WO2016046397A1 - Peptides cycliques contre la protéine membranaire externe a (ompa) pour le traitement d'infections provoquées par des pathogènes à gram négatif - Google Patents

Peptides cycliques contre la protéine membranaire externe a (ompa) pour le traitement d'infections provoquées par des pathogènes à gram négatif Download PDF

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Publication number
WO2016046397A1
WO2016046397A1 PCT/EP2015/072166 EP2015072166W WO2016046397A1 WO 2016046397 A1 WO2016046397 A1 WO 2016046397A1 EP 2015072166 W EP2015072166 W EP 2015072166W WO 2016046397 A1 WO2016046397 A1 WO 2016046397A1
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WIPO (PCT)
Prior art keywords
seq
peptide
pro
cyclic peptide
use according
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PCT/EP2015/072166
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English (en)
Inventor
Younes Smani
Jerónimo PACHÓN DÍAZ
Ernest GIRALT LLEDÓ
Meritxell Teixidó Turá
Núria BAYÓ PUXAN
Jordi VILA ESTAPÉ
Xavier VILA FARRÉS
Original Assignee
Servicio Andaluz De Salud
Fundació Institut De Recerca Biomèdica (Irb Barcelona)
Hospital Clínic De Barcelona
Institut D'investigacions Biomèdiques August Pi I Sunyer (Idibaps)
Universidad De Sevilla
Universitat De Barcelona
Fundació Privada Institut De Salut Global Barcelona
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Publication of WO2016046397A1 publication Critical patent/WO2016046397A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • OmpA anti-outer membrane protein A
  • 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].
  • 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.
  • 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.
  • £. 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.
  • the most studied virulence factor in A. baumannii is the outer membrane protein A
  • 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.
  • 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.
  • 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].
  • 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.
  • 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 ].
  • 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].
  • OmpA can stimulate the murine dendritic cells to release pro-inflammatory cytokines [Torres et al. Infect. Immun. 2006; 74: 2676-85].
  • 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.
  • 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.
  • 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
  • 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,
  • the peptide is a cyclic hexapeptide.
  • 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.
  • 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).
  • 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).
  • 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).
  • amino acids are in D- or L-enantiomer form. It means aminoacids can all be D, all L or combinations of L and D.
  • the peptide is a peptide of formula (I).
  • the peptide is a peptide of formula (II).
  • the peptide is a peptide of formula (III).
  • the peptide is a peptide of formula (IV).
  • the peptide is a peptide of formula (VI).
  • the peptide is a peptide of formula (VII).
  • 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).
  • the peptide is a peptide of formula (X).
  • the peptide is a peptide of formula (XI).
  • the peptide is a peptide of formula (XII).
  • the peptide is a peptide of formula (XIII).
  • the peptide is a peptide of formula (XIV).
  • the peptide is a peptide of formula (XV).
  • the peptide is a peptide of formula (XVI).
  • the peptide is a peptide of formula (XVII).
  • 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.
  • the peptide of the invention is cyclic.
  • the peptide is a cyclic hexapeptide.
  • 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.
  • 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).
  • the peptide is a peptide of formula (I).
  • 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).
  • the peptide is a peptide of formula (IV).
  • compositions 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.
  • 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.
  • 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 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
  • 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
  • an implantable medical device comprising a cyclic peptide, or a combined preparation as defined above.
  • the medical device may be a catheter of a prosthesis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 1 (0.25 and 0.5 mg/ml)
  • SEQ ID NO: 5 0.5 mg/ml
  • FIG. 4 Effect of SEQ ID NO: 1 on the interaction of A baumannii, P. aeruginosa and £ coli with fibronectin.
  • 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. 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.
  • 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.
  • 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
  • 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).
  • the peptide of the invention is cyclic.
  • 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.
  • 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).
  • 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).
  • 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
  • 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.
  • the peptide of the invention is a cyclic peptide.
  • 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.
  • 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.
  • the peptide compound of sequence SEQ ID NO:1 has the formula (Formula
  • 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.
  • the peptide compound of sequence SEQ ID NO:2 has the formula (Formula II)
  • 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.
  • the peptide compound of sequence SEQ ID NO:3 has the formula (Formula Hi)
  • 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.
  • the peptide compound of sequence SEQ ID NO:4 has the formula (Formula iv)
  • 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.
  • the peptide compound of sequence SEQ ID NO:5 has the formula (Formula V).
  • 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.
  • the peptide compound of sequence SEQ ID NO:6 has the formula (Formula VI)
  • 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.
  • the peptide compound of sequence SEQ ID NO:7 has the formula (Formula VII)
  • 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.
  • the peptide compound of sequence SEQ ID NO:8 has the formula (Formula VIII)
  • 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.
  • the peptide compound of sequence SEQ ID NO:9 has the formula (Formula IX)
  • 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.
  • the peptide compound of sequence SEQ I D NO: 1 1 has the formula (Formula XI)
  • 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.
  • the peptide compound of sequence SEQ I D NO:13 has the formula (Formula XI II)
  • 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.
  • the peptide compound of sequence SEQ I D NO:15 has the formula (Formula XV)
  • 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.
  • the peptide compound of sequence SEQ ID NO:17 has the formula (Formula XVII)
  • 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.
  • the peptide compound of sequence SEQ ID NO:18 has the formula (Formula XVIII)
  • enantiomers 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.
  • enantiomer means a single enantiomer that is sustantially excempt of the opposite enantiomer and/ or any other stereoisomer.
  • sustantially 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.
  • 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.
  • 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.
  • pharmaceutically acceptable salt 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,
