WO2019162302A1 - Nc8bêta de plantaricine tronqué destiné à être utilisé dans le traitement ou la prophylaxie d'une infection bactérienne - Google Patents

Nc8bêta de plantaricine tronqué destiné à être utilisé dans le traitement ou la prophylaxie d'une infection bactérienne Download PDF

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WO2019162302A1
WO2019162302A1 PCT/EP2019/054166 EP2019054166W WO2019162302A1 WO 2019162302 A1 WO2019162302 A1 WO 2019162302A1 EP 2019054166 W EP2019054166 W EP 2019054166W WO 2019162302 A1 WO2019162302 A1 WO 2019162302A1
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seq
pharmaceutical composition
plnc8
ytlgikilw
polypeptide
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PCT/EP2019/054166
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English (en)
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Torbjörn BENGTSSON
Hazem KHALAF
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Bengtsson Torbjoern
Khalaf Hazem
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • 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

  • This invention pertains in general to the field of treatment of infections. More particularly the invention relates to a use of a truncated b-chain of the bacteriocin PLNC8 ⁇ for the prevention and/or treatment of infections.
  • HAI Hospital-acquired infection
  • a nosocomial infection is an infection that is acquired in a hospital or other health care facility. Such an infection can be acquired in hospital, nursing home, rehabilitation facility, outpatient clinic, or other clinical settings. Infection is spread to the susceptible patient in the clinical setting by various means. Health care staff can spread infection, in addition to contaminated equipment, bed linens, or air droplets. It is estimated that 6 million patients in the EU and USA contract a HAI per year, resulting in up to 150 000 deaths annually.
  • ESKAPE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter
  • MRS A Metal-resistant Staphylococcus aureus
  • VRE Vancomycin- resistant Enterococcus
  • Streptococcus spp Escherichia coll.
  • Bacteriocins constitute a promising potential alternative or complement to traditional antibiotics and have several advantages such as low risk of resistance development, limited effects on normal flora and beneficial effects on human tissue.
  • Bacteriocins are a group of bacterially produced peptides used to fight other bacteria. Bacteriocins may have a net positive charge and express amphipathic structures that interact with negatively charged microbial membranes and kill microbes usually through pore-forming mechanisms. These mechanisms are more difficult to evade by developing resistance, compared to metabolic enzymes, which usually are targets for conventional antibiotics.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSE methicillin-resistant Staphylococcus epidermidis
  • VRE vancomycin- resitant Enterococcus
  • the present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above-identified deficiencies in the art and disadvantages singly or in any combination and solves at least the above mentioned problems.
  • a polypeptide X having 14 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with YTLGIKIL W S AYKH (SEQ ID NO 3) is thus provided.
  • the polypeptide X is a truncated form of the b-chain of the bacteriocin PLNC8 ⁇ .
  • Such a polypeptide has been shown to have antimicrobial activity.
  • the antimicrobial activity is i.a. mediated by lysis and fragmentation of the cell wall of the bacteria.
  • the polypeptide X comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising
  • VPTSVYTLGIKILWSAYKH SEQ ID NO 6 PTSVYTLGIKILWSAYKH SEQ ID NO 7 TSVYTLGIKILWSAYKH SEQ ID NO 8 SVYTLGIKILWSAYKH SEQ ID NO 9 V YTLGIKILW S AYKH SEQ ID NO 10 YTLGIKIL W S AYKH SEQ ID NO 3 YTLGIKILW S AYKHR SEQ ID NO 11 YTLGIKILW S AYKHRK SEQ ID NO 12 YTLGIKILW S AYKHRKT SEQ ID NO 13 YTLGIKILW S AYKHRKTI SEQ ID NO 14 YTLGIKILW S AYKHRKTIE SEQ ID NO 15 YTLGIKILW S AYKHRKTIEK SEQ ID NO 16 YTLGIKILW S AYKHRKTIEKS SEQ ID NO 17 YTLGIKILW S AYKHRKTIEKSF SEQ ID NO 18
  • At least 90% of the amino acids in said polypeptide X are D-amino acid residues.
  • Such peptides are more stable and less sensitive to proteolytic cleavage compared to their corresponding L-variants.
  • a pharmaceutical composition comprising a polypeptide X according to the invention is also provided.
  • the pharmaceutical composition may be formulated for administration as a single dose or as multiple doses, such as two, three, four, five or even more doses.
  • the pharmaceutical composition may further comprise a polypeptide Y having 15 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with DLTTKLWSSWGYYLG (SEQ ID NO 4).
  • the polypeptide Y is a truncated form of the a-chain of the bacteriocin PLNC8 ⁇ .
  • the polypeptide Y comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising:
  • At least 90% of the amino acids in peptide X and/or peptide Y are D-amino acid residues.
  • Such peptides are more stable and less sensitive to proteolytic cleavage compared to their corresponding L-variants.
  • the pharmaceutical composition further comprises at least one antibiotic.
  • the peptide(s) and the antibiotic act synergistically and enhance the effect of each other.
  • the antibiotic is selected from the group consisting of antibiotics that inhibit bacterial cell wall synthesis, antibiotics that inhibit nucleic acid synthesis and antibiotics that inhibit protein synthesis.
  • peptide X and peptide Y are present in a in a molar ratio of from between 5:1 to 1 :20, preferably 1 :1 to 1 :7, most preferably 1 :1.
  • the pharmaceutical composition comprises between 0.1 to 50 mM of peptide X and/or peptide Y.
  • the pharmaceutical composition comprises the antibiotic in an amount of between 0.0019 and 50 pg/ml.
  • the pharmaceutical composition is formulated as a solution, a cream, a gel, or an ointment or formulated in immobilized form as a coating on a device.
  • the pharmaceutical composition is formulated as a composition, which when applied to a surface forms a coating substantially coating the part of the device on which the pharmaceutical composition is applied.
  • the coating may be resistant or substantially resistant to water and certain solvents.
  • the coating may also be resistant or substantially resistant to abrasion and/or wear.
  • the coating when applied to e.g. a handle, the coating will not need to be applied after each use of the handle, but will resist abrasion and/or wearing for a longer time period, such as several hours up to several days or weeks, depending on the frequency of interaction with other objects.
  • the pharmaceutical composition is formulated as a gel, wherein the gel further comprises gelatine and glycerol.
