeruginosa (10 ⁸ cfu/ml): PA01 strain (D), and £ coli (10 ⁸ cfu/ml): ATCC 25922 strain (E) were pretreated with SEQ ID NO: 1 at 0.25 and 0.5 mg/ml or with SEQ ID NO: 5 at 0.5 mg/ml for 30 min, and incubated with 10 μg/ml fibronectin pre-coated 96-wells plates for 2 h. Representative results of three independent experiments are shown. P<0.05: ^* between untreated bacteria and treated bacteria with SEQ ID NO: 1 . Fig. 5. Effect of SEQ ID NO: 1 on the cell death caused by A baumannii and P.

aeruginosa. A549 cells were infected for 24 h by A. baumannii (10 ⁸ cfu/ml): ATCC 17978 strain (A), 77 strain (B) and 1 13-16 strain (C), and by P. aeruginosa (10 ⁸ cfu/ml): PA01 strain (D), pretreated with SEQ ID NO: 1 (0.062, 0.125, 0.25 and 0.5 mg/ml) or with SEQ ID NO: 5 (0.5 mg/ml) for 30 min. The cellular viability was determined by MTT assay. Representative results of three independent experiments are shown. P<0.05: ^* between infected A549 cells, and infected A549 cells with SEQ ID NO: 1 treatment.

Fig. 6. Effect of SEQ ID NO: 1 on biofilm formation by A baumannii, P. aeruginosa and £ coli. Biofilm formation was determined for 24 h by crystal violet staining of standard

(ATCC 19606 and ATCC 17978) and clinical (77, C4, C5, C12, IB1 , IB2, HC1 , and HC2) A. baumannii strains (A), standard P. aeruginosa strain PA01 (B), and standard £ coli strain ATCC 1053 (C), pretreated with SEQ ID NO: 1 (0.25 and 0.5 mg/ml) for 30 min. Representative results of three independent experiments are shown. P<0.05: ^* between untreated bacteria and treated bacteria with SEQ ID NO: 1. C: colonizing, IB: bacteremic infecting, HC: blood culture.

Fig. 7. Effect of the inhibitors SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 on the adherence of A baumannii ATCC 17978 (A), P. aeruginosa Pa01 (B), and £ co// ^' ATCC 25922 (C) to A549 cells. A549 cells were infected for 2 h by A baumannii ATCC 17978 strain (10 ⁸ cfu/ml), P. aeruginosa Pa01 strain (10 ⁸ cfu/ml), and £. coli ATCC 25922 (10 ⁸ cfu/ml) pretreated with these inhibitors at 250 g/ml for 30 min. Representative results of two independent experiments are shown.

Fig. 8. Effect of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1 , and SEQ ID NO: 14 vs. SEQ ID NO: 1 on the adherence of A baumannii, P. aeruginosa and £ coli to A549 cells. A549 cells were infected for 2 h by A baumannii ATCC 17978 strain (10 ⁸ cfu/ml) (A), by P. aeruginosa PA01 strain (10 ⁸ cfu/ml) (B), and by £ co// ^' ATCC 25922 strain (10 ⁸ cfu/ml) (C), pretreated with SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1 , and SEQ ID NO: 14 (31.25, 62.5, 125, and 250 Mg/ml) or with SEQ ID NO: 5 (31 .25, 62.5, 125, and 250 Mg/ml) for 30 min. Representative results of two independent experiments are shown.

Fig. 9. Effect of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 on the adherence of A baumannii, P. aeruginosa and £ coli to A549 cells. A549 cells were infected for 2 h by A baumannii ATCC 17978 strain (10 ⁸ cfu/ml) (A), by P. aeruginosa PA01 strain (10 ⁸ cfu/ml) (B), and by £ co// ^' ATCC 25922 strain (10 ⁸ cfu/ml) (C), pretreated with SEQ ID NO: 16, SEQ ID NO: 17, , and SEQ ID NO: 18 (31.25 and 62.5 250 Mg/ml) or with SEQ ID NO: 5 (31.25 and 62.5 g/ml) for 30 min. Representative results of one independent experiments are shown. Fig. 10. SEQ ID NO:1 potentiates the colistin activity against colistin-sensitive and colistin- resistant A baumanniii in vitro. (A) Time-kill curves of ATCC 17978 and 1 1 strains in presence of SEQ ID NO:1 or SEQ ID NO:5 (0, 12.5 or 125 Mg/ml), colistin (sub-MIC) alone or in combination with SEQ ID NO:1 , SEQ ID NO:5 or colistin, for 24 h. (B) Time-kill curves of ATCC 17978 and 1 1 strains in presence of different combination between SEQ ID NO:1 , SEQ ID NO:5 or colistin, and SEQ ID NO:1 , SEQ ID NO:5 or colistin added for second time 4 h after bacterial addition, for 24 h. Col: colistin.

DETAILED DESCRIPTION OF THE INVENTION The authors of the present invention have demonstrate that the use of such peptides block the adherence/invasion of the host by Gram negative bacteria, in particular A baumannii, P. aeruginosa and E. coli, as well as the cytotoxicity caused by these pathogens. So, the present invention provides peptides inhibiting OmpA for use as a medicament in the treatment of the infections caused by Gram negative microorganisms.

As mentioned above, a first aspect of the invention refers to a cyclic peptide having 4-8 amino acids independently selected from the group consisting of: an aromatic amino acid selected from the group consisting of Trp, 3Pal, 2Nal,1 Nal, Phe, Phe(4-CF3), Tyr, Tyr(P03H2), Tyr(Me), 5FTrp, 6FTrp and Bip; Pro or a proline derivative selected from the group consisting of Pro, Pro(4-NH ₂ ), Pro(3-NH ₂ ), Pro(N3), Acylated 4-NH ₂ -Pro, Acylated

3-NH ₂ -Pro, Alkylated 4-NH ₂ -Pro, Alkylated 3-NH ₂ -Pro, Ala, Pip and /V-MeAla; a positively charged amino acid selected from the group consisting of Arg, hArg, Arg(Me), Orn, Lys, hLys, Dab Dap and His; Ser; and Cys, for use as medicament, wherein when the selected amino acid is Cys, there are two in the cyclic peptide and are forming a disulfide bridge. Peptide of the invention from now on, able to block OmpA.

3Pal is beta-(3-pyridyl)-alanine; 2Nal: Beta-(2-naphtyl)-alanine; 1 Nal is Beta-(l -naphtyl)- alanine; Bip is 4-phenyl-phenylalanine; Pro(N3) is Proline (4-azido); Pip is 2- piperidinecarboxylic acid; hArg is homoarginine; hLys is homolysine; Dab is alpha, gama- Diaminobutiric acid; Dap is alpha, beta-Diaminopropionic acid; 5FTrp is 5-fluorotryptophan; 6FTrp is 6-fluorotryptophan; Tyr(P03H2) is Phosphotyrosine;

Tyr(Me) is 0-methyl)-tyrosine; Phe(4-CF3) is (4-trifluoromethyl)-phenylalanine; Orn is Ornitine; and Arg(Me) is Arg(N-methyl).

In this document we understand by cyclic peptides a cycle between the /V-terminal and the C-terminal.

In a preferred embodiment of this aspect of the invention, the peptide of the invention is cyclic. In a preferred embodiment of this aspect of the invention, the peptide is a cyclic hexapeptide. More preferably, the peptide is selected from the list including the peptide sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 or any of their salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. Even more preferably the peptide is selected from the peptides of SEQ ID NO: 1 formula (I), SEQ ID NO: 2 formula (II), SEQ ID NO: 3 formula (III), SEQ ID NO: 4 formula (IV), SEQ ID NO: 8 formula (VIII), SEQ ID NO: 10 formula (X), SEQ ID NO: 11 formula (XI),SEQ ID NO: 14 formula (XIV) and SEQ ID NO: 18 formula (XVIII). And even more preferably the peptide is selected from the peptides of SEQ ID NO: 1 formula (I), SEQ ID NO: 2 formula (II), SEQ ID NO: 3 formula (III), SEQ ID NO: 4 formula (IV). Even more preferably the peptide is SEQ ID NO: 1 formula (I).