  • the preferred salts of acid addition pharmaceuticallytransporte 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
  • 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.
  • 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.
  • composition is a
  • 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:
  • the active ingredient is an antimicrobial, and more preferably an antibiotic.
  • the active ingredient is selected from imipenem, meropenem, ciprofloxacine, cefepime, tigecicline, colistin, amoxiciline/clavulanic, or any of their combinations.
  • 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
  • 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.
  • medicine or medicament refers to any substance used to prevent, diagnose, relieve, treat or cure any disease in human or animals.
  • 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
  • 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.
  • excipients such as diluents, carriers and similars
  • additives such as stabilizing agents, preservatives, solubilizing agents, buffer solutions and similar, as many as desired.
  • excipients of the formulation polyvinylpyrrolidone, gelatin, hydrocellulose, arabic gum, polyethilenglycol, mannitol, sodium chloride or sodium citrate are included.
  • water that contains, at least, one or more buffer constituents, and also stabilizing agents, preservatives and solubilizing agents can be used.
  • 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.
  • compositions of the present 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.
  • 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.
  • 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.
  • the pharmaceutical form of the invention is a plaster.
  • the pharmaceutical form is selected from a solution, suspension or an emulsion.
  • 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.
  • 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.
  • colloids are materials formed by a dispersed phase (matrix) and a dispersant phase (filler).
  • dispersing phase is water, it is called “hydrocolloid”. It can be
  • 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, ....
  • 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.
  • 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.
  • a mammal including a human, suffering from or being susceptible to a an infection caused by Gram negative pathogens
  • 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.
  • Salmonella enterritis and typhoid fever
  • Yersinia plague
  • Vibrio cholera
  • P. aeruginosa nosocomial infections or in patients with cystic fibrosis
  • Klebsiella pneumoniae pneumoniae
  • the Gram negative pathogen belongs to Phylum Proteobacteria, and more preferably to
  • 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.
  • 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.
  • 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.
  • A. baumannii species 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 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.
  • EEC enteropathogenic
  • ETEC enterotoxigenic
  • ECIS enteroinvasive
  • amino acid sequence refers to a polymeric form of amino acids of any length, which can be coding or non-coding, chemically or biochemically modified.
  • amino acids it will consider the utilization of L-amino acids, except those indicated as D-amino acid.
  • 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.
  • 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.
  • Peptide compounds were purified by HPLC using a Symmetry Ci 8 column (100 x 30 mm x 5 ⁇ - ⁇ , 100 A, Waters), flow 10 mL/min, solvents: A: H 2 0 with 0.1 % TFA; B: MeCN with
  • the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.
  • the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.
  • Example 1.4 Preparation of cyclo[Arg-Pro-D-Trp-Arg-Pro-D-Trp] (SEQ ID NO:4)
  • the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.
  • the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.
  • Example 1.8 Preparation of cyclo[Trp-D-Pro(4-NH 2 )-Arg-Trp-D-Pro-Arg] (SEQ ID NO:8)
  • the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.
  • 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.
  • the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.
  • the peptide was cleaved from the resin and lyophilized. Cyclization was performed following the described method.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 1 we studied the effect of SEQ ID NO: 1 on the adherence of A. baumannii, P. aeruginosa and £ coli to A549 cells (figure 2).
  • 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.
  • 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.
  • A. baumannii (10 8 cfu/ml): ATCC 17978, 77 and 1 13-16 strains
  • P. aeruginosa (10 8 cfu/ml): PA01 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mice we are interested to use the inhibitor SEQ I D NO: 1 as directed treatment against infections caused by A baumannii.
  • mice 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.
  • MLD minimal lethal dose
  • SEQ ID NO: 1 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.
  • 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).
  • the PAMPA assay 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.
  • 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.
  • 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.
  • 4 ⁇ 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%),
  • phosphatidylinositol (PI; 4.1 %), phosphatidic acid (0.8%), and other compounds (30.9%).
  • Samples containing the peptides (195 ⁇ _) were added to the donor compartments, (three replicates). Afterwards, acceptor wells were placed above the donor plate and filled with 200 ⁇ _ 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 ⁇ 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.
  • 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 ⁇ _ 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.
  • 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.
  • 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 ⁇ g 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).
  • 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).
  • hexapeptide for use as medicament.
  • 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.
  • 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.
  • composition following the previous clauses, wherein the composition is a
  • compositions for use as medicament.
  • composition or the combined preparation comprises a pharmaceutically acceptable vehicle, for use as medicament.
  • composition according to any of the clauses 8-10, wherein the composition also comprises another active ingredient for use as medicament.
  • 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
  • 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
  • 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.
  • 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
  • Enterobacteriaceae and more preferably to the Escherichia genus, for use as
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Communicable Diseases (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des peptides contre la protéine externe A (OmpA) pour le traitement d'infections provoquées par des pathogènes à Gram négatif, en particulier Acinetobacter baumanii, Escherichia coli ou Pseudomonas aeruginosa. Parmi les hexapeptides cycliques préférés figurent le tryptophan, la proline et l'arginine, et un hexapeptide cyclique préféré possède la séquence c- (WPRWPR). L'invention concerne également des variants de ceux-ci avec des acides aminés modifiés, des acides aminés de la série D ou un ordre différent des acides aminés dans l'hexapeptide cyclique.
PCT/EP2015/072166 2014-09-25 2015-09-25 Peptides cycliques contre la protéine membranaire externe a (ompa) pour le traitement d'infections provoquées par des pathogènes à gram négatif WO2016046397A1 (fr)