  • the gel may comprise hyaluronic acid.
  • the pharmaceutical composition is formulated in immobilized form as a coating on a device, wherein the device is chosen from the group consisting of a wound dressing, an orthopedic implant, a dental implant, a urinary catheter and an urinary stent.
  • the device is provided with an antibacterial coating, which exerts its effect on the surroundings during a longer period of time, such as hours, days or even weeks.
  • This may be advantageous, since the antibiotic effect is only exerted locally and not systemically.
  • infections that are commonly associated with implants, catheters and stents may be counteracted locally, without the need of administrating antibiotics systemically.
  • a pharmaceutical composition for use in the treatment or prophylaxis of a bacterial infection wherein the pharmaceutical composition is a pharmaceutical composition according to the invention is also provided.
  • the bacterial infection may be caused by gram-negative bacteria.
  • the bacterial infection may be caused by gram-positive bacteria.
  • the bacterial infection is caused by
  • Staphylococcus spp including MRSA, MRSE
  • Streptococcus spp e.g. S. mutans, S. constellatus, S. anginosus
  • Enterococcus faecium including VRE
  • Klebsiella pneumoniae Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and/or Escherichia coli.
  • the bacterial infection is caused by
  • Staphylococcus spp such as S. mutans, S. constellatus, S. anginosus.
  • the bacterial infection is caused by Staphylococcus spp and/or Streptococcus spp.
  • the bacterial infection may be caused by Escherichia coli.
  • the bacterial infection may be caused by Enterococcus ssp.
  • the bacterial infection may be caused by Pseudomonas aeruginosa.
  • the bacterial infection may be caused by Porphyromonas gingivalis.
  • Such bacteria are a common cause of hospital-acquired infection (HAI).
  • ESKAPE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter
  • MRS A Metal-resistant Staphylococcus aureus
  • VRE Vancomycin-resistant Enterococcus
  • Streptococcus spp Escherichia coli.
  • the composition is administered locally on the site of infection, such as topically.
  • a use of a pharmaceutical composition according to the invention in coating at least part of a device to limit colonization of bacteria on the surface of the device is also provided.
  • the device is a medical device, such as a prosthesis or a wound dressing.
  • the bacteria are Staphylococcus spp
  • Streptococcus spp e.g. S. mutans, S. constellatus, S.
  • the bacterial infection is caused by
  • Staphylococcus spp Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus.
  • the bacteria are Staphylococcus spp and/or Streptococcus spp.
  • a polypeptide for use in the treatment or prophylaxis of a bacterial infection wherein the polypeptide is a polypeptide according to the invention, is also provided.
  • Figure 1 shows PLNC8 ⁇ markedly inhibits the growth and survival of different strains of S. aureus and S. epidermidis. Different Staphylococcus species were cultured for 20 h in the presence of increasing concentrations of PLNC8 ⁇ (1 : 1).
  • S. epidermidis was generally more susceptible to PLNC8 ⁇ than S. aureus.
  • FIG. 2 The molar ratio of PLNC8 a and PLNC8 b is critical for optimal antimicrobial activity.
  • S. epidermidis ATCC 12228 was exposed to different molar ratios of PLNC8 a and b for 20 h.
  • a molar ratio of 1 : 1 between PLNC8 a and PLNC8 b is most efficient at inhibiting and killing S. epidermidis.
  • Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for different molar ratios of PLNC8 a and b. *Thc highest total concentration of the peptides was kept constant at 50 mM, while the concentrations of PLNC8 a and b individually were altered to obtain different molar ratios.
  • PLNC8 ⁇ is effective at disrupting S. epidermidis biofilms.
  • the biofilm positive strain S. epidermidis RP62A was allowed to form biofilms followed by removal of suspended bacteria and then incubation with PLNC8 ⁇ , PLNC8 a or PLNC8 b for 1 h.
  • PLNC8 ⁇ is most efficient and rapid at disrupting biofilms of S. epidermidis.
  • A- CF release was recorded after exposure of liposomes with increasing concentrations of L- or D-variants of PLNC8 a, b or ⁇ (1 :1).
  • B- S. epidermidis ATCC 12228 was incubated with increasing concentrations of PLNC8 a and b, alone or in combination (1 : 1), for 20 h.
  • Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for PLNC8 a, b and ⁇ are indicated.
  • FIG. 5 The L- and D-variant of PLNC8 ⁇ rapidly permeabilize the plasma membrane of S. epidermidis.
  • FIG. 6 The D-form of PLNC8 ⁇ is more stable and less sensitive to proteolytic cleavage.
  • the peptides (100mM) a) L-PLNC8 ⁇ b) D-PLNC8 ⁇ c) E-REM38 ⁇ d) ⁇ -REM38b were treated with Trypsin (5mM) in Ammonium Bicarbonate buffer (50mM) for 16 h at 37°C before being acified (2.5% TFA), dried, suspended in H20 +0.1% TFA, desalted (ZipTip) and analyzed by MALDI-ToF MS. Number above the peaks indicate molecular weights (Da) and number in brackets sequences of amino acids. Full-length a- and b-peptide, 1-29 and 1-34, respectively.
  • FIG. 7 Both the L- and D-form of PLNC8 ⁇ display a low hemolytic activity. Human erythrocytes were incubated with different concentrations (0.5-50 mM) of L- or D-variant of PLNC8 ⁇ , b or ⁇ (1 :1) for 1 h. In (B) this is shown for and truncated forms ⁇ 1-15, ⁇ 1-22, b7-20, b1-20, b7-34. Hemolytic activity was determined after incubation of human erythrocytes with the peptides for 1 h.
  • Figure 8 Amino acid sequences of truncated peptides of PLNC8 a and PLNC8 b.
  • MIC Minimum inhibitory concentration
  • MMC minimum bactericidal concentration
  • FIG. 11 Antimicrobial activity of a combination of truncated PLNC8 a and PLNC8 b.
  • FIG. 12 Morphological effects of PLNC8 ⁇ on S. epidermidis using TEM and SEM.
  • PLNC8 a caused massive bleb formation and PLNC8 b induced bacterial lysis shown by an extracellular release of intracellular content.
  • PLNC8 ⁇ was most efficient causing complete bacterial lysis.