SEQ ID NO 1 = cy cl o [Trp- D- P ro-Arg-Trp- D- P ro-Arg]

SEQ ID NO 2 = cyclo [Arg-D-Pro-Trp-Arg-D-Pro-Trp]

SEQ ID NO 3 = cyclo [D-Arg-Pro-Trp-D-Arg-Pro-Trp]

SEQ ID NO 4 = cyclo [Arg-Pro-D-Trp-Arg-Pro-D-Trp]

SEQ ID NO 5 = Ac-Trp-D-Pro-Arg-Trp-D-Pro-Arg-OH (lineal control)

SEQ ID NO 6 = cyclo [Arg-D-Pro-Trp-Trp-D-Pro-Arg]

SEQ ID NO 7 = cyclo [Trp-D-Pro-Trp-Arg-D-Pro-Arg]

SEQ ID NO 8 = cyclo [Trp-D-Pro(4-NH ₂ )-Arg-Trp-D-Pro-Arg]

SEQ ID NO 9 = ^: cyclo [Tyr-D-Pro-Ala-Tyr-D-Pro-Ala]

SEQ ID NO 10 = cyclo [3Pal-D-Pro-Arg-3Pal-D-Pro-Arg]

SEQ ID NO 11 = cyclo [2Nal-D-Pro-Arg-2Nal-D-Pro-Arg]

SEQ ID NO 12 = cyclo [2Nal-D-Pro-Arg-3Pal-D-Pro-Arg]

SEQ ID NO 13 = cyclo [3Pal-D-Pro-Arg-Trp-D-Pro-Tyr]

SEQ ID NO 14 = cyclo [Trp-D-Pro-Dab-Trp-D-Pro-Dab]

SEQ ID NO 15 = cyclo [Trp-D-Pro-Lys-Trp-D-Pro-Lys] SEQ ID NO: 16 = cyclo [Arg-D-Pro-Cys-Arg-D-Pro-Cys] with a disulphide bridge between Cys

SEQ ID NO: 17 = cyclo [Ala-D-Pro-Cys-Ala-D-Pro-Cys] with a disulphide bridge between Cys

SEQ ID NO: 18 = cyclo [Ser-D-Pro-Cys-Ser-D-Pro-Cys] with a disulphide bridge between Cys

In a particular embodiment of the present invention, the cyclic peptide having 4-8 amino acids residues is independently selected from tryptophan, proline and arginine, from now on called peptide of the invention, for the use as medicament. In a preferred embodiment, the peptide of the invention is a cyclic peptide. In another preferred embodiment, it is a cyclic hexapeptide, and in a more preferred embodiment it is a cyclic hexapeptide of sequence SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or any of their pharmaceutically acceptable salts, prodrugs, derivatives or analogues, or any of their combinations.

In this document, we understand by SEQ ID NO:1 a cyclic hexapeptide of sequence cyclo[Trp-D-Pro-Arg-Trp-D-Pro-Arg], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:1 has the formula (Formula

I)-

Formula I

In this document, we understand by SEQ ID NO:2 a cyclic hexapeptide of sequence cyclo[Arg-D-Pro-Trp-Arg-D-Pro-Trp], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:2 has the formula (Formula II)

Formula II

In this document, we understand by SEQ ID NO:3 a cyclic hexapeptide of sequence cyclo[D-Arg-Pro-Trp-D-Arg-Pro-Trp], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:3 has the formula (Formula Hi)

Formula III In this document, we understand by SEQ ID NO:4 a cyclic hexapeptide of sequence cyclo[Arg-Pro-D-Trp-Arg-Pro-D-Trp], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:4 has the formula (Formula iv)

Formula IV

In this document, we understand by SEQ ID NO:5 an hexapeptide of sequence Ac-Trp-D- Pro-Arg-Trp-D-Pro-Arg-OH used as lineal control, or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:5 has the formula (Formula V).

Formula V In this document, we understand by SEQ ID NO:6 a cyclic hexapeptide of sequence cyclo[Trp-D-Pro-Arg-Arg-D-Pro-Trp], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:6 has the formula (Formula VI)

Formula VI

In this document, we understand by SEQ ID NO:7 a cyclic hexapeptide of sequence cyclo[Trp-D-Pro-Trp-Arg-D-Pro-Arg], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:7 has the formula (Formula VII)

Formula VII In this document, we understand by SEQ ID NO:8 a cyclic hexapeptide of sequence cyclo[Trp-D-Pro(4-NH ₂ )-Arg-Trp-D-Pro-Arg], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:8 has the formula (Formula VIII)

Formula VIII

In this document, we understand by SEQ ID NO:9 a cyclic hexapeptide of sequence cyclo[Tyr-D-Pro-Ala-Tyr-D-Pro-Ala], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:9 has the formula (Formula IX)

Formula IX In this document, we understand by SEQ I D NO: 10 a cyclic hexapeptide of sequence cyclo[3Pal-D-Pro-Arg-3Pal-D-Pro-Arg], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ I D NO:10 has the formula (Formula X)

Formula X

In this document, we understand by SEQ I D NO: 1 1 a cyclic hexapeptide of sequence cyclo[2Nal-D-Pro-Arg-2Nal-D-Pro-Arg], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ I D NO: 1 1 has the formula (Formula XI)

Formula XI In this document, we understand by SEQ I D NO: 12 a cyclic hexapeptide of sequence cyclo[2Nal-D-Pro-Arg-3Pal-D-Pro-Arg], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ I D NO:12 has the formula (Formula XII)

Formula XI I

In this document, we understand by SEQ I D NO: 13 a cyclic hexapeptide of sequence cyclo[3Pal-D-Pro-Arg-Trp-D-Pro-Tyr], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ I D NO:13 has the formula (Formula XI II)

Formula XII I In this document, we understand by SEQ I D NO: 14 a cyclic hexapeptide of sequence cyclo[Trp-D-Pro-Dab-Trp-D-Pro-Dab], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ I D NO:14 has the formula (Formula XIV)

Formula XIV

In this document, we understand by SEQ I D NO: 15 a cyclic hexapeptide of sequence cyclo[Trp-D-Pro-Lys-Trp-D-Pro-Lys], or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ I D NO:15 has the formula (Formula XV)

Formula XV In this document, we understand by SEQ ID NO:16 a bicyclic hexapeptide of sequence cyclo[Arg-D-Pro-Cys-Arg-D-Pro-Cys] with a disulphide bridge between cysteines, or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:16 has the formula (Formula XVI)

Formula XVI In this document, we understand by SEQ ID NO:17 a bicyclic hexapeptide of sequence cyclo[Ala-D-Pro-Cys-Ala-D-Pro-Cys] with a disulphide bridge between cysteines, or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:17 has the formula (Formula XVII)

Formula XVII

In this document, we understand by SEQ ID NO:18 a bicyclic hexapeptide of sequence cyclo[Ser-D-Pro-Cys-Ser-D-Pro-Cys] with a disulphide bridge between cysteines, or any of its salts, stereoisomers, enantiomers, prodrugs, derivatives or analogues, or any of their combinations. In a particular embodiment, the peptide compound of sequence SEQ ID NO:18 has the formula (Formula XVIII)

Formula XVIII

In this document we understand by "inhibition of OmpA" or "blocking of OmpA" a blockage of the channel of OmpA that avoids the interaction of this channel or porins with the proteins of the host.

Compounds that have the same atomic composition and connectivity but are mirrow images between them are enantiomers. A mixture of the enantiomers is call a racemic mixture. In this document the term "enantiomer" means a single enantiomer that is sustantially excempt of the opposite enantiomer and/ or any other stereoisomer. Also, the term "sustatially excempt of" used in this document refers that the ratio among

enantiomers is as example 85:15 and, preferably, 90:10, more preferably 95:5 and even more preferably, 99:1 or higher.

Any of the peptides of the invention and preferably the peptide of SEQ ID NO:1 SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:1 1 , SEQ ID NO:14 and SEQ ID NO: 18 could be found either as a single enantiomer or, alternatively, in the racemic form.

As it is used in this document, the term "derivative" includes either compounds

pharmaceutically acceptable, it means, derivatives of the compounds with formula (I), (II), (III), (IV), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII) and/or

(XVIII) that could be used in the preparation of a medicament, as well as, pharmaceutical derivatives not acceptable, as this could be useful in the preparation of acceptable pharmaceutical derivatives. Also in the scope of this invention, prodrugs of compounds of formula (I), (II), (III), (IV), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII) and/or (XVIII) are found. The term "prodrug" as it is used herein includes any derivatives of compounds of formula (I) (II), (III), (IV), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII) and/or (XVIII), as an example, esters, including esters and amides of carboxilic acids, esters of amino acids, phosphate esters, sulphonate esters of metalic salts, etc... carbamates, amides, etc. that when administered to an individual is able to provide, directly or indirectly, the compound of formula (I), (II), (III), (IV), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII) and/or (XVIII) to the individual. Advantageously, this derivative is a compound that increases the bioavailability of the compound of formula (I), (II), (III), (IV), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII) and/or (XVIII) when it is administered to an individual or that it increases the release of the compounds of formula (I), (II), (III), (IV), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII) and/or (XVIII) in a biological compartment of the individual. The preparation of this prodrug could be achieve using conventional methods known by the experts in the field.

The expression "pharmaceutically acceptable salt" as it is used herein, refers to salts of the compounds of the previous formulas that are sustantially non toxic for the living organisms. The typical pharmaceutically acceptable salts include the salts prepared by reaction of the present invention with a mineral or organic acid or an inorganic base. These salts are known as salts of acid addition and salts of base addition.

The acids commonly used to form the salts of acid addition are mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and similars, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid and similars. Examples of such pharmaceutically acceptable salts are sulfate, pirosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,

dihydrogenphosphate, metaphosphate, pirophosphate, chloride, bromide, iodide, acetate, propionate, decanate, caprylate, acrylate, formiate, isobutirate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- 1 ,4-dionate, hexyne-1 ,6-dionate, benzoate, chlorobenzoate, methylbenzoate,

dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,

xylenosulfonate, phenylacetate, phenylpropionate, phenylbutirate, citrate, lactate, γ- hydroxybutirate, glycolate, tartrate, methanosulfonate, propanosulfonate, naphthalene-1 - sulfonate, naphthalene-2-sulfonate, mandalate and similars. The preferred salts of acid addition pharmaceutically apropiate are the ones formed with mineral acids such as hydrochloric acid and hydrobromic acid, and the ones formed with organic acids such as maleic acid and methanesulfonic acid. The salts of base addition include those derived from inorganic bases, such as

hydroxides, carbonates, bicarbonates of amonium or alkali metals or alkaline earth metals, and similars. Bases useful to prepare the salts of the invention in this way include sodium hydroxide, potasium hydroxide, amonium hydroxide, potasium carbonate, sodium carbonate, sodium bicarbonate, potasium bicarbonate, calcium hydroxide, calcium carbonate, and similars. The forms of sodium salt and potasium salt are particularly preferred.