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CN115837016A (zh) * 2023-02-04 2023-03-24 四川大学华西医院 一种aoa-2-pxb@lnp脂质纳米粒及制备方法

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GB2408263A (en) * 2003-11-14 2005-05-25 Pacgen Biopharmaceuticals Inc Antimicrobial peptides with reduced hemolysis and methods of their use.
WO2011095218A1 (fr) * 2010-02-05 2011-08-11 Polyphor Ag Peptidomimétiques fixés au brin matrice ayant une activité modulatrice des cxcr7
WO2012083226A1 (fr) * 2010-12-17 2012-06-21 The Penn State Research Foundation Identification d'inhibiteurs d'une réponse au stress bactérien

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N V PRASADARAO ET AL: "Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells", INFECTION AND IMMUNITY, 1 January 1996 (1996-01-01), UNITED STATES, pages 146 - 153, XP055226145, Retrieved from the Internet <URL:http://iai.asm.org/content/64/1/146.full.pdf> [retrieved on 20151105] *
VEBER D F ET AL: "A POTENT CYCLIC HEXAPEPTIDE ANALOGUE OF SOMATOSTATIN", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 292, 2 July 1981 (1981-07-02), pages 55 - 58, XP000579804, ISSN: 0028-0836, DOI: 10.1038/292055A0 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115837016A (zh) * 2023-02-04 2023-03-24 四川大学华西医院 一种aoa-2-pxb@lnp脂质纳米粒及制备方法
CN115837016B (zh) * 2023-02-04 2024-02-27 四川大学华西医院 一种aoa-2-pxb@lnp脂质纳米粒及制备方法

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