  • the truncated forms of PLNC8 b, b ⁇ -20 and b7-20, induced fragmentation of the bacterial cell wall and in combination with PLNC8 ⁇ S. epidermidis went through lysis.
  • PLNC8 ⁇ in a formula is effective against S. epidermidis and retains its activity after long-term storage. Bacterial lysis was visualized by studying the uptake of Sytox Green by S. epidermidis ATCC 12228 exposed to a gel containing different concentrations (5-100 mM) of PLNC8 ⁇ . The activity of the formula with 100 mM PLNC8 ⁇ was also tested on blood-agar plates with S. epidermidis, at time zero and after long-term storage at 4°C. A gradient of PLNC8 ⁇ was created by spreading the gel over the agar surface with a plastic loop. Inhibition of bacterial growth is demonstrated by the translucent areas.
  • S. epidermidis isolated from prosthetic joint infections, including heterogeneous glycopeptide intermediate S. epidermidis (hGISE), was exposed to L- PLNC8 ⁇ for 20 h and MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were determined.
  • hGISE heterogeneous glycopeptide intermediate S. epidermidis
  • PFNC8 ⁇ acts synergistically with antibiotics. Synergistic antimicrobial effects between antibiotics and F- or D-PFNC8 ⁇ .
  • S. epidermidis strain 154 was exposed to F-PFNC8 ⁇ or D-PFNC8 ⁇ (3.1 mM), a serial dilution of teicoplanin, vancomycin, rifampicin and gentamicin, alone or in their combination with 3.1 mM F-PFNC8 ⁇ or D-PFNC8 ⁇ .
  • PFNC8 b and truncated PFNC8 a markedly amplify the inhibitory effects of teicoplanin against S. epidermidis. Synergistic antimicrobial effects between teicoplanin and PFNC8 ⁇ . S. epidermidis (strain 154) was exposed to a serial dilution of teicoplanin or full-length/truncated PLNC8 ⁇ alone or in their combination (serial dilution of teicoplanin and 6.25 mM full-length/truncated PLNC8 ⁇ ), and
  • FIG. 17 PLNC8 ⁇ markedly permeabilizes and kills different species of Streptococcus. Streptococcus spp (S. mutans (Sm), S. constellatus (Sc) and S. anginosus (Sa)) were treated with 5mM PLNC8 ⁇ for 2 min, followed by analysis of uptake of Sytox Green. S. constellatus and S. anginosus were more susceptible to PLNC8 ⁇ than S. mutans.
  • PLNC8 ⁇ causes rapid membrane permeabilization on liposomes.
  • CD-spectroseopy of PLNC8 ⁇ CD measurements of (A) L- PLNC8 ⁇ and (B) D-PLNC8 ⁇ (100mM each) without (dashed) and with (solid) liposomes (0.5mg/ml, ⁇ 660mM) in PBS. Three repeats with PBS as background.
  • Liposome containing samples were incubated for at least 30 min prior to measurements.
  • HAI Hospital-acquired infection
  • peptides derived from a L. plantarum NC8 bacteriocin optionally used together with antibiotics.
  • HAI Hospital-acquired infection
  • the invention is not limited to this application but these peptides may be applied to many other uses, including for example disinfection and coating of surfaces.
  • HAI Hospital-acquired infection
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSE Staphylococcus epidermidis
  • Lactobacillus species indeed may be able to contribute to solving these problems, from its ability to suppress pathogens primarily through expression and secretion of certain bacteriocins.
  • Lactobacillus plantarum is a highly versatile lactic acid bacterium found in saliva and gastrointestinal tract as well as fermented vegetables, meat and dairy products.
  • L. plantarum NC8 has been used as a model strain in many laboratories worldwide, and is a naturally plasmid-free L. plantarum strain.
  • L. plantarum NC8 has previously been shown to produce a two-peptide bacteriocin, PLNC8 ⁇ , classified as a class lib bacteriocin. The inventors have previously shown that PLNC8 ⁇ is efficient against the periodontal pathogen Porphyromonas gingivalis and stimulates cell proliferation (1,2).
  • the idea of the invention is to exploit the antibacterial effects of bacteriocin PFNC8c 3 for the prevention and treatment of acute and chronic infections, such as periodontitis, wound infections, implant-associated infections and urinary tract infections.
  • Products based on bacteriocins can be of enormous importance in health care, with improved public health and a positive impact on the social economy.
  • PLNC8 ⁇ markedly inhibited the growth and the survival of all bacterial strains (Fig. 1).
  • a bio film is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharides, proteins and extracellular DNA.
  • Staphylococcus biofilms are not totally impervious to antibiotics, and certain fluorescently tagged antimicrobials (such as daptomycin) have been shown to penetrate the biofilms of S. aureus and S. epidermidis by diffusion.
  • Truncation of a polypeptide will result in structural and chemical changes of the molecule, why truncated peptides of PLNC8 a and PLNC8 b, respectively, were constructed in sequences of 6-7 amino acids, to correspond to the number of amino acids required for formation of an alpha helix (shown in Fig. 8).
  • the effects of truncated PLNC8 a and PLNC8 b were evaluated in both a liposome system (resembling bacteria) and on S. epidermidis.
  • Truncated peptides of PLNC8 b were shown to have antimicrobial activity. The antimicrobial activity is i.a. mediated by lysis and fragmentation of the cell wall of the bacteria.
  • Truncated PLNC8 b peptides of the invention are referred to as polypeptide X.
  • a polypeptide X having 14 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with YTLGIKIL WSAYKH (SEQ ID NO 3) is provided.
  • the polypeptide X is a truncated form of the b-chain of the bacteriocin PLNC8 ⁇ .
  • fragments with potent antimicrobial activity were PLNC8 b fragments comprising amino acids 1-34 (full-length), 7-34, 1-20 and 7-20. It seems that peptide b-sequences b7-13 and b14-20, or parts thereof, are crucial for the antibacterial effects of PLNC8 b. Furthermore, fragments retaining the b7-13 and b14-20 sequences were even more efficient when combined with b1-6. Thus, the peptide b1-20 is the most effective in inhibiting S. epidermidis. It was surprisingly found that growth of S.
  • epidermidis was most efficiently inhibited by either sequence b1-20 (SEQ ID NO 5) or b7-20 (SEQ ID NO 3), respectively, and these truncated peptides were as effective, or even more effective, than the full-length native PLNC8 b (1-34) (Fig. 9).