The preferred embodiments mentioned for the medical use of the cyclic peptide are also preferred embodiments of the new cyclic peptides as products per se.

A second aspect of the invention refers to a pharmaceutical composition, from now on composition of the invention, that comprises at least a peptide of the invention, in the elaboration or preparation of the medicament, or alternatively, to a composition that comprises at least a peptide of the invention for its use as medicament.

In a preferred embodiment of this aspect of the invention, the composition is a

pharmaceutical composition. In another preferred embodiment of this aspect, the composition comprises a pharmaceutical acceptable carrier. Even in a more preferred, the composition also comprises another active ingredient. In another more preferred embodiment the active ingredient is an antimicrobial, and more preferably an antibiotic. In another preferred embodiment, the active ingredient is selected from imipenem, meropenem, ciprofloxacine, cefepime, tigecicline, colistin, amoxiciline/clavulanic, or any of their combinations. Another aspect of the invention refers to a combined preparation that comprises:

A) a component A that comprises a peptide of the invention, and

B) a component B that comprises another active ingredient.

In a preferred embodiment of this aspect of the invention, the active ingredient is an antimicrobial, and more preferably an antibiotic. In another preferred embodiment, the active ingredient is selected from imipenem, meropenem, ciprofloxacine, cefepime, tigecicline, colistin, amoxiciline/clavulanic, or any of their combinations.

It is important to highlight that the term "combined preparation" or also called

"juxtaposition", in this document, means that the components of the combined preparation do not need to be found as a union, for example in a true composition, to be accessible to the combined application, separated or sequential. In this way, the expression

"juxtaposed" implies that a true combination is not needed, in view of the physical separation of the components.

As it is used herein, the term "active ingredient", "active substance", "pharmaceutically active substance" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or another different effect in the diagnostic, cure, mitigation, treatment, or prevention of a disease, or that it affects to the structure and function of the human body or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present in it in a planned modified form that provides the specific activity or effect.

The term "medicine or medicament", as it is used in this document, refers to any substance used to prevent, diagnose, relieve, treat or cure any disease in human or animals. In the context of the present invention, the disease is an infection by organisms Gram negative, and preferably of the genus Escherichia, of the genus Pseudomonas or the genus Acinetobacter. The peptides from the present invention could be formulated or formed in a

pharmaceutical composition that includes, at least, a peptide from the present invention together with one or more pharmaceutical carriers, including excipients, such as diluents, carriers and similars, and additives, such as stabilizing agents, preservatives, solubilizing agents, buffer solutions and similar, as many as desired. Among the excipients of the formulation polyvinylpyrrolidone, gelatin, hydrocellulose, arabic gum, polyethilenglycol, mannitol, sodium chloride or sodium citrate are included. For the injection and other formulations of liquid administration, it is preferred water that contains, at least, one or more buffer constituents, and also stabilizing agents, preservatives and solubilizing agents can be used. For the formulations of solid administration any of the different thickening agents can be used, for stuffing, to increase volume and vehicle, such as, starchs, sugars, fatty acids and similars. The pharmaceutical formulations, also the use of different formulations that allow the controlled release, slow release, sustained release, and additives are considered, in a way that the dose could be formulated to have the distribution of the peptide of the present invention during a period of time.

As much the compositions of the present invention, as the combined preparation or the pharmaceutical forms of the invention, could be formulated for its administration to an animal, and more preferably to a mammal, including human, in a variety of forms known in the state of the tecnique. In this way, it could be, without limitation, in sterile aqueuos solution or in biological fluids, such as serum. The aqueuos solutions could be buffered or non buffered and have additional active or inactive components. The additional components include salts to modulate ionic strength, preservatives, including, but not limited to, antimicrobial agents, antioxidants, quelating agents, and similars, and nutrients including glucose, dextrose, vitamines and minerals. Alternatively, the compositions could be prepared for its administration in its solid form. The compositions could be combined with various vehicles or inert excipients, including but not limited to, agglutinative agents such as microcristalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or cornstarch; lubricants such as magnesium stearate, sliding agents such as colloidal silica; sweeteners such as sacarose or sacarine; flavouring agents such as mint or methyl salicylate. Therefore, other aspect of the invention refers to a pharmaceutical form that comprises the composition of the invention, or the peptide of the invention.

In a preferred embodiment of this aspect of the invention, the pharmaceutical form of the invention is selected from the list including plaster, pomade, paste, cream, solution, suspension, emulsion, lotion, liniment, gel, hydrogel, hydrocolloid, foam, powder, or any their combinations. In a more preferred realization, the pharmaceutical form of the invention is a plaster. In an even more preferred realization, the pharmaceutical form is selected from a solution, suspension or an emulsion. In this report, the term ^" pharmaceutical form ^" means the mixture of one or more active ingredients with or without additives presenting physical characteristics for its adequate dosage, conservation, administration and bioavailability.

A ^" plaster ^" or ^" patch ^" is a pharmaceutical form consisting in solid or semisolid form that contains the active ingredient(s) and additive(s), and spread on a cloth, plastic or scotch tape, which serves as a support and protection, in addition to have an occlusive effect and macerating action that allow the direct contact with the skin and softens with body temperature.

A ^" ointment ^" or ^" pomade ^" is a pharmaceutical form consisting in a soft consistence preparation that contains the active ingredient(s) and additive(s) incorporated in an appropriate base giving it masse and consistence. It adhered and applied on the skin and mucous membranes. This base can be liposoluble or water soluble, generally is anhydrous or with a maximum of 20 percent of water. Also, it called hydrophilic ointment when contain a washable or removable base with water.

A ^" paste ^" is a pharmaceutical form consisting in a semisolid form that contains the active ingredient(s) and additive(s), made with high concentrations of insoluble powders (20 to 50 percent), fat or aqueous bases, absorbents or weak abrasives combined with soaps. A ^" cream ^" is a pharmaceutical form consisting in a liquid or semisolid preparation that contains the active ingredient(s) and additive(s) necessary to obtain an emulsion, generally oil in water with a water content exceeding 20 percent.

A ^" solution ^" is a pharmaceutical form consisting in a liquid transparent and homogenous preparation obtained by dissolution of the active ingredient(s) and additive(s) in water, and which is utilized for external or internal use. In the case of injectable solutions, ophthalmic and otic, these solutions must be sterile. The term ^" solution ^" includes solvent.

A ^" suspension ^" is a pharmaceutical form that consists in a disperse system composed by two phases, which contain the active ingredient(s) and additive(s). One of the both phases, the continuous or external is generally a liquid or a semisolid, and the dispersed or internal phase is constituted of insoluble solids (active ingredients) but dispersible in the external phase. In the case of injectable suspension, this last must be sterile. A ^" emulsion ^" is a pharmaceutical form that consists in a heterogeneous system, generally formed by two immiscible liquids together; in which the dispersed phase is composed by small globules distributed in the carrier in which they are immiscible. The dispersed phase is also known as internal and the dispersion medium is known as external or continuous phase. It exist emulsions type water/oil or oil/water, and may be presented as semisolids or liquids. The active ingredient(s) and additive(s) can be in the external or internal phase. A ^" lotion ^" is a pharmaceutical form that can be presented as solution, suspension or emulsion, that contains the active ingredient(s) and additive(s), and whose dispersant agent is predominantly water. A ^" liniment ^" is a pharmaceutical form consisting in liquid presentation, solution or emulsion that contains the active ingredient(s) and additive(s), whose carrier is aqueous, alcoholic or oily.

A ^" jelly ^" is a pharmaceutical form consisting in semisolid colloid that contains the active ingredient(s) and additive(s), whose water soluble base is generally composed by gums such as tragacanthn. Other bases used are glycerine, pectin, alginates, boro-glycerie compounds, synthetic derivatives or natural substances as carboxymethyl cellulose.

A ^" foam ^" is a pharmaceutical form consisting in semisolid preparation composed by two phases: one liquid carrying the active ingredient(s) and additive(s), and other gaseous carrying propellant gas to leave the product as cloud form.

A ^" gel ^" is a pharmaceutical form consisting in semisolid preparation that contains the active ingredient(s) and additive(s), solids in a liquid which can be water, alcohol or oil forming a network of trapped particles in the liquid phase.

The hydrogels are colloidal systems with solid appearance as heat coagulated albumin, cooling gelled gelatin, etc. One of the hydrogels properties is to swell and increase in volume by water and substances absorption in which dissolved, a common property to all tissues of organisms formed by colloidal materials.

The ^" colloids ^" are materials formed by a dispersed phase (matrix) and a dispersant phase (filler). When the dispersing phase is water, it is called "hydrocolloid". It can be

characterized by coagulation (transformed from solution to solid gel) when the dispersed phase is abundant, and by flocculation (transformed from gel to solution) when the dispersed phase is low.

The drugs according to the invention containing the peptide of the invention as active ingredient are usually prepared according to conventional methods and are administered in a pharmaceutically adequate form. Such compositions, pharmaceutical forms and/or their formulations may be administered to an animal including a mammal and therefore man in a variety of forms including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intrathecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.