  • the polypeptide X comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising
  • a pharmaceutical composition comprising a polypeptide X according to the invention is also provided.
  • the pharmaceutical composition may be formulated for administration as a single dose or as multiple doses, such as two, three, four, five or even more doses.
  • Truncated PLNC8 a peptides of the invention are referred to as polypeptide Y.
  • truncated PLNC8 b fragments When combining the truncated PLNC8 b fragments with a PLNC8 a peptide, it was revealed that a combination of truncated b7-34, b7-20, b ⁇ -20, or b1-34 showed effect on MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) against S. epidermidis, as shown in figure 9. As can be seen, truncated b7-20, b ⁇ -20 were especially effective, both in themselves and in combination a PLNC8 a peptide.
  • the pharmaceutical composition may further comprise a polypeptide Y having 15 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with DLTTKLWSSWGYYLG (SEQ ID NO 4).
  • the polypeptide Y is a truncated form of the a-chain of the bacteriocin PLNC8 ⁇ .
  • polypeptide Y comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising:
  • DLTTKLWSSWGYYLGKKARWNLKHPYVQ SEQ ID NO 25 DLTTKLWS S WG Y YLGKKARWNLKHP YV SEQ ID NO 26 DLTTKLWS SWGY YLGKKARWNLKHP Y SEQ ID NO 27 DLTTKLWS SWGY YLGKKARWNLKHP SEQ ID NO 28 DLTTKLWS S WG Y YLGKKARWNLKH SEQ ID NO 29 DLTTKLW S S W GYYLGKKARWNLK SEQ ID NO 30 DLTTKLW S S WGYYLGKKARWNL SEQ ID NO 31.
  • polypeptide Y comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising:
  • the innovation pertains to truncated peptides from plantaracin NC8 ⁇ (PLNC8 ⁇ ) a and b peptides (e.g. comprising the functional motifs in a1-15, ⁇ 1-22, b ⁇ -20 and b7-20). It was found that antibacterial properties are retained, or even enhanced, while higher diffusion rates in bacterial films are obtained when peptide size decreases. This since the diffusion coefficients increase strongly as the system size increases, enabling smaller peptides to penetrate further into a biofilm. In a gel, such as a bacterial biofilm, diffusion is even more affected by particle size, since larger particles will also have a higher risk of becoming entrapped in pores of the gel.
  • Bacterial biofilms may also seek to combat antibiotics by a reaction with the antimicrobial agent. Similarly, infections are often associated with high proteolytic activity caused by both bacteria and the body's immune system, which means that antimicrobial peptides or proteins may be inactivated.
  • D- PLNC8 ⁇ , D-PLNC8b, L-PLNC8 ⁇ and T-PLNC8b were exposed to trypsin and the presence of proteolytic fragments was analyzed with MALDI-TOF mass spectrometry (Fig. 6). While trypsin generated several fragments of both the a- and b-peptide of L-PLNC8, no obvious fragmentation was observed of the a- and b-peptide of D-PLNC8. Thus, the D- variants are more resistant to trypsin-mediated degradation than the L-variants.
  • PLNC8 ⁇ the L- and D-variant
  • lysis of erythrocytes isolated from human whole blood was investigated. However, no hemolytic activity was observed (Fig. 7). As such, there is no toxic effect on human cells.
  • At least 90% of the amino acids in said polypeptide X are D-amino acid residues.
  • At least 90% of the amino acids in peptide X and/or peptide Y are D-amino acid residues.
  • D-variants are increased stability and less sensitivity to proteolytic cleavage compared to their corresponding L-variants. This results in a longer lifetime of the D-variant peptides and thus prolonged antibacterial effect.
  • Non-natural or modified amino acids may also be introduced that enable convenient coupling chemistries, including click-chemistry approaches.
  • bacteriocins can also be modified with either N- or C-terminal azide groups to enable copper-free click reaction with e.g. cyclooctyne conjugated polymers.
  • biodegradable polymers such as hyaluronic acid (HA)
  • the release rate will be dependent on the hydrolysis rate of the biopolymer backbone and can be tuned to a certain extent by using different polymers.
  • hyaluronidase is expressed by S. aureus, as a virulence factor, degrading polysaccharides between cells and thereby enabling spreading of the infection.
  • the biodegradeable polymer is HA, the release rate of the peptides will increase in the presence of S. aureus.
  • PLNC8o$ is a two peptide bacteriocin, so in order to investigate the role of the PLNC8 a chain and PLNC8 b chain, respectively, in the inhibitory and bactericidal action of the bacteriocin, the effects of different molar ratios between the peptides on S. epidermidis were studied. It was found that a molar ratio of 1 : 1 is most efficient at inhibiting and killing S. epidermidis (Fig. 2). However, a ratio of between PLNC8 a chain and PLNC8 b chain of 1 : 1 to 1 :7 also showed a good effect.
  • the first and second peptides are present in a in a molar ratio of from between 5 : 1 to 1 :20, preferably 1 :1 to 1 :7, most preferably 1 :1.
  • combination therapy combinations of antimicrobial agents are utilized for the prevention of the development of resistance and to shorten the length of treatment time. It was investigated whether combinations of PLNC8 ⁇ together with different traditional antibiotics would be effective in the treatment of S. epidermidis. In Fig. 15, results are summarized for PLNC8 ⁇ together with Rifampicin and
  • PLNC8 ⁇ decreased MIC and MBC of teicoplanin more than lO-fold against S. epidermidis (Fig. 15).
  • a combination of PLNC8 ⁇ and rifampicin was found to be even more effective.
  • MIC and MBC of rifampicin was lowered more than lOO-fold when treating S. epidermidis in the presence of L-PLNC8 ⁇ or D-PLNC8 ⁇ .
  • L-PLNC8 ⁇ decreased MIC and MBC of gentamicin 15-30 fold against S. epidermidis.
  • L-PLNC8 ⁇ or D-PLNC8 ⁇ lowered MIC and MBC of vancomycin 2-fold. (Fig. 15 and Table 3).
  • MRSE Staphylococcus epidermidis
  • ESKAPE includes six pathogenic bacterialspecies (Enterococcusfaecium, Staphylococcusaureus, Klebsiellapneumoniae,
  • ESKAPE pathogens are prioritized by WHO to promote research and development of new antimicrobials, since multidrugresistance is a serious threat to global public health. Infections caused by these pathogens are often hospital-acquired, and pose a particular threat to patients requiring medical devices, such as catheters, ventilators and implants.
  • Enterococcus faecium and addition of sub-MIC concentrations significantly enhanced the effects of different antibiotics. This is shown in table 6 below.
  • PLNC8 b together with truncated PLNC8 a, PLNC8 a together with truncated PLNC8 b and truncated PLNC8 a together with truncated PLNC8 b markedly amplify the inhibitory effects of teicoplanin against S. epidermidis.
  • the pharmaceutical composition further comprises at least one antibiotic.
  • the peptide(s) and the antibiotic act synergistically and enhance the effect of each other.
  • antibiotics from the three largest groups of antibiotics were selected and tested; antibiotics that inhibit bacterial cell wall synthesis, antibiotics that inhibit nucleic acid synthesis and antibiotics that inhibit protein synthesis.
  • the combination of antibiotics and PLNC8 ⁇ provides a powerful synergistic effect, and reduces (up to 100 times) MIC and MBC for antibiotics from the three classes.
  • Fig. 15 teicoplanin, vancomycin, rifampicin, and gentamicin were evaluated.
  • the antibiotic is selected from the group consisting of antibioticts that inhibit bacterial cell wall synthesis, antibiotics that inhibit nucleic acid synthesis and antibiotics that inhibit protein synthesis.
  • the antibiotic is selected from a group consisting of rifampicin, gentamicin, teicoplanin and vancomycin.
  • the antibiotic is selected from a group consisting of rifampicin, gentamicin, and teicoplanin.
  • the composition may comprise between 10 nM to 50 mM of the first peptide and/or of the second peptide. As shown in Fig. 15, concentrations within the micromolar range effectively reduce S. epidermidis in the presence of an antibiotic.
  • the pharmaceutical composition comprises between 0.1 to 50 mM of peptide X and/or peptide Y.
  • MIC and MBC of rifampicin was lowered more than lOO-fold when treating S. epidermidis in the presence of L-PLNC8 ⁇ or D-PLNC8 ⁇ , resulting in an effective amount already at 0.0019 pg/ml.
  • the pharmaceutical composition comprises the antibiotic in an amount of at between 0.002 pg/ml to 50 pg/ml, such as at least 0.01 pg/ml to 5 pg/ml, such as at least 0.1 pg/ml to 1 pg/ml, such as at least 0.8 pg/ml.
  • the pharmaceutical composition comprises the antibiotic in an amount of at least 0.78 pg/ml, such as at least 0.097 pg/ml such as at least 0.0019 pg/ml, such as at least 0.0097 pg/ml.
  • the antibiotic is vancomycin in an amount of at least 0.78 pg/ml.
  • the antibiotic is teicoplanin in an amount of at least 0.097 pg/ml.
  • the antibiotic is rifampicin in an amount of at least 0.0019 pg/ml.
  • the antibiotic is gentamicin in an amount of at least 0.0097 pg/ml.
  • the antibiotic is a combination of at least two of vancomycin, teicoplanin, rifampicin and gentamicin.
  • antibiotics treatment may lead to unwanted side effects, such as affecting or even destroying the protective flora or stimulating the development of antibiotics resistance. Such treatment may also lead to changes in the intestinal bacterial composition, which may result in superinfection by fungi and other infective organisms.
  • Peptides X and Y may beneficially be administered locally, in the form of a solution, a cream, a gel or in immobilized form (as described further under coating below).
  • the formulations may further include a solvent and/or a variety of excipients, for instance to stabilize the peptides and suppress aggregation, such as solubilizers, surfactants, bulking agents (such as carbohydrates), thickeners (such as polymers) to increase solution viscosity, preservatives, vehicles, salts or sugars to stabilize proteins and to obtain physiological tonicity and osmolality and/or buffering agents to control pH.
  • excipients for instance to stabilize the peptides and suppress aggregation, such as solubilizers, surfactants, bulking agents (such as carbohydrates), thickeners (such as polymers) to increase solution viscosity, preservatives, vehicles, salts or sugars to stabilize proteins and to obtain physiological tonicity and osmolality and/or buffering agents to control pH.
  • the composition is formulated as a solution, a cream, a gel, or an ointment or formulated in immobilized form as a coating on a device.
  • the pharmaceutical composition is for use in the treatment or prophylaxis of a bacterial infection.
  • the composition is administered locally on the site of infection, such as topically.
  • peptides from PLNC8 ⁇ may be linked or associated with a supporting material.
  • PLNC8 ⁇ was loaded in a formula (gel) consisting of gelatin and glycerol.
  • PLNC8 ⁇ in the gel rapidly lysed S. epidermidis and the PLNC8 ⁇ -containing gel totally inhibited the growth of the bacteria on agar plates (Fig. 13).
  • the activity of PLNC8 ⁇ in the gel was stable after long-term storage at 4°C for at least 180 days.
  • the composition is formulated as a gel, wherein the gel further comprises gelatine and glycerol.
  • the effect of formulating the composition as a gel is to provide a localized, long-term antibacterial effect.
  • the bacterial infection is caused by Staphylococcus spp, Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus.
  • the bacterial infection is caused by Staphylococcus spp and/or Streptococcus spp.
  • Bacterial infection and inflammation is sometimes linked to implants, caused by the bacterial adherence and colonization in the implant area.
  • Treatment may include removing dead tissue, antibiotics, and improved hygiene.
  • Preventive measures include polishing the implant surface, to minimize bacterial adherence, which is a time consuming and costly procedure. Implant coating or treatment with antibacterial material would minimize these incidences and avoid the high-cost of producing a highly polished surface on implant.
  • a coating comprising the X and Y peptide of the invention (i.e. truncated bacteriocin NC8 ⁇ ), possibly together with an antibiotic, may be used to impart bacterial resistance to a coating for an implant.
  • such a coating may be used for any medical device, or part of a medical device, where bacterial colonization on the surface should be prevented.
  • the medical device may also be a band-aid comprising the first and second peptide (i.e. pepides X and Y) and/or antibiotic of the invention.