The dosage to obtain a therapeutically effective amount depends on a variety of factors of mammal, such as age, weight, sex, tolerance, .... As utilized in this description, the term "therapeutically effective amount" refers to the amount of the peptide of the invention, prodrugs, peptide derivatives or analogues to produce the desired effect, and generally will be determined, among other causes, by the characteristics of such prodrugs, derivatives or analogues and the therapeutic effect to be achieved. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in these compositions are those known by the experts. Another aspect of the invention refers to the use of the peptide of the invention, the combined preparation, the composition of the invention which is a pharmaceutical composition or the pharmaceutical form of the invention in the development of a medicament for the treatment, alleviation and/or prevention of infections caused by Gram negative bacilli. Alternatively, it refers to the peptide of the invention, the composition of the invention or the pharmaceutical form of the invention for its use in the treatment, alleviation and/or prevention of infections caused by Gram negative pathogens. This aspect can also be formulated as a method of treatment and/or prophylaxis of a mammal, including a human, suffering from or being susceptible to a an infection caused by Gram negative pathogens, said method comprises the administration to said patient of a therapeutically effective amount of the the peptide of the invention, the combined preparaton, the composition of the invention which is a pharmaceutical composition, or the pharmaceutical form of the invention as defined above, together with pharmaceutically acceptable excipients or carriers. In this report, the term of "Gram negative organisms", "Gram negative pathogens" or

"Gram negative bacteria" means any bacteria that have double cell membrane (one external and other internal). In microbiology, Gram negative bacteria are known to those not stained by dark blue or violet using Gram staining, and present a weak pink color, where the name "Gram-negative" or also "gramnegative". This feature is closely linked to the structure of the cell envelope, which reflects a natural type of bacterial organization. They are one of the main groups of bacteria, and when referred as taxon, the name of Negibacteria [Cavalier-Smith. Biol. Direct. 2006; 1 : 19] or Didermata are also utilized. Within the Gram negative pathogens is the Gammaproteobacteria class. In this class, many important pathogens are present such as Salmonella (enteritis and typhoid fever), Yersinia (plague), Vibrio (cholera), P. aeruginosa (nosocomial infections or in patients with cystic fibrosis) and Klebsiella pneumoniae (pneumonia).

Therefore, in a preferred realization of this aspect of the invention, the Gram negative pathogen belongs to Phylum Proteobacteria, and more preferably to

Gammaproteobacteria class.

In other more preferred realization, the Gram negative pathogen belongs to

Enterobacteriales order and most preferably to the Enterobacteriaceae family. In a further preferred realization, it belongs to the Escherichia genus, and in a particular realization to the E. coli species.

In other more preferred realization, the Gram negative pathogen belongs to

Pseudomonadales order and most preferably to the Pseudomonadaceae family. In a further preferred realization, it belongs to the Pseudomonas genus, and in a particular realization to the P. aeruginosa species.

In a preferred realization of this aspect of the invention, the bacteria of the

Pseudomonadales order belongs to Moraxellaceae family. More preferably, the bacteria of the Pseudomonadales order belongs to Acinetobacter genus. In a further preferred realization, the bacteria of the Pseudomonadales order belongs A. baumannii species.

In this report, A. baumannii species [Bouvet and Grimont Int. J. Syst. Bacteriol. 1986; 36: 228-40], refers to Gram negative bacilli species belonging Proteobacteria phylum.

Acinetobacter species are strictly aerobic non-fermenting bacilli, non-motile, oxidase- negative, that are present in pairs in the microscope. They are widely distributed in nature, and are important in the soil and contribute to its mineralization. A. baumannii is a cellular organism of the Bacteria domain; Proteobacteria Phylum; Gammaproteobacteria class; Pseudomonadales order; Moraxellaceae family and Acinetobacter genus.

P. aeruginosa [Schroeter 1872; Migula 1900 Int. J. Syst. Bacteriol. 1980; 30: 225-420] is a Gram negative bacteria, aerobic, with unipolar motility. It is an opportunistic pathogen in humans and plants. P. aeruginosa infects the lungs and respiratory tracts, the urinary tracts, the tissues (wounds), and also causes sepsis (disseminate infection in the body). Pseudomonas can cause pneumonia in patients, which sometimes require mechanical support to overcome such pneumonia. It is one of the most common microorganisms isolated in many studies. E. coli [Migula 1895; Castellani and Chalmers 1919; Int. J. Syst. Evol. Microbiol. 2003, 53, 807-10] is an Enterobacteria that is found generally in animal intestines, and therefore in sewage, but it can be found everywhere, since it is a ubiquitous organism. Although the most strains are safe and live in the intestines of healthy humans and animals, this strain produces a powerful toxin and can cause severe diseases such as hemolytic uremic syndrome. Six strains according to their pathogenic capacity are distinguished:

enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (ECIS),

enterohemorrhagic (EHEC), enteroaggregative (EAEC) and diffusely adherent (ECAD) £. coli. The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used here interchangeably, and refer to a polymeric form of amino acids of any length, which can be coding or non-coding, chemically or biochemically modified. Regarding to the nomenclature of the amino acids, it will consider the utilization of L-amino acids, except those indicated as D-amino acid.

Throughout the description and claims, the word "include" and its variants are not intended to exclude other technical features, additives, components or steps. For the experts, other objects, advantages and features of the invention will arise in part from the description and practice of the invention. The following examples and figures are provided as an illustration, and are not intended to be limiting of the present invention.

EXAMPLES OF THE INVENTION Example 1. Peptide synthesis

Protected amino acids and resins were supplied by: Luxembourg Industries (Tel-Aviv, Israel), Neosystem (Strasbourg, France), Calbiochem-Novabiochem AG (Laufelfingen, Switzerland), Bachem AG (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz,

Germany). Other reagents and solvents used are summarized in Table 1 .

Table 1 Commercial supplier Reagents and solvents

piperidine, DBU, toluene, acetic anhidride, a-ciano-4-

Aldrich

hydroxicinnamic acid (ACH),

Applied GL Biochem

1 -hydroxy-7-azabenzotriazole (HOAt)

Shangai

KaliChemie trifluoroacetic acid (TFA)

benzotriazol-1 -yl-oxytripyrrolidinophosphonium

Novabiochem

hexafluorophosphate (PyBOP)

dichloromethane (DCM), dimethylformamide (DMF), methanol

Scharlab

(MeOH), tert-butylmethylether

SDS MeCN

Iris Biotech (1 H-7-Azabenzotriazole-1 -yl)-oxy-tris-pyrrolidino-phosphonium hexafluorophosphat (PyOAP)

Sigma Aldrich Triisopropylsilane (TIS)

Sigma Aldrich 2,3,4,6,7,8,9,10-Octahydropyrimidol[1 ,2-a]azepine (DBU)

Carlo Erba Toluene

Carlo Erba Piperidine

General considerations about the synthesis. Solid-phase peptide elongation and other solid-phase manipulations were carried out manually in polypropylene syringes fitted with a polyethylene porous disk. Solvents and soluble reagents were removed by suction. Washings between different synthetical steps were carried out with dimethylformamide (DMF) (5 x 0.5 min) and dichloromethane (DCM) (5 x 0.5 min) using 10 mL of solvent/g of resin each time.

Identification tests: The test used for the identification and control of the synthesis were the following:

A) Kaiser colorimetric assay for the detection of solid-phase bound primary amines (E. Kaiser et al., Anal. Biochem. 1970, vol. 34, pp. 595-598);

B) p-nitrophenyl ester test for secondary amines bound to solid-phase (A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791 ).

Resin initial conditioning:The 2-chlorotrityl resin was conditioned by washing with DCM (5 x 30 s) and DMF (5 x 30 s) and finally DCM (5 x 30 s). Fmoc group removal: Removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was done with 20% (v/v) piperidine in DMF using a treatment of 30 s followed by two treatments of 10 minutes each. Two additional treatments with DBU , toluene, piperidine, DMF (5%, 5%, 20%, 70%) (2 x 5 min) were performed to ensure the removal of the Fmoc group from secondary amines.

Coupling methods: The coupling of the first amino acid to the 2-chlorotrityl resin was performed by adding the protected amino acid (0.7 eq.), to the resin in DCM followed by DI EA (12 eq.). The mixture was allowed to react with intermittent manual stirring for 1 .5 h. Afterwards methanol (0.8 ml/g of resin) were added and leaved for 15 min. The solvent was removed by suction and the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The extent of coupling was checked by Kaiser colorimetric assay.

Coupling of the second and following amino acids was performed as follows: Protected amino acid (4 eq.), PyBOP (4 eq.), HOAt (12 eq.) were dissolved in DMF and added sequentially to the resin, subsequently DI EA was added (12 eq.). The mixture was allowed to react with intermittent manual stirring for 1 .5 h. The solvent was removed by suction and the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The extent of coupling was checked by Kaiser colorimetric assay. In the case of coupling onto proline the coupling reaction was carried out twice under the same conditions. The extent of coupling was checked by the p-nitrophenyl ester colorimetric assay.

Disulphide bridge formation: In the case of peptides having an extra disulphide bridge, after the deprotection of the /V-terminus of the last amino acid, the resin was washed several times with DMF, DCM and DMF. The formation of the disulfide bond took place when 2 x 15 min of 5 eq. I2 were added on the resin. It was the first cyclization and was followed by cleavage of a small amount of resin and H PLC and HPLC-MS to ensure the bridge formation. Cleavage from the resin: It was carried out by treating resin with TFA 2% in DCM ( 5 x 30 s). The filtrates were collected over water and DCM was evaporated by N ₂ . The residue was resuspended in a mixture H ₂ 0:MeCN (1 : 1 ) and lyophilized.