  • first and second peptide i.e. pepides X and Y
  • antibiotic of the invention This would help facilitate local administration on a wound or infection site.
  • the bacteriocin and antibiotic may either be tethered to a polymeric scaffold via a flexible linker or physically entrapped in a biopolymeric matrix, its bactericidal property will be retained, or even improved because of its high local concentration
  • the composition is formulated in immobilized form as a coating on a device, wherein the device is chosen from the group consisting of a wound dressing, an orthopedic implant, a dental implant, a urinary catheter and an urinary stent.
  • a pharmaceutical composition is used in coating at least part of a device to limit colonization of bacteria on the surface of the device.
  • the device is a medical device, such as a prosthesis or a wound dressing.
  • the bacterial infection is caused by
  • Staphylococcus spp including MRSA, MRSE
  • Streptococcus spp e.g. S. mutans, S. constellatus, S. anginosus
  • Enterococcus faecium including VRE
  • Klebsiella pneumoniae Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and/or Escherichia coli.
  • the bacteria are Staphylococcus spp, Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus. In one further embodiment, the bacteria are Staphylococcus spp and/or Streptococcus spp.
  • truncated b in combination with full length or truncated a, truncated a and in combination with truncated or full length b have a rapid and direct effect on different pathogens without expressing any toxic effects on surrounding human cells.
  • the combination enhances, 2-130 fold, the effect of and sensitivity to antibiotics.
  • Substitution of L-amino acids of PLNC8 ⁇ / ⁇ by D-amino acids does not change the anti-bacterial effects of the bacteriocin.
  • the D-form of PLNC8 ⁇ / ⁇ is much more stable against proteolytic cleavage and is thus adapted for a therapeutical use in vivo.
  • truncated b in combination with full length or truncated a, truncated a in combination with truncated or full length b, optionally in D/L form, optionally together with antibiotics, is very well suited for the prevention or treatment of infections.
  • This combination can be administered locally in soluble form in gels (ointments, creams) and in immobilized form, e.g. on wound dressings, orthopedic implants, dental implants, urinary catheters and stents, and act antibacterially with no cytotoxic side effects.
  • Such a combination provides the following advantages: It acts very fast (seconds to minutes); is effective and very potent (nano-micromolar doses); has a wide anti-bacterial spectrum - both against gram-negative and gram-positive bacteria;
  • Staphylococcus aureus CCUG 35601 MRSA, Culture Collection, University of Gothenburg
  • Staphylococcus aureus ATCC 29213 MSSA, ATCC, Manassas, VA
  • Staphylococcus epidermidis ATCC 12228 ATCC, Manassas, VA
  • RP62A RP62A
  • N15 and 10 clinical isolates of Staphylococcus epidermidis that have previously been characterized. Isolated Escherichia coli, Enterococcus faecium (including VRE), Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter spp and Acinetobacter baumannii were obtained from Orebro University hospital.
  • Isolated Streptococcus mutans, Streptococcus constellatus and Streptococcus anginosus were obtained from Malmo University.
  • the bacteria were grown on Luria-Bertani (LB) agar plates, supplemented with 5% defibrinated horse blood, and incubated at 37°C overnight. Single colonies were inoculated into 5 ml of LB broth and incubated on a shaker (300 rpm) at 37°C overnight. The bacterial concentration was determined by viable count and adjusted to correlate with approximately 10 9 CLU/ml.
  • DLTTKLW S S WGYYLGKKARWNLKHP YV QL -COOH DLTTKLW S S WGYYLGKKARWNLKHP YV QL -COOH
  • PLNC8 ⁇ H2N- SVPTSVYTLGIKILWSAYKHRKTIEKSFNKGFYH-COOH
  • scrambled-PLNC8 ⁇ H2N-TWLKY GHGDAKLWSWSKPLNLTFRY QYVK-COOH
  • scramblcd-PLNC8 ⁇ H2N-LKLWNTYGTFSRFYTSKSEVKIAHGIKSIHVPYK-COOH
  • truncated forms of PLNC8 ⁇ and PLNC8 ⁇ were synthesized using conventional Fmoc chemistry on a Pair automated peptide synthesizer (Protein Technologies, Inc) in a 100 pmol scale.
  • Peptide elongation was performed using a four-fold excesses of amino acid (Iris biotech gmbh) and activator (TBTU, Iris biothech gmbh) and using an eight- fold excesses of base (DIPEA). Fmoc removal was accomplished by treatment with
  • Liposomes were prepared by dry film formation, hydration and finally extrusion through a polycarbonate membrane to form monodisperse large unilamellar vesicles.
  • the lipids l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1- palmitoyl-2-oleoyl-sn-glycero-3 -phosphatidylcholine (POPC) (Avanti Polar Lipids, Alabaster, USA) were mixed at molar ratios 1 :99, 5:95 and 10:90 while dissolved in chloroform.
  • a dry lipid film was formed by evaporation of the chloroform by nitrogen flow and overnight lyophilization.
  • the film was hydrated with either 10 mM phosphate buffer (PB) pH 7 or 10 mM phosphate buffer saline (PBS) pH 7, and the solution was vortexed for 1 min and put on a shaker for 1 h before extruded 21 times through a 100 nm pore-sized polycarbonate membrane.
  • PB phosphate buffer
  • PBS phosphate buffer saline
  • the lipid film was hydrated with buffer (PBS) containing self-quenching concentration (50 mM) of 5(6)-carboxyfluorescein (CF) (Sigma Aldrich) and liposomes were prepared as described above. Removal of unencapsulated CF was done by gel filtration using a PD- 25 column (GE Healthcare, Uppsala, Sweden) and liposomes with encapsulated CF were eluted with PBS.
  • PBS buffer
  • CF 5(6)-carboxyfluorescein
  • Liposomes were diluted to 25 mM (total lipid concentration) in PBS, followed by additions of 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 mM of the L-form or D-form of PLNC8 a and b, separately and combined, and truncated forms of PLNC8 a, separately and combined with PLNC8 b, and truncated forms of PLNC8 b, separately and combined with PLNC8 a.
  • a final addition of 0.5 % Triton X-100 was made at the end of all measurements and the total amount of CF (100% release) was estimated after 15 min incubation.