Cyclization in solution: For the cyclization, the solvent used was DCM/DMF (98:2), PyAOP (2 eq.) was solubilized in DMF and at the same time the peptide was resuspended in

DCM at a concentration of 5 mM. Afterwards both solutions were combined, the volumes adjusted and 6 eq. of DI EA were added. The reaction was checked by HPLC and completed in 2-3 h.

Acetylation of /V-terminal: Once we have the amino /V-terminal group free, acetic anhydride (50 eq.) and DI EA (50 eq.) were added, after 30 min resin was washed with DCM.

Elimination of side-chain protecting groups: Once the cyclization has been performed, the solution was concentrated and the oil obtained was treated with TFA (95%), H ₂ 0 (2.5%) and TIS (2.5%) (2 h) to the eliminate the side-chain protecting groups. Afterwards TFA was evaporated under N ₂ and ie f-butyl methyl ether was added to precipitate the peptide product and the mixture was centrifuged during 10 min at 3500 x G, the supernatant was discarded and the process repeted three times. Finally the pellet was resuspended in a mixture of H ₂ 0:MeCN (1 : 1 ) and lyophilized.

The identity of the different peptide compounds synthesized was confirmed using mass spectrometry "Matrix Assisted Laser Desorption/lonization-Mass Spectrometry" (MALDI) (Instrument: MALDI Voyager DE RP time-of-flight (TOF) PE Biosystem) or HPLC-MS (Instrument: Waters model Alliance 2695, quaternary pump, UVA is detector model 2998, ESI-MS model Micromass ZQ and software Masslynx version 4.1 ) using a Symmetry 300 Ci ₈ (150 x 3.9 mm x 5 μηη), 300 A column using a 1 mL/min flow; solvents: A: H ₂ 0 with 0.1 % formic acid; B: MeCN with 0.07 % formic acid. Their purity was checked by reverse phase HPLC using a Symmetry Ci ₈ column (150 x 4.6 mm x 5 μηη, 100 A, Waters) and a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; solvents: A: H ₂ 0 with 0.045% TFA; B: MeCN with 0.036% TFA, Instrument: Waters model Alliance

2695 constituted by a quaternary pump, a diode array detector model 966, controlled by the software millenium version 3.5.

Peptide compounds were purified by HPLC using a Symmetry Ci ₈ column (100 x 30 mm x 5 μη-ι, 100 A, Waters), flow 10 mL/min, solvents: A: H ₂ 0 with 0.1 % TFA; B: MeCN with

0.1 % TFA, Instrument: Waters system with a quaternary pump, Simple Manager 2700 autoinjector, UVA is detector model 2487 and a Fraction collector I I, controlled by the software Masslynx version 3.5. Example 1.1 : Preparation of cyclo[Trp-D-Pro-Arg-Trp-D-Pro-Arg] (SEQ ID NO:1 )

The synthesis of this peptide has been performed as described above. N° Coupling Protected amino acids Quantity

1 Fmoc-Arg(Pbf)-OH 45.4 mg

2 Fmoc-D-Pro-OH 135 mg

3 Fmoc-Trp(Boc)-OH 210.6 mg

4 Fmoc-Arg(Pbf)-OH 259.5 mg

5 Fmoc-D-Pro-OH 135 mg

6 Fmoc-Trp(Boc)-OH 210.6 mg

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 2.99 min in a 20-70% MeCN gradient. Mass spectrometry (MALDI-TOF): [M+H]+: 879.29 Da. Yield: 80%.

Example 1.2: Preparation of cyclo[Arg-D-Pro-Trp-Arg-D-Pro-Trp]

(SEQ ID NO:2)

The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method. Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.35 min in a 0-100% MeCN gradient. Mass spectrometry (MALDI-TOF): [M+H]+: 879.29 Da. Yield: 80%

Example 1.3: Preparation of cyclo[D-Arg-Pro-Trp-D-Arg-Pro-Trp] (SEQ ID NO:3)

The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.75 min in a 10-60% MeCN gradient. Mass spectrometry (MALDI-TOF): [M+H]+: 879.29 Da. Yield: 84%

Example 1.4: Preparation of cyclo[Arg-Pro-D-Trp-Arg-Pro-D-Trp] (SEQ ID NO:4)

The synthesis of this peptide has been performed as described above.

N° Coupling Protected amino acids Quantity

1 Fmoc-D-Trp(Boc)-OH 36.9 mg

2 Fmoc-Pro-OH 135 mg

3 Fmoc-Arg(Pbf)-OH 259.5 mg

4 Fmoc-D-Trp(Boc)-OH 210.6 mg 5 Fmoc-Pro-OH 135 mg

6 Fmoc-Arg(Pbf)-OH 259.5 mg

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.60 min in a 0-100% MeCN gradient. Mass spectrometry (MALDI-TOF): [M+H]+ 879.29 Da. Yield: 30%

Example 1.5: Preparation of Ac-Trp-D-Pro-Arg-Trp-D-Pro-Arg-OH (lineal control) (SEQ ID NO:5)

The synthesis of this peptide has been performed as described above.

The peptide was acetylated following the described method and then cleaved from the resin and lyophilized. Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.5 min in a 0-100% MeCN gradient. Mass spectrometry (MALDI-TOF): [M+H]+: 939.51 Da. Yield: 69% Example 1.6: Preparation of cyclo[Trp-D-Pro-Arg-Arg-D-Pro-Trp] (SEQ ID NO:6) The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.37 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 880.34 Da. Yield: 12%

Example 1.7: Preparation of cyclo[Trp-D-Pro-Trp-Arg-D-Pro-Arg] (SEQ ID NO:7)

The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method. Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.32 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 880.3 Da. Yield: 39%

Example 1.8: Preparation of cyclo[Trp-D-Pro(4-NH ₂ )-Arg-Trp-D-Pro-Arg] (SEQ ID NO:8)

The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method. Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.07 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 895.52 Da. Yield: 22% Example 1.9: Preparation of cyclo[Tyr-D-Pro-Ala-Tyr-D-Pro-Ala] (SEQ ID NO:9)

The synthesis of this peptide has been performed as described above.

N° Coupling Protected amino acids Quantity

1 Fmoc-Ala-OH 36.3 mg

2 Fmoc-D-Pro-OH 101.2 mg 3 Fmoc-Tyr(tBu)-OH 137.8 mg

4 Fmoc-Ala-OH 93.4 mg

5 Fmoc-D-Pro-OH 101.2 mg

6 Fmoc-Tyr(tBu)-OH 137.8 mg

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method. Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.78 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 663.9 Da. Yield: 39% Example 1.10: Preparation of cyclo[3Pal-D-Pro-Arg-3Pal-D-Pro-Arg] (SEQ ID NO:10)

The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 3.06 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 804.25 Da. Yield: 24%

Example 1.11 : Preparation of cyclo[2Nal-D-Pro-Arg-2Nal-D-Pro-Arg] (SEQ ID NO:11 ) The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.80 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 902.36 Da. Yield: 7%

Example 1.12: Preparation of cyclo[2Nal-D-Pro-Arg-3Pal-D-Pro-Arg] (SEQ ID NO:12)

The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method. Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 3.81 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 427.30 Da. Yield: 56%

Example 1.13: Preparation of cyclo[3Pal-D-Pro-Arg-Trp-D-Pro-Tyr] (SEQ ID NO:13)

The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.04 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 849.23 Da. Yield: 36%

Example 1.14: Preparation of cyclo[Trp-D-Pro-Dab-Trp-D-Pro-Dab] (SEQ ID NO:14)

The synthesis of this peptide has been performed as described above.

N° Coupling Protected amino acids Quantity

1 Fmoc-Dab (Boc)-OH 51.4 mg

2 Fmoc-D-Pro-OH 101.2 mg

3 Fmoc-Trp(Boc)-OH 157.9 mg

4 Fmoc-Dab(Boc)-OH 132.2 mg 5 Fmoc-D-Pro-OH 101.2 mg

6 Fmoc-Trp(Boc)-OH 157.9 mg

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method. Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.23 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 768.16 Da. Yield: 39% Example 1.15: Preparation of cyclo[Trp-D-Pro-Lys-Trp-D-Pro-Lys] (SEQ ID NO:15)

The synthesis of this peptide has been performed as described above.

The peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: RP-HPLC: lineal gradient of MeCN in water in 8 min using a Sunfire Ci ₈ column (100 x 4.6 mm x 3.5 μηη, 100 A, Waters), flow 1 mL/min; retention time: 4.29 min in a 0-100% MeCN gradient. Mass spectrometry (HPLC-MS): [M+H]+: 824.25 Da. Yield: 37%

Example 1.16: Preparation of cyclo[Arg-D-Pro-Cys-Arg-D-Pro-Cys] with a disulphide bridge between Cys (SEQ ID NO:16) The synthesis of this peptide has been performed as described above. N° Coupling Protected amino acids Quantity

1 Fmoc-Cys(Acm)-OH 45.6 mg

2 Fmoc-D-Pro-OH 101.2 mg

3 Fmoc-Arg(Pbf)-OH 194.6 mg

4 Fmoc-Cys(Acm)-OH 165.8 mg

5 Fmoc-D-Pro-OH 101.2 mg

6 Fmoc-Arg(Pbf)-OH 194.6 mg

The disulphide bridge was formed on solid phase and then the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: UPLC retention time 1 .50 min in a using a SunFire C18 column (2.1 x 50 mm x 1 .7 μηι, 100 A, Waters) in a 3.6 min linear gradient from 0 to 100% of B in A (A=H ₂ 0 with 0.045% TFA; B=MeCN with 0.036% TFA) and detection at 220 nm. Mass spectrometry (HPLC-MS): [M+H]+: 71 1 .57 Da. Yield: 1.3%

Example 1.17: Preparation of cyclo[Ala-D-Pro-Cys-Ala-D-Pro-Cys] with a disulphide bridge between Cys (SEQ ID NO:17) The synthesis of this peptide has been performed as described above.

The disulphide bridge was formed on solid phase and then the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method. Product characterization: UPLC retention time 1 .20 min in a using a SunFire C18 column (2.1 x 50 mm x 1 .7 μηι, 100 A, Waters) in a 3.6 min linear gradient from 0 to 100% of B in A (A=H ₂ 0 with 0.045% TFA; B=MeCN with 0.036% TFA) and detection at 220 nm. Mass spectrometry (HPLC-MS): [M+H]+: 541 .19 Da. Yield: 3.5%

Example 1.18: Preparation of cyclo [Ser-D-Pro-Cys-Ser-D-Pro-Cys] with a disulphide bridge between Cys (SEQ ID NO:18) The synthesis of this peptide has been performed as described above.

The disulphide bridge was formed on solid phase and then the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.

Product characterization: UPLC retention time 1 . 04 min in a using a SunFire C18 column (2.1 x 50 mm x 1 .7 μηι, 100 A, Waters) in a 3.6 min linear gradient from 0 to 100% of B in A (A=H ₂ 0 with 0.045% TFA; B=MeCN with 0.036% TFA) and detection at 220 nm. Mass spectrometry (HPLC-MS): [M+H]+: 573.18 Da. Yield: 3.1 %

Example 2. In vitro activity assay

In this study, we used a non cyclic hexapeptide ((SEQ I D NO: 5) linear peptide) as negative control composed by tryptophan, proline, and arginine (Ac-Trp-DPro- Arg-Trp-D- Pro-Arg-OH). The structure of this peptide is represented in the formula (V):

Formula (V)

First, we studied the effect of different inhibitors SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 on the adherence of A baumannii ATCC 17978 strain to human lung epithelial cells (A549) (figure 1 ). Following the protocol described previously by the inventors [Smani et al. J Biol Chem 2012; 287: 26901 -10], the A549 cells (n = 3 / group) were infected for 2 h by A. baumannii ATCC 17978 strain (10 ⁸ cfu/ml). This strain was pretreated with SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 (0.25, 0.5 and 1 mg/ml) for 30 min. As shown in the figure 1 , the treatment with SEQ ID NO: 1 or SEQ ID NO: 3 was more effective, and reduced significantly and dose dependent the adherence of this strain to A549 cells. In contrast, the treatment with the control peptide SEQ ID NO: 5 (0.25, 0.5 and 1 mg/ml) for 30 min had no effect on the adherence of A baumannii ATCC 17978 strain to A549 cells. The treatment with SEQ ID NO: 1 was then selected for the rest of in vitro and in vivo experiments due to its major effect in the reduction of adherence of ATCC 17978 strain to A549 cells in comparison with the treatment with SEQ ID NO: 3. In addition, we studied the effect of SEQ ID NO: 1 on the adherence of A. baumannii, P. aeruginosa and £ coli to A549 cells (figure 2). The A549 cells (n = 3 / group) were infected for 2 h by A. baumannii (10 ⁸ cfu/ml): ATCC 17978, 77 and 1 13-16 strains, by P. aeruginosa (10 ⁸ cfu/ml): PA01 strain, and by £ coli (10 ⁸ cfu/ml): ATCC 25922 strain.

These strains were pretreated with SEQ ID NO: 1 (0.25 and 0.5 mg/ml) or SEQ ID NO: 5 (0.5 mg/ml) for 30 min. As shown in the figure 2, the treatment with SEQ ID NO: 1 reduced significantly and dose dependent the adherence of these strains to A549 cells. In contrast, the treatment with the control peptide SEQ ID NO: 5 (0.5 mg/ml) had no effect on the bacterial adherence to A549 cells.

Moreover, bacterial adherence was also studied with immunofluorescence assay [Smani et al. J Biol Chem 2012; 287: 26901 -10] (figure 3). A549 cells (n = 3 / group) grown on coverslips in 24-wells plate were infected for 2 h by A. baumannii (10 ⁸ cfu/ml): ATCC 17978, 77 and 1 13-16 strains, by P. aeruginosa (10 ⁸ cfu/ml): PA01 strain, and by £ coli (10 ⁸ cfu/ml): ATCC 25922 strain. These strains were pretreated with SEQ ID NO: 1 (0.5 mg/ml) for 30 min. The bacterial and fibronctin staining were determined using specific antibodies against OMPs of A. baumannii, P. aeruginosa and £. coli (green staining), and human fibronectin (red staining). The A549 cells attached by one or more bacterial colonies were considered positive. As shown in the figure 3, the treatment with SEQ ID NO: 1 reduced significantly the adherence of these strains to A549 cells.

In parallel, the inventors were interested in the study of the SEQ ID NO: 1 effect on the interaction of A. baumannii, P. aeruginosa and £ coli with fibronectin. A. baumannii (10 ⁸ cfu/ml): ATCC 17978, 77 and 1 13-16 strains, P. aeruginosa (10 ⁸ cfu/ml): PA01 strain, and

£ coli (10 ⁸ cfu/ml): ATCC 25922 strain were pretreated for 30 min with SEQ ID NO: 1 (0.062, 0.125, 0.25 or 0.5 mg/ml) or with SEQ ID NO: 5 (0.5 mg/ml), and incubated for 2 h in 96-wells plate precoated with 10 μg/ml fibronectin (figure 4). Likewise in the figures 1 and 2, the treatment with SEQ ID NO: 1 reduced significantly and dose dependent the interaction of these strains with fibronectin. In contrast, the treatment with the control peptide SEQ ID NO: 5 (0.5 mg/ml) had no effect on this interaction.

Furthermore, we studied the protective effect of SEQ ID NO: 1 on the cell death caused by A. baumannii, P. aeruginosa and £ coli. A549 cells (n = 3 / group) were infected for 24 h by A. baumannii (10 ⁸ cfu/ml): ATCC 17978, 77 and 1 13-16 strains, by P. aeruginosa (10 ⁸ cfu/ml): PA01 strain, and by £ coli (10 ⁸ cfu/ml): ATCC 25922 strain. These strains were pretreated with SEQ ID NO: 1 (0.062, 0.125, 0.25 and 0.5 mg/ml) or by SEQ ID NO: 5 (0.5 mg/ml) for 30 min. The cell viability was determined by MTT assay. The treatment with SEQ I D NO: 1 protected significantly the A549 cells from death caused by A baumannii and P. aeruginosa strains (figure 5), but not that caused by £ coli strain (data not shown). In contrast, the treatment with the control peptide SEQ I D NO: 5 (0.5 mg/ml) had not significant effect on the cell death caused by these strains.

In addition, we studied the effect of SEQ I D NO: 1 on the biofilm formation by standard and clinical strains of A. baumannii, P. aeruginosa and £ coli on abiotic surfaces for 24 h. Violet crystal staining assay was used as described previously by the inventors [Smani et al. Clin. Microbiol. Infect. 2012; 18: E1 -4]. The treatment of these strains with SEQ I D NO: 1 (0.25 and 0.5 mg/ml) reduced significantly the production of biofilm in round bottom microwell plates (figure 6) and in polyvinyl endotracheal tubes (data not shown).

Also, we determined the effect of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ I D NO: 9, SEQ I D NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ I D NO: 13, SEQ I D NO: 14 and SEQ I D NO: 15 on the adherence of A. baumannii ATCC 17978 strain, P. aeruginosa Pa01 strain, and £ coli ATCC 25922 strain to human lung epithelial cells (A549) (figure 7). Following the protocol described previously by the inventors [Smani et al. J Biol Chem 2012; 287: 26901 -10], the A549 cells (n = 3 / group) were infected for 2 h by A baumannii ATCC 17978 strain (10 ⁸ cfu/ml), P. aeruginosa Pa01 strain (10 ⁸ cfu/ml), and £. co// ^' ATCC 25922 strain (10 ⁸ cfu/ml). These strains were pretreated with SEQ I D NO: 6, SEQ I D NO: 7, SEQ ID NO: 8, SEQ I D NO: 9, SEQ I D NO: 10, SEQ I D NO: 1 1 , SEQ I D NO: 12, SEQ I D NO: 13, SEQ I D NO: 14 and SEQ I D NO: 15 at 250 g/ml for 30 min. As shown in the figure 7A, the treatment with SEQ ID NO: 6, SEQ I D NO: 8, SEQ I D NO: 10, SEQ I D NO: 1 1 , and SEQ I D NO: 14 were more effective, and reduced the adherence of A baumannii ATCC 17978 strain to A549 cells. In contrast, the treatment with SEQ I D NO: 7, SEQ I D NO: 9, SEQ I D NO: 12, SEQ I D NO: 13, and SEQ I D NO: 15 had no effect on the adherence of A baumannii ATCC 17978 strain to A549 cells. In the figure 7B, the treatment with SEQ I D NO: 6, SEQ ID NO: 7, SEQ I D NO: 8, SEQ ID NO: 9, SEQ I D NO: 10, SEQ I D NO: 1 1 , SEQ ID NO: 12, SEQ I D NO: 13, SEQ ID NO: 14 and SEQ I D NO: 15 were more effective, and reduced the adherence of Pa01 strain to A549 cells. In the figure 7C, the treatment with SEQ ID NO: 8, SEQ I D NO: 10, SEQ I D NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 were more effective, and reduced the adherence of £ coli ATCC 25922 strain to A549 cells. In contrast, the treatment with SEQ I D NO: 6, SEQ ID NO: 7, SEQ I D NO: 9, and SEQ I D NO: 12 had no effect on the adherence of £ coli ATCC 25922 strain to A549 cells. Based to these data, and to compare to the effect of SEQ I D NO: 1 , we selected for the next experiment the peptides SEQ ID NO: 8, SEQ I D NO: 10, SEQ I D NO: 1 1 , and SEQ I D NO: 14 which presented significant effect against the adherence of the three pathognes to A549 cells. For this, we studied the dose-dependent effect of SEQ I D NO: 8, SEQ I D NO: 10, SEQ I D NO: 1 1 , and SEQ I D NO: 14 (31 .25, 62.5, 125 yig/m\) versus the dose- dependent effect of SEQ I D NO:1 (31 .25, 62.5, 125 Mg/ml) on the adherence of A

baumannii ATCC 17978, P. aeruginosa Pa01 and £ coli ATCC 25922 strains to A549 cells. In the figure 8A, we demonstrated that SEQ ID NO: 8 and SEQ ID NO: 14 (with minor impact) reduced dose-dependently the adherence of A baumannii ATCC 17978 to A549 cells more than SEQ ID NO: 1 . Meanwhile SEQ I D NO: 10 and SEQ I D NO: 1 1 reduced also the adherence of A baumannii ATCC 17978 to A549 cells, but their effects were lower than SEQ I D NO: 1 . In figure 8B, we demonstrated that SEQ ID NO: 8, SEQ I D NO: 10, SEQ I D NO: 1 1 , and SEQ ID NO: 14 reduced dose-dependently the

adherence of P. aeruginosa Pa01 to A549 cells more than SEQ I D NO: 1 . In figure 8C, we demonstrated that SEQ I D NO: 8, SEQ I D NO: 1 1 , and SEQ I D NO: 14 reduced dose- dependently the adherence of £ coli ATCC 25922 to A549 cells more than SEQ I D NO: 1 . Meanwhile SEQ I D NO: 10 reduced also the adherence of £ coli ATCC 25922 to A549 cells, but its effect was lower than SEQ I D NO: 1 . From these data, we can conclude that SEQ ID NO: 8 and SEQ I D NO: 14 present better effect than SEQ ID NO: 1 on the adherence of A baumannii, P. aeruginosa and £. coli. In contrast, SEQ I D NO: 10 and SEQ ID NO: 1 1 present better effect than SEQ I D NO: 1 only on the adherence of P.

aeruginosa.

Example 3. In vivo assay

In mice, we are interested to use the inhibitor SEQ I D NO: 1 as directed treatment against infections caused by A baumannii. For this, we studied first the toxicity of SEQ ID NO: 1 in vivo. Healthy female C57BL/6 mice (n = 6 / group) were injected intraperitoneally (ip.) by a unique dose of SEQ I D NO: 1 diluted in 0.5 ml NaCI 0.9%. We administered different doses (20, 40, 80 and 160 mg/kg). Mice survival was monitored for 7 days. As shown in table 1 , the doses of SEQ I D NO: 1 (10, 20 and 40 mg/kg) were not toxic. In contrast, the dose of SEQ I D NO: 1 (80 and 100 mg/kg) induced 20% and 100% mice mortality, respectively. The lethal dose (LD)0, LD50 and LD100 were 40, 85 and 160 mg/kg, respectively. Table 1. Toxicity of SEQ ID NO: 1.

In the order to know the effective dose 50 (ED50) of the inhibitor SEQ ID NO: 1 , we calculated the ED50 using a murine peritoneal sepsis model. Groups of six female C57BL/6 mice were inoculated ip. by the minimal lethal dose (MLD) of A. baumannii ATCC 17978 (3.2 log cfu/ml). Two hours after the bacterial inoculation, mice were injected ip. with NaCI 0.9% or with SEQ ID NO: 1 (10, 20 or 40 mg/kg). The mice survival was monitored for 7 days. As shown in table 2, the dose of SEQ ID NO: 1 (20 mg/kg) reached 83.33% of mice survival in 7 days post-infection by A. baumannii. In contrast, the dose of SEQ ID NO: 1 (10 and 40 mg/kg) reached only 66.67 and 50% of the mice survival, respectively.

Table 2. DE50 of SEQ ID NO: 1 1 in a murine peritoneal sepsis model caused by A. baumannii ATCC 17978 strain.

To determine the therapeutic efficacy of the SEQ ID NO: 1 in a murine peritoneal sepsis model during 3 days, 3 groups of 6 female C57BL/6 mice were treated with a unique dose of SEQ ID NO:1 (10 mg/kg, group 1 ), with two doses of SEQ ID NO: 1 (10 mg/kg/d, group 2), or with three doses of SEQ ID NO: 1 (10 mg/kg/d, group 3). The first dose of SEQ ID NO: 1 was administered in mice 2 h post-bacterial inoculation with MLD of ATCC 17978 strain. In addition, group of six femal C57BL/6 mice control was untreated by SEQ ID NO: 1 but received the same bacterial inoculation.

The treatment with SEQ ID NO: 1 at 24, 48 and 72 h reduced significantly the bacterial concentration in spleen to 8.79 ± 0.16, 6.9 ± 1.32 y 5.48 ± 1 .34 log cfu/g, respectively, in comparison with the control group without treatment with SEQ ID NO: 1 and infected by ATCC 17978 (9.52 ± 0.17 log cfu/g). Moreover, the treatment with SEQ ID NO: 1 at 24, 48 and 72 h reduced significantly the bacterial concentration in lungs to 9.21 ± 0.28, 6.81 ± 1 .31 , y 5.97 ± 1 .46 log cfu/g, respectively, in comparison with the control group without treatment with SEQ ID NO: 1 and infected by ATCC 17978 (9,77 ± 0,17 log cfu/g) (Table 3). These results demonstrate that SEQ ID NO: 1 is able to protect against the infections caused by A. baumannii.

Table 3. Therapeutical efficacy of SEQ ID NO: 1 in a murine peritoneal sepsis model caused by A baumannii ATCC 17978 strain.

Example 4. PAMPA assay

The PAMPA assay, introduced by Kansy et al., allows the parallel evaluation of passive diffusion transport of various compounds through a mixture of lipids, thus emulating the biological barrier of interest. A selected lipid mixture is deposited onto a filter, which is divided into two compartments. Lipids are chosen in function of the composition of the barrier. The compartments above and below the filter contained only buffer and the molecule to test in buffer, respectively. Magnetic stirring was applied for 4 h in donor wells. This approach almost totally reduced the unstirred water layer (UWL). Afterwards, each well was quantified by UV-absorption after injection into a RP-HPLC system. Time and concentration used were optimized to achieve a satisfactory relation signal-to-noise during quantification and to prevent back-diffusion, i.e. the experiment was performed while the transport rate was constant. Finally, propranolol (a β-adrenergic receptor blocker with high penetration) was used as a positive control.

The formula for P _e calculation is shown in the next equation: where f is the running time (4 h), C _A (t) is the concentration of the compound in the acceptor well at time t, and C _D (t ₀ ) is the compound concentration in the donor well before running the PAMPA assay (t ₀ = 0 h). Permeability is considered excellent with values > 4.0 (- 10 ^"6 ) cm/s, uncertain between 1 .0 and 4.0 (- 10 ^"6 ) cm/s and poor with values below 1 .0 (- 10 ^"6 ) cm/s.

Transport (%) values were obtained by dividing the amount in the acceptor well at time f, C _A (t), and in the donor well at time zero, C _D (t ₀ ), multiplied by 100. Membrane retention was calculated from the difference between the initial amount, C _D (t ₀ ), and the amounts in donor, C _D (t), and acceptor, C _A (t), compartments at the end of the experiment (f = 4 h). The buffer (System Solution) was prepared from the commercial concentrated solution (plON) by dissolving 5 mL with 200 mL of water. The pH (2.4) was adjusted to 7.4 by using a 0.5 M NaOH solution. Then, 1 -propanol (20%) was added to the solution.

The samples were dissolved with the System Solution containing 20% 1 -propanol (1 mL). Peptides were assayed at concentrations adjusted to allow the best quantification by RP- HPLC. Propranolol was used as a positive control. The PAMPA sandwich (96-transwell plate) was separated into the donor and acceptor plates, and the stirring magnets were added to the donor compartments. Next, 4μί of a phospholipid mixture (Porcine Brain Polar Lipid Extract; 20 mg/mL in dodecane) was added to the membrane, located at the bottom of the acceptor compartments. This phospholipid mixture comprised phosphatidylcholine (PC; 12.6%),

phosphatidylethanolamine (PE; 33.1 %), phosphatidylserine (PS; 18.5%),

phosphatidylinositol (PI; 4.1 %), phosphatidic acid (0.8%), and other compounds (30.9%). Samples containing the peptides (195 μΙ_) were added to the donor compartments, (three replicates). Afterwards, acceptor wells were placed above the donor plate and filled with 200 μΙ_ of System Solution (20% 1 -propanol).