  • the CF release is presented as percentage release for each time interval (measurements taken every minute). The percentage CF release is calculated as 100 x (F - F 0 )/(F T - F 0 ) where Fo is the initial fluorescence intensity of CF before peptide addition, F is the fluorescence intensity of CF at time point t and F T is the maximum fluorescence after the addition of Triton X-100. Results are shown in Fig. 4.
  • the broth microdilution method was used to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Two-fold serial dilutions of the peptides were used and the final concentrations ranged from 0.097-50 mM. The final concentrations of the antibiotics vancomycin and teicoplanin ranged from 0.097-50 Lig/ml, while rifampicin ranged from 0.0019-1 ⁇ g/ml and gentamicin 0.0097-5 Lig/m 1. The effect of bacteriocin-antibiotic combinations was accomplished by using the same concentration series of antibiotics with a constant concentration of bacteriocins (3.1 mM) in all the wells.
  • the MIC was determined visually and spectroscopically (620 nm) as the first concentration that completely inhibited bacterial growth. All concentrations that resulted in complete inhibition of bacterial growth were cultured (10 m ⁇ ) on blood- agar plates, and the lowest concentration where no growth was observed on agar represented the MBC. All experiments were repeated at least three times.
  • the fluorescent dye Sytox® Green which can only cross damaged membranes and fluoresce upon binding to nucleic acids, was used to study the antimicrobial activity of PLNC8 ⁇ on S. epidermidis, S. aureus (MSSA, MRSA) and Streptococcus spp. .
  • the bacteria were washed and resuspended in Krebs-Ringer Glucose buffer (KRG) (120 mM NaCl, 4.9 mM KC1, 1.2 mM MgS0 4 , 1.7 mM KH 2 P0 4 , 8.3 mM Na 2 HP0 4 , and 10 mM glucose, pH 7.3) and incubated in the presence or absence of different combinations of PLNC8 ⁇ in 96-well microtiter plates for 2 min. Images were captured with Olympus BX41 at 40x magnification.
  • KRG Krebs-Ringer Glucose buffer
  • Electron microscopy was used to visualize the damage of bacteria caused by PLNC8 ⁇ . Briefly, bacteria were pelleted and washed with Krebs-Ringer Glucose buffer (KRG) (120 mM NaCl, 4.9 mM KC1, 1.2 mM MgS0 4 , 1.7 mM KH 2 P0 4 , 8.3 mM Na 2 HP0 4 , and 10 mM glucose, pH 7.3). The bacteria were then treated with different concentrations of PLNC8 ⁇ in a ratio of 1 : 1 for 5 min, followed by fixation in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.3. Critical point drying was applied for specimens for SEM coated with Gold using a Sputter coater.
  • KRG Krebs-Ringer Glucose buffer
  • Specimens for TEM were washed in 0.1M phosphate buffer, postfixed in 2% osmium tetroxide in 0.1M phosphate buffer for 2 hours and embedded into LX- 112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (approximately 50-60 nm) were cut by a Leica ultracut UCT/ Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Hitachi HT 7700 (Tokyo, Japan). Digital images were taken by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Munster, Germany). Representative images of three independent experiments can be seen in Fig. 12.
  • Bacteriocins are often unstructured in solution but typically adopt a more ordered secondary structure when bound to the bacterial cell membrane as a result of membrane partitioning.
  • Circular dichroism spectroscopy measurements were performed on a Chirascan (Applied Photophysics, United Kingdom) using a 1 mm cuvette at room temperature. A wavelength scan of 195-280 nm was recorded 3 times for each sample, averaged and baseline corrected using PB buffer (pH 7.4, 10 mM). In all samples, the concentration of each peptide was 30 mM, prepared in PB buffer. In experiments with liposomes the final lipid concentration was 660 mM (0.5 mg/ml). To compensate for the different total peptide concentrations used, the averaged data were converted to mean residue ellipticity (MRE).
  • MRE mean residue ellipticity
  • the hemolytic activity of the peptides was investigated by collecting blood from healthy volunteers in heparinized vacutainers. The blood was centrifuged at 600 xg for 5 min and the erythrocyte pellet was washed three times in PBS. The cells were then suspended in PBS and added to 96-well plates (15% erythrocyte suspension/well), containing the peptides with two-fold serial dilution. The plates were incubated for 1 h at 37°C followed by centrifugation for 5 min at 900 xg and measurement of the supernatants at 540 nm. Haemolytic activity (%) was calculated by subtracting the negative control from all values and normalization against the positive control (0.5% Triton X-100), that was set to 100%. All experiments, each in duplicate, were repeated three times.
  • S. epidermidis RP62A was inoculated into 5 ml of LB broth and incubated on a shaker at 37°C overnight.
  • the bacterial culture was diluted 1 :100 into fresh media and 100 m ⁇ of bacterial suspension per well was added in a 96-well microtiter plate and incubated statically at 37°C for 20 h.
  • the wells were washed three times by submerging the plate into a container with distilled water to remove unattached cells.
  • Fresh LB media was added to each well (100 m ⁇ ) followed by addition of the peptides in different concentrations. The plate was incubated statically for 1 h.
  • Detached material in the wells were transferred to a new microtiter plate for absorbance measurements at 620 nm.
  • the remaining attached bio films were stained with 0.1% crystal violet for 15 min before the plate was washed four times in distilled water as mentioned above and allowed to dry at room temperature for 2 h.
  • the crystal violet was solubilized in 30% acetic acid for 15 min and the absorbance quantified at 550 nm. Each experiment, with three replicates, was repeated three times.
  • PLNC8 ⁇ markedly inhibited the growth and the survival of all bacterial strains (Fig. 1).
  • the antibacterial activity of PLNC8 ⁇ may in vivo be restricted by proteolytic activity exerted by proteases from both bacteria and human cells.
  • proteases from both bacteria and human cells.
  • the L-form of amino acids that normally occurs in peptides such as PLNC8 ⁇
  • the effects of the L- and D-variants of PLNC8 ⁇ were tested on both a liposome system (resembling bacteria) and on S. epidermidis. It was found that the D- variant of PLNC8 ⁇ was almost as effective in destroying liposomes and inhibiting and/or killing S. epidermidis as the L-variant (Fig. 4). Furthermore, the perturbation of the plasma membrane of S. epidermidis was equally rapid (2 min) for the L- and D- variant, respectively, of PLNC8 ⁇ (Fig. 5).