The PAMPA plate was placed into a GUTBOX (containing wet sponges) for 4 h at room temperature. Agitation was maintained in 25 μηη of unstirred water layer (UWL). After the incubation time, the donor and the acceptor plates were separated, and the samples were collected from both and placed into separate tubes. The integrity of the samples (donor and acceptor wells and time zero solutions) was identified by MALDI-TOF spectroscopy. The samples were also analyzed by RP-HPLC, and P _e was calculated from the integrated chromatographic peaks.

We studied the permeability, transport and membrane retention of different inhibitors SEQ ID NO: 1 , SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1 and SEQ ID NO: 14, results are shown in table 4.

Example 5. Synergic activity with colistin

Time-kill kinetic assays of colistin-sensitive (Col-S) ATCC 17978 strain or colistin-resistant (Col-R) 1 1 strain were conducted on Moeller Hinton Broth cation-adjusted in presence of SEQ ID NO:1 (0, 12.5 or 125 g/ml), SEQ ID NO:5 (125 g/ml), colistin (sub-MIC) alone or in combination with SEQ ID NO:1 , SEQ ID NO:5 or colistin were performed in duplicate as previously described [Smani et al. J. Infect. Dis. 201 1]. Moreover, in some conditions SEQ ID NO:1 , SEQ ID NO:5 or colistin was added for second time 4 h after bacterial addition. Drugs free broth was evaluated in parallel as a control, and cultures were incubated at 37 °C. Viable counts were determined by serial dilution at 0, 2, 4, 8, and 24 h after adding the SEQ ID NO:1 , SEQ ID NO:5 or colistin, and plating 100 μΙ_ of control, test cultures, or dilutions at the indicated times onto sheep blood agar plates. Plates were incubated for 24 h, and after colony counts, the Iog10 of viable cells (cfu/ml) was determined. We examined the ability of SEQ ID NO:1 in combination with colistin to kill Col-S ATCC 17978 strain and Col-R 1 1 strain in a time course assay. 125 g/ml of SEQ ID NO:1 in combination with 0.25 g/ml of colistin, a colistin sub-MIC for the ATCC 17978 strain, showed greater killing of the ATCC 17978 strain than colistin alone by decreasing the bacterial cell count after 24 h by 5.54 log CFU/ml. Combination of 12.5 μg ml SEQ ID NO:1 , a SEQ ID NO:1 Cmax in mice treated with 10 mg/kg SEQ ID NO:1 , and 0.25 g ml colistin showed the same reduction in bacterial cell count by 5.41 log CFU/ml respect to colistin after 24 h (Fig. 10A). Furthermore, combination of 125 μg/ml SEQ ID NO:1 with 8 μg/ml colistin, a colistin sub-MIC for 1 1 strain, showed excellent killing of 1 1 strain by decreasing the bacterial cell count respect to colistin after 2 and 4 h by 3.22 and 2.16 log cfu/ml, respectively, however bacteria showed regrowth after this time. With 12.5 μg/ml of SEQ ID NO:1 limited activity of SEQ ID NO:1 combined with 8 μg/ml of colistin was observed (Fig. 10A). In a control experiment, SEQ ID NO:1 , SEQ ID NO:5 or

combinations between SEQ ID NO:5 and 0.25 or 8 μg/ml colistin to ATCC 17978 or 1 1 strains, respectively, had no effect on their growth (Fig. 10A).

Because colistin half-life in bacterial culture broth is 4 h [Owen et al. J. Antimcrob.

Chemother. 2007, Bergen et al. Antimicrob. Agents Chemother. 2010], we cannot rule out the possibility that the observed bacterial regrowth with strain 1 1 in presence of MV5 and 8 μg/ml colistin (Fig. 1A) could be due to colistin degradation in the bacterial culture broth. To compensate this degradation, we added a second dose of 4 μg/ml of colistin 4 h post- incubation with SEQ ID NO:1 and 8 μg/ml of colistin. In this last case, greater activity and synergy between SEQ ID NO:1 and colistin were observed (Fig. 1 B). In the control experiment, addition of a second dose of SEQ ID NO:5 or SEQ ID NO:1 4 h post- incubation with SEQ ID NO:5 or SEQ ID NO:1 and 8 μg/ml of colistin did not affect significantly the regrowth of 1 1 strain (Fig. 1 B).

Solubility assay The compounds SEQ ID No8, SEQ ID No10 and SEQ ID No14 show clear solution at concentration of 20 mg/mL in aqueous buffer (Hank's balanced salt solution, pH7).

The compounds SEQ ID No1 and SEQ ID No1 1 show clear solution at concentration of 1 .25 mg/mL in aqueous buffer (Hank's balanced salt solution, pH7).

REFERENCES CITED IN THE APPLICATION

- E. Kaiser et al., Anal. Biochem. 1970, vol. 34, pp. 595-598 - A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791

CLAUSES:

1 . - A cyclic peptide having 4-8 amino acids independently selected from tryptophan, proline and arginine, for use as medicament.

2. - A peptidic compound according to clause 1 , wherein the peptide is a cyclic

hexapeptide, for use as medicament.

3. - A peptidic compound according to any of the clauses 1 -2, wherein the amino acids are either in the enantiomer D form or enantiomer L form, for use as medicament. 4.- A peptidic compound according to any of the clauses 1 -3, wherein

the hexapeptide is an hexacyclic of sequence SEQ ID NO:1 , SEQ ID NO:2, SEQ I D NO:3, SEQ ID NO:4 or any of their salts, stereoisomers, enantiomers, prodrugs, derivatives, analogues or any of their combinations, for use as medicament. 5.- A peptidic compound according to any of the clauses 1 -4, wherein the selected peptide is the peptide of sequence SEQ ID NO: 1 or any of their salts, stereoisomers, enantiomers, prodrugs, derivatives, analogues or any of their combinations, for use as medicament.

6.- A peptidic compound according to any of the clause 1 -5, wherein the selected peptide is the peptide of sequence SEQ ID NO: 1 and Formula I , for use as medicament.

7 - A combined preparation that comprises a peptidic compound according to any of the clauses 1 -6 and another active ingredient . 8.- A composition that comprises at least a peptidic compound according to any of the clauses 1 -6, or the combined preparation according to clause 7, for use in the elaboration of a medicament.

9.- A composition following the previous clauses, wherein the composition is a

pharmaceutical composition, for use as medicament. 10. - A composition and/or of the combined preparation according to any of the clauses 8- 9, wherein the composition or the combined preparation comprises a pharmaceutically acceptable vehicle, for use as medicament. 1 1 .- A composition according to any of the clauses 8-10, wherein the composition also comprises another active ingredient for use as medicament.

12. - A composition and/or of the combined preparation according to any of the clauses 8-

1 1 , wherein the other active ingredient is an antimicrobial compound, preferably an antibiotic and even more preferably selected from the list consisting of: imipenem, meropenem, ciprofloxacin, cefepime, tigecyclin, colistin, amoxicillin/clavulanic acid, or any of their combinations, for use as medicament

13. - A pharmaceutical form that comprises a peptidic compound according to any of the clauses 1 -6, or a combined preparation according to clause 7, or a composition or a combined preparation according to any of the clauses 8-12.

14. - A pharmaceutical form according to clause 13, that is selected from the list consisting of: poultice, ointment, plaster, cream, solution, suspension, emulsion, lotion, liniment, gel, hydrogel, hydrocolloid, foam, powder, or any of their combinations.

15. - A peptidic compound according to any of the clauses 1 -6, a combined preparation according to clause 7, a composition or a combined preparation according to any of the clauses 8-12, or a pharmaceutical form according to any of the clauses 13-14, for use in the elaboration of a medicament.

16. - A peptidic compound according to any of the clauses 1 -6, a combined preparation according to clause 7, a composition or a combined preparation according to any of the clauses 8-12, or a pharmaceutical form according to any of the clauses 13-14, for use in the elaboration of a medicament for the treatment, relief and/or prevention of infections caused by Gram negative pathogens.

17. - A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 16, wherein the Gram negative pathogen belongs to Phylum

Proteobacteria, for use as medicament. 18.- A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 16-17, wherein the Gram negative pathogen belongs to

Gammaproteobacteria class, for use as medicament. 19.- A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 16-18, wherein the Gram negative pathogen belongs to

Enterobacterials order, for use as medicament.

20. - A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 19, wherein the Gram negative pathogen belongs to the family

Enterobacteriaceae, and more preferably to the Escherichia genus, for use as

medicament.

21 . - A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 19, wherein the Gram negative pathogen belongs to E. coli specie, for use as medicament.

22. - A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 16-18, wherein the Gram negative pathogen belongs to

Pseudomonadales order, for use as medicament.

23. - A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 22, wherein the Gram negative pathogen belongs to the family Pseudomonadaceae, and more preferably to the Pseudomonas genus, for use as medicament.

24. - A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 23, wherein the Gram negative pathogen belongs to P. aeruginosa specie, for use as medicament.

25. - A peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 22, wherein the Gram negative pathogen belongs to the family Moraxellaceae, and more preferably to the Acinetobacter genus, for use as medicament. 26.- The use of a peptidic compound, a combined preparation, a composition, a pharmaceutical form according to clause 25, wherein the Gram negative pathogen belongs to A. Baumannii specie, for use as medicament.