  • D-PLNC8 ⁇ , D-PLNC8 ⁇ , L-PLNC8 ⁇ and L-PLNC8 ⁇ were exposed to trypsin and the presence of proteolytic fragments was analyzed with MALDI-TOF mass spectrometry (Fig. 6). While trypsin generated several fragments of both the a- and b-peptide of L-PLNC8, no obvious fragmentation was observed of the a- and b-peptide of D-PLNC8.
  • Truncated forms of PLNC8 ⁇ express antibacterial activities similar to the native bacteriocin or are even more effective.
  • Truncated peptides of PLNC8 a and PLNC8 b, respectively, were constructed in sequences of 6-7 amino acids corresponding to the number of amino acids needed for formation of an alpha helix (figure 8). The effects of truncated PLNC8 a and PLNC8 b were tested on both a liposome system (resembling bacteria) and on S. epidermidis.
  • the peptide b-sequences b7-13 and b14-20 are crucial for the effects of PLNC8 b and are more efficient when combined with b1-6. Thus, the peptide b1-20 is most effective in inhibiting S. epidermidis.
  • PLNC8 ⁇ was used with a supporting material.
  • PLNC8 ⁇ was loaded in a formula (gel) consisting of gelatin and glycerol.
  • PLNC8 ⁇ in the gel rapidly lysed S. epidermidis and the PLNC8 ⁇ -containing gel totally inhibited the growth of the bacteria on agar plates (Fig. 13).
  • the activity of PLNC8 ⁇ in the gel was stable after long-term storage at 4°C for at least 180 days.
  • Heterogeneous glycopeptide intermediate S. epidermidis is common in prosthetic joint infections (PJIs). Glycopeptide treatment, such as treatment with vancomycin and teicoplanin, is not sufficient in many cases of PJIs.
  • PLNC8 ⁇ effectively inhibits different strains of S. epidermidis isolated from PJIs, including S. epidermidis (hGISE) (figure 14). The D-form of PLNC8 ⁇ is almost as effective as the L-form in inhibiting strain S. epidermidis 154 (Fig. 15).
  • Combination therapy is utilized both to prevent the development of antibiotic resistance and to shorten the length of treatment. The effect in the treatment of S.
  • epidermidis of the combination of L-PLNC8 ⁇ or D-PLNC8 ⁇ with different antibiotics belonging to different classes was also shown: the cell wall synthesis inhibitors vancomycin and teicoplanin, the nuclic acid synthesis inhibitor rifampicin and the protein synthesis inhibitor gentamicin.
  • L-PLNC8 ⁇ and D-PLNC8 ⁇ decreased MIC and MBC of teicoplanin more than lO-fold against S. epidermidis (Fig. 15).
  • a combination of PLNC8 ⁇ and rifampicin was even more effective.
  • MIC and MBC of rifampicin was lowered more than lOO-fold when treating S. epidermidis in the presence of L-PLNC8 ⁇ or D-PLNC8 ⁇ (Fig. 15).
  • L-PLNC8 ⁇ decreased MIC and MBC of gentamicin 15 -30 fold against S. epidermidis.
  • L-PLNC8 ⁇ or D-PLNC8 ⁇ lowered MIC and MBC of vancomycin 2-fold.
  • a combination of the truncated ⁇ -peptide 1-22 with full-length b-peptide decreased MIC and MBC of teicoplanin more than lO-fold against S. epidermidis (figure 15), i.e. the same effects as with PLNC8 ⁇ (figure 14).
  • ⁇ 1-22 and b1-20 lowered MIC and MBC of teicoplanin approximately 4-fold, however, full-length ⁇ - peptide and b1-20 had no effects (figure 16).
  • the full- length and truncated PLNC8 b and PLNC8 a markedly amplify the inhibitory and bactericidal effects of teicoplanin and rifampicin against S. epidermidis.
  • a combination of the truncated ⁇ -peptide 1-22 with full-length b-peptide decreased MIC of rifampicin approximately 4-fold against S. epidermidis. ⁇ 1-22 and b1-20, respectively full-length ⁇ -peptide and b1-20, have two-fold effect (Fig. 16).
  • PLNC8 ⁇ markedly permeabilizes and kills different species of Streptococcus. Streptococcus spp (S. mutans (Sm), S. constellatus (Sc) and S. anginosus (Sa)) were treated with 5mM PLNC8 ⁇ for 2 min, followed by analysis of uptake of Sytox Green. S. constellatus and S. anginosus were more susceptible to PLNC8 ⁇ than S. mutans. References

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Abstract

L'invention concerne un polypeptide X ayant 14 à 28 acides aminés et comprenant une séquence d'acides aminés ayant au moins 90 %, 95 %, 96 %, 97 %, 98 % ou 99 % d'identité de séquence (% SI) avec SEQ ID NO 3. L'invention concerne également une composition pharmaceutique du polypeptide X, la composition pharmaceutique peut en outre comprendre un polypeptide Y ayant de 15 à 28 acides aminés et comprenant une séquence d'acides aminés ayant au moins 90 %, 95 %, 96 %, 97 %, 98 % ou 99 % d'identité de séquence (% SI) avec SEQ ID NO 4. La composition pharmaceutique peut être utilisée dans le traitement ou la prophylaxie d'une infection bactérienne.
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SE2151338A1 (en) * 2021-10-29 2023-04-30 Curenc Ab Truncated peptides

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ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
BENGTSSON, T.; ZHANG, B.; SELEGARD, R.; WIMAN, E.; AILI, D.; KHALAF, H.: "Dual action bacteriocin PLNC8 αβ through inhibition of Porphyomonas gingivalis infection and promotion of cell proliferation", PATHOGENS AND DISEASE, 12 June 2017 (2017-06-12)
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MALDONADO ANTONIO ET AL: "Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 69, no. 1, 1 January 2003 (2003-01-01), pages 383 - 389, XP002563184, ISSN: 0099-2240, DOI: 10.1128/AEM.69.1.383-389.2003 *
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SE2151338A1 (en) * 2021-10-29 2023-04-30 Curenc Ab Truncated peptides
WO2023075675A1 (fr) * 2021-10-29 2023-05-04 Curenc Ab Peptide antibactérien